Proteomic profiling of regenerated urinary bladder tissue with stem cell seeded scaffold composites in a non-human primate bladder augmentation model

Abstract

Urinary bladder insult can be caused by environmental, genetic, and developmental factors. Depending upon insult severity, the bladder may lose its ability to maintain capacity and intravesical pressures resulting in renal deterioration. Bladder augmentation enterocystoplasty (BAE) is employed to increase bladder capacity to preserve renal function using autologous bowel tissue as a “patch.” To avoid the clinical complications associated with this procedure, we have engineered composite grafts comprised of autologous bone marrow mesenchymal stem cells (MSCs) with CD34+ hematopoietic stem/progenitor cells (HSPCs) co-seeded onto a pliable synthetic scaffold [POCO; poly(1,8-octamethylene-citrate-co-octanol)] or a biological scaffold (SIS; small intestinal submucosa) to regenerate bladder tissue in a baboon bladder augmentation model. We set out to determine the protein expression profile of bladder tissue that has undergone regeneration with the aforementioned stem cell seeded scaffolds along with baboons that underwent BAE. Data demonstrate that POCO and SIS grafted animals share high protein homogeneity between native and regenerated tissues while BAE animals displayed heterogenous protein expression between the tissues following long-term engraftment. We posit that stem cell seeded scaffolds can recapitulate tissue that is almost indistinguishable from native tissue at the protein level and may be used in lieu of procedures such as BAE.

1 |. Introduction

Urinary bladder dysfunction can arise from neurologic conditions such as myelodysplasia, spina bifida (SB) exhibited in pediatric patients, spinal cord injury (SCI), traumatic brain injury (TBI), cerebral palsy, and military-based trauma. [1,2]. For combat military personnel, bladder injury is often a result of penetrating trauma [3]. Between October 2001 through January 2008, US military personnel who served overseas were found to exhibit bladder injury at rate of 21.3% (or 189) of the recorded 887 who had unique genitourinary (GU) injuries [4]. In a study by Kronstedt et al who analyzed the data set from the Department of Defense Trauma Registry (DoDTR) from January 1, 2007 to March 17, 2020, of the 2,584 GU combat injured soldiers who required surgery, 1,090 (42%) required bladder repairs. Additionally, in a study of 530 veterans enrolled in Trauma Infectious Disease Outcomes Study (TIDOS), 89 patients acquired genitourinary injuries from deployment within the period of 2009 – 2014. Of the 89 patients, 19 (21%) of the patients had bladder injury and a majority (52.6%) of these patients had UTIs [5]. The goal in managing a severely dysfunctional bladder includes preserving physiological function of the bladder so that urine can be stored under low pressure while maintaining its ability to void efficiently under volitional control. When conservative management fails, including the use of medicines or catheterization, surgical intervention in the form of urinary bladder diversion procedures or bladder augmentation enterocystoplasty (BAE) is often employed for the treatment of severe bladder conditions [6,7]. The ideal clinical outcome of these types of procedures is to increase bladder capacity and compliance by reducing intravesical pressure in order to protect renal function and improve on quality-of-life metrics [8].

BAE employs the use of autologous intestinal tissue including the ileum, gastric segments, or colonic segments. Unfortunately, this highly invasive surgical procedure poses unwanted long-term issues [1,6,8,9,10]. The ileum and colon promote the formation of bladder calculi at a rate of 3–52.5% and occurring as early as 5 months post-BAE [1,11,12]. The gastric segment is the last option and has a reduced risk of calculi but poses its unique complications such as hematuria-dysuria syndrome and increased risk of malignancy [9]. Additionally, other clinical issues arise over time including, excessive mucus production, electrolyte imbalances, and perforation can occur in all cases [13]. These issues are treatable in most cases but forces patients to perform intermittent self-catheterization to prevent bladder infection, calculi, and urinary tract infections. These issues can still arise even with ideal management, yet efficacy varies patient to patient. Although BAE is somewhat effective, the 10-year risk rate of re-do surgery is as high as 43.9% for spina bifida patients, for example [14].

Within the context of this study, we describe the proteomic profiling of three unique grafts used for bladder augmentation. These include the current gold standard for BAE (ileum), small intestininal submucosa (SIS) (a widely utilized biological scaffold) [15,16], and the highly reproducible synthetic scaffold, poly(1,8-octamethylene-citrate-co-octanol) (POCO) within the context of a non-primate (baboon) bladder augmentation model. Both the SIS and POCO scaffolds were co-seeded with autologous bone marrow derived mesenchymal stem cells (MSCs) with CD34+ hematopoietic stem/progenitor cells (HSPCs) prior to graft implantation [13,17,18]. Baboons underwent partial cystectomy and were then independently grafted with either autologous ileum in Enterocystoplasty (E), cell-seeded SIS (CS-SIS), or cell-seeded POCO (CS-POCO). At the conclusion of the study, native and regenerated (or ileum-augmented) bladder tissues were collected. The proteomic profiles of regenerated or ileum-augmented versus native bladder were analyzed and compared. This study is the first of its kind to demonstrate proteomic profiling in a large bladder tissue deficit, baboon bladder augmentation model that bears significant phylogenetic resemblance to human counterparts.

2 |. MATERIALS AND METHODS

2.1 |. Baboon bladder augmentation procedure

The study design for the bladder augmentation in the baboon (Papio anubis) model was described previously [12]. Briefly, a 50–65% bladder cystectomy was performed in the animal cohorts and the bladder deficit was augmented with either ileum (enterocystoplasty; E), cell-seeded (bone marrow derived, autologous MSCs and CD34+ HSPCs) biological scaffold (CS-SIS), and cell-seeded biodegradable and elastomeric scaffold (CS-POCO); n=3 baboons/group. Tissue-centric analyses utilized samples at 24M (CS-POCO); 24M (one animal at 27M, E); 26–29M (CS-SIS).

2.2 |. Protein and peptide purification

50 mg of tissue from each sample was homogenized in 1 ml lysis buffer containing 8 M urea, 1% SDS, in 50 mM HEPES pH 8.5, and HALT protease inhibitor cocktail (Thermo Fisher Scientific). The tissue extract was centrifuged at 3000g for 15 min to eliminate tissue debris and the supernatant was transferred to a new tube. 200 ug of protein from each sample was purified from impurities and lipids by methanol-chloroform precipitation and resuspended in 6M guanidine in 100 mM triethylammonium bicarbonate (TEAB). Proteins were reduced with 1 mM DTT and alkylated with 5 mM iodoacetamide, and were further diluted with 100 mM TEAB to minimize the guanidine hydrochloride concentration to less than 1.5 M before digestion with trypsin/lys-C protease mix, MS Grade, 1:50 ratio, (Thermo Fisher Scientific) overnight at 37°C. The digest was then acidified with formic acid to a pH of ∼2–4 and desalted by using C18 HyperSep cartridges. The purified peptide solution was dried and quantified using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific). An equal amount of peptide (∼50 μg) from each sample was then used for isobaric tandem mass tag (TMT) labeling as per the manufacturer’s instructions (Thermo Fisher Scientific). After two hours of incubation at room temperature, the reaction was quenched with hydroxylamine at a final concentration of 0.3% (v/v). Isobaric-labeled samples were then combined and lyophilized. The combined isobaric labeled peptide samples were then fractionated by Pierce High pH Reversed-Phase Peptide Fractionation Kit to eight fractions per the manufacturer’s protocol. Fractions were then dried using a speed vac and reconstituted in LC-MS sample buffer (5% acetonitrile, 0.125% formic acid) until LC-MS/MS analysis and concentration were assessed using Micro BCA.

2.3 |. MS/MS tandem mass spectrometry

Purified peptides, 1.0 ug each, were loaded onto a Vanquish Neo UHPLC system (Thermo Fisher Scientific) with a heated trap and elute workflow with a c18 PrepMap, 5mm, 5μM trap column (P/N 160454) in a forward-flush configuration connected to a 25cm Easyspray analytical column (P/N ES802A rev2) 2μM, 100A, 75um x 25 with 100% Buffer A (0.1% formic acid in water) and the column oven operating at 35°C. Peptides were eluted over a 240 min gradient, using 80% acetonitrile, 0.1% formic acid (buffer B), starting from 2.5% to 10% over 10 min, to 30% over 140 min, to 40% over 60 min, to 65% over 18 min, then to 99% in 5 min and kept at 99% for 7 min, after which all peptides were eluted. Spectra were acquired with an Orbitrap Eclipse Tribrid mass spectrometer with FAIMS Pro interface (Thermo Fisher Scientific) running Tune 3.5 and Xcalibur 4.5 and using TMTpro SPS-MS3 RTS FAIMS method implemented in the Xcaliber software with Real Time search filter (RTS) for MS3 triggering. For all acquisition methods, spray voltage set to 1900V, and ion transfer tube temperature set at 300°C, FAIMS switched between CVs of −40 V, – 55 V, and −70 V with cycle times of 1.0s. MS1 detector set to orbitrap with 120 K resolution, wide quad isolation, mass range = normal, scan range = 400–1600 m/z, max injection time = 50 ms, AGC target = 300% microscans = 1, RF lens = 30%, without source fragmentation, and datatype = positive and centroid. Monoisotopic precursor selection was set to included charge states 2–6 and reject unassigned. Dynamic exclusion was allowed n = 1 exclusion for 40 s with 10ppm tolerance for high and low. An intensity threshold was set to 5000. Precursor selection decision = most intense. MS2 settings include isolation window = 0.7, scan range = auto normal, collision energy = 30% CID, scan rate = turbo, max injection time = 35 ms, AGC target = 1 × 104, Q = 0.25. In MS3, an on-line real-time search algorithm (Orbiter) was used to trigger MS3 scans for quantification. MS3 scans were collected in the Orbitrap using a resolution of 50,000, scan range 100–500, notch number =10, activation type HCD=55%, maximum injection time of 200 ms, and AGC of 200%. Isobaric tag loss exclusion was set to TMT pro reagent.

2.4 |. MS/MS data analysis

Raw data were analyzed using Proteome Discoverer 2.5 (Thermo Fisher Scientific) using the “PWF Tribrid TMTpro Quan SPS MS3 SequestHT Percolator” and “CWF Comprehensive Enhanced Annotation Reporter Quan” methods implemented in the PD2.5 software. The data were searched against the Baboon UniProt Protein Sequence Database [Papio anubis (species) Proteome Taxon ID 9555]. The search parameters included precursor mass tolerance of 10 ppm and 0.6 Da for fragments, allowing two missed trypsin cleavages, acetylation(+42.011 Da), Met-loss / −131.040 Da (M), and Met-loss+Acetyl (−89.030 Da (M) as N-terminal dynamic modification and carbamidomethylation (Cys), TMTpro / +304.207 Da in any N-terminus, TMTpro / +304.207 Da (K) as a static modification. Percolator PSM validation was used with the following parameters: strict false discover rate (FDR) of 0.01, relaxed FDR of 0.05, maximum ΔCn of 0.05, and validation based on q-value. Reporter ion quantitation was using the method TMTpro 18plexlotsWC320807, 18-plex Tandem Mass Tag^® of Proteome Sciences plc method implemented on the proteome discoverer software and general quantification settings used with following settings, Peptides to Use: Unique + Razor; Consider Protein Groups for Peptide Uniqueness set as True; Normalization based on Total Peptide Amount; Scaling Mode set as none, low abundance peptides were removed by filtering out proteins with less than 3 PSMs, Protein ratio was calculated based on protein abundance and t-test were used for class comparison. Differentially expressed proteins were selected based on p-value < 0.05 and log₂ fold change > 1.0 or < −1.0, or a 2-fold change. A positive fold change indicated a higher expression in the grafted or regenerated tissue compared to native tissue, while a negative fold change a higher expression in the native tissue compared to grafted or regenerated tissue. For uncharacterized proteins or proteins with unknown function presented in the manuscript, the UniProt Accession number was search against UniProt database https://www.uniprot.org/ and the NCBI Gene database https://www.ncbi.nlm.nih.gov/gene/. If required, more information was obtained with regards to protein identity by matching the amino acid sequence of the protein on the NCBI BLAST alignment program https://blast.ncbi.nlm.nih.gov/Blast.cgi.

2.5 |. Data normalization

Data was normalized by calculating the total sum of the abundance values for each channel over all the peptides identified. The channel with the highest total abundance was considered as a reference and correction was made for the abundance values in all other channels by a constant factor so that total abundance in all channels were the same.

3 |. RESULTS

3.1 |. Box plot distribution

In our study, we had examined the tissues collected from the native bladder tissue and compared against its own grafted tissue. There were three baboons in each of the three study groups, and 2 tissue samples from each animal, either grafted (for E group) or regenerated (for CS-POCO and CS-SIS) and native tissue samples, resulting in a total of 18 tissue samples as shown in Figure 1. The box plot was created to visualize the variation in abundances of mass spectrometric signals across different samples and conditions. As shown in the figure, the boxed area in each sample contained data that fell within Interquartile Range (IR), or 25–75% of the data range which includes data from Q1 to Q3, with the indicated median being at Q2. Any data points outside of the bars of the upper or lower region were single data points that are Q3 + 1.5IR or Q3 – 1.5IR, respectively. As shown in the figure, the IR for all 18 samples was similar and consistent across all samples indicating a uniform distribution of the data and suggested a tightly grouped result set. Since the median of the data was similar across all samples, there were minimal number of outliers and minimal variations variables or shifts amongst the samples.

Figure 1:

Box Plot Distribution of 18 tissue samples examined. For each study group-Entero, CS-POCO, and CS-SIS, there were 3 animals and for each animal the grafted or regenerated tissue was studied along with its matched native tissue sample. The colored box indicated the Interquartile Range of 25–75% of the data range with a line in the middle indicating the median. The outliers were single proteins shown for each of the sample.

3.2 |. Volcano plots

Proteomic profiles of the regenerated (or grafted) bladder tissue vs. the native bladder tissue for each animal were generated using our baboon bladder augmentation model. The data for the three animals were then averaged within each three groups (E, CS-POCO, and CS-SIS) of bladder augmentation. We analyzed a total of 5292 possible proteins and examined their expression patterns in the tissues within the aforementioned groups and determined the ratio of expression between the regenerated vs. native for each tissue graft type. The entire proteomic database for all 5292 proteins is included in the supplemental section, which contained expression levels for each tissue as well as differential expression levels for each regenerated/grafted bladder tissue type when compared to its own bladder native tissue.

For visualization of differential protein expression pattern, the proteomics data from the three groups was represented in our volcano plots, with the p-value graphed against the log₂ fold ratio. Figures 2A, 2B, and 2C showed volcano plots of Entero: Grafted vs. Native, CS-POCO: Regenerated vs. Native, and CS-SIS: Regenerated vs. Native, respectively, comparing grafted tissue to its native tissue. For consideration for differential protein expression the p-value was less than 0.05 while the log₂-fold change was either ≥ 1 or ≤ −1. As indicated on the volcano plots, the areas where the proteins that were indicated as differentially expressed had log₁₀ p-value > 1.3 (or p-value < 0.05), and log₂ fold ratio ≥ 1 for the proteins expression in the grafted or regenerated tissue to be higher than its native tissue (as marked by pink box) or log₂ fold ratio ≤ −1 for protein expression in the grafted or regenerated tissue to be lower than its native tissue (marked in blue box). As shown in Figure 2A the E group with the autograft ileum, when comparing the grafted tissue to the native tissue, of the 5292 proteins that were surveyed, 160 proteins were expressed at higher level in the native tissue compared to the E grafted tissue at a log₂ fold ratio ≤ −1, both at p-value < 0.05. The information of these 160 proteins were included in Table 1A. Conversely, 259 proteins had higher expression in the E grafted tissue than the native tissue at a log₂ fold ratio ≥ 1 and are detailed in Table 1B. In Figure 2B, the CS-POCO study group, no protein in the generated tissue had significant overexpression over the native tissue, while 2 proteins had log₂ fold ratio ≤ −1 or higher expression level in native tissue than CS-SIS regenerated tissue, at significant level of p-value < 0.05. In Figure 2C, the CS-SIS study group, there was 1 protein that had a log₂ fold ratio ≥ 1 (expressed in higher levels in CS-SIS regenerated tissue than its native tissue) and 5 proteins that had log₂ fold ratio ≤ −1 (expressed at higher level native protein than CS-SIS regenerated tissue), also all had a p-value < 0.05. The differently expressed proteins are shown in the tables in the following section, where Table 1A and Table 1B include differentially expressed proteins in the E group as represented in Figure 2A, while Table 1C and Table 1D correspond to Figure 2B and Figure 2C, respectively.

Figure 2:

Volcano Plots showing the differentially expressed protein distribution of the grafted or regenerated tissue vs. the native tissue. Each protein is shown as a dot in the graph. Proteins in the green colored box indicated proteins that were highly expressed in the native tissue vx. the grafted or regenerated tissue with log2 fold ratio of < −1 and p-value < 0.05. Proteins in the pink colored box indicated proteins that were highly expressed in the grafted or regenerated tissue vs. the tissue with log2 fold ratio of > 1 and p-value < 0.05. (A) Entero: Grafted vs. Native tissue showed many differentially expressed proteins in both grafted and native tissue. (B) CS-POCO: Regenerated vs. Native tissue showed the least number of proteins differentially expressed in either tissue. (C) CS-SIS: Regenerated vs. Native showed 4 proteins highly expressed in the Native tissue and 1 protein in the Regenerated tissue.

Table 1A.

Enterocystoplasty- Ileal grafted proteins < Native proteins

UniProt Accession #	Gene Symbol	Description	Biological Process	Cellular Component	Molecular Function	Abundance Ratio (log2): Ileal graft/Native	Abundance Ratio P-Value: Ileal graft/Native
A0A8I5QZL2	PCP4	Purkinje cell protein 4	calmodulin dependent kinase signaling pathway;positive regulation of neuron differentiation	protein-containing complex	calcium ion binding;calmodulin binding	−4.09	0.034
A0A096NPC2	TRIM29	Tripartite motif containing 29	negative regulation of protein localization to nucleus;negative regulation of transcription by RNA polymerase II	cytoplasm	identical protein binding;p53 binding;zinc ion binding	−3.55	0.039
A0A2I3M5V3	AGTR2	Type-2 angiotensin II receptor	stress response;signal transduction;other biological processes	plasma membrane;other membranes	other molecular function	−3.24	0.038
A0A2I3LWV5	HPSE2	Heparanase 2 (inactive)		membrane	hydrolase activity, acting on glycosyl bonds	−3.13	0.012
A0A096NZQ7	SBSPON	SMB domain-containing protein		cellular anatomical entity		−2.99	0.011
A0A2I3M6J6	FHL1	LIM zinc-binding domain-containing protein	muscle organ development		metal ion binding	−2.81	0.022
A0A2I3N649	MPP2	MAGUK p55 scaffold protein 2	cell proliferation;cell signal transduction;signaling pathways			−2.76	0.013
A0A096MRC5	NDUFA4	NDUFA4 mitochondrial complex associated	NADH dehydrogenase activity and oxidoreductase activity	membrane		−2.68	0.024
A0A8I5NB52	EGFLAM	EGF like, fibronectin type III and laminin G domains		extracellular region	calcium ion binding	−2.67	0.031
A0A8I5N6C2	CDK5RAP1	CDK5 regulatory subunit associated protein 1		anchoring junction;cytoskeleton	structural molecule activity	−2.52	0.022
A0A096NLE0	SRL	Sarcalumenin		membrane	GTP binding	−2.50	0.050
A0A8I5NLI7	KLHL41	Kelch like family member 41		BBSome		−2.46	0.013
A0A2I3LFN3	CNDP1	Carnosine dipeptidase 1	proteolysis;regulation of protein metabolic process	cytosol	dipeptidase activity	−2.43	0.038
A0A8I5MXU6	PARMl	Prostate androgen-regulated mucin-like protein 1		cytosol;early endosome;Golgi apparatus late endosome;nucleoplasm;plasma membrane		−2.35	0.031
A0A096N640	P2RY14	Purinergic receptor P2Y14	signal transduction	other membranes	other molecular function	−2.33	0.008
A0A096NNR5	PTGIS	Prostacyclin synthase (Prostaglandin 12 synthase)	apoptotic signaling pathway; cellular response to hypoxia, to interleukin-1, to interleukin-6; negative regulation of inflammatory response, of NF-kappaB transcription factor activity, of nitric oxide biosynthetic process; positive regulation of angiogenesis, of execution phase of apoptosis, of peroxisome proliferator activated receptor signaling pathway; prostaglandin biosynthetic process	caveola;endoplasmic reticulum membrane;nucleus	heme binding;hydroperoxy icosatetraenoate dehydratase activity; iron ion binding; monooxygenase activity; oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen;prostaglandin-l synthase activity	−2.33	0.017
A0A0A0MW42	GNG7	Guanine nucleotide-binding protein subunit gamma	signal transduction	plasma membrane;other membranes	other molecular function	−2.31	0.017
A0A096ND84	EFEMP2	EGF containing fibulin extracellular matrix protein 2	cell organization and biogenesis;other biological processes	non-structural extracellular;extracellular matrix	other molecular function	−2.29	0.028
A0A096NCU7	SLITRK6	SLIT and NTRK like family member 6	cell organization and biogenesis;developmental processes;other biological processes	plasma membrane;other membranes;other cell component		−2.29	0.034
A0A096NUF7	FBLNS	Fibulin-5	cell organization and biogenesis	non-structural extracellular	other molecular function	−2.16	0.013
A0A096NHT4	PI16	Peptidase inhibitor 16	negative regulation of cell growth involved in cardiac muscle cell development	extracellular space		−2.14	0.042
A0A096NR13	TPBG	Trophoblast glycoprotein	cell-cell signaling;cell organization and biogenesis;developmental processes;other biological processes	plasma membrane;other membranes;ER/Golgi;other cell component		−2.14	0.048
A0A096NNW4	PCOLCE	Procollagen C-endopeptidase enhancer		extracellular region	collagen binding;heparin binding; peptidase activator activity	−2.12	0.005
A0A2I3M360	ITIH5	Inter-alpha-trypsin inhibitor heavy chain 5	hyaluronan metabolic process;negative regulation of peptidase activity	extracellular region	serine-type endopeptidase inhibitor activity	−2.06	0.006
A0A096NPR2	PRELP	Proline and arginine rich end leucine rich repeat protein	cellular senescence			−2.06	0.046
A0A096NFV1	TACSTD2	Tumor associated calcium signal transducer 2		membrane		−2.03	0.033
A0A2I3MTL4	DYSF	Dysferlin		cytoplasmic vesicle membrane;plasma membrane	metal ion binding	−2.00	0.020
A0A2I3LMG0	PTPRU	Protein-tyrosine-phosphatase	cell adhesion;other biological processes	other membranes;other cell component	other molecular function	−1.89	0.001
A0A096NKR2	FBLN1	Fibulin-1	cell organization and biogenesis;other biological processes	non-structural extracellular	enzyme regulator activity;other molecular function	−1.86	0.008
A0A096NXG3	TGM2	Transglutaminase 2	protein metabolism;other metabolic processes;transport;developmental processes;signal transduction;other biological processes	extracellular matrix;plasma membrane;other membranes;cytosol;mitochondrion;ER/Golgi;nucleus;other cell component	other molecular function	−1.85	0.039
A0A2I3LCG8	ADH7	Alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide	alcohol metabolic process		oxidoreductase activity;zinc ion binding	−1.85	0.048
A0A096P1P0	KANK1	KN motif and ankyrin repeat domains 1	cell cycle OR cell proliferation;cell organization and biogenesis;other biological processes	plasma membrane;other membranes;other cell component	other molecular function	−1.79	0.020
A0A2I3LKZ6	FBLN1	Fibulin-1	cell organization and biogenesis; protein metabolism; other metabolic processes; stress response; other biological processes	non-structural extracellular;extracellular matrix	signal transduction activity or receptor binding;enzyme regulator activity;extracellular structural activity;other molecular function	−1.78	0.006
A9X179	ENPP1	Ectonucleotide pyrophosphatase/phosphodiesterase 1	3’-phosphoadenosine 5’-phosphosulfate metabolic process; ATP metabolic process; bone mineralization; cellular response to insulin stimulus;gene expression; generation of precursor metabolites and energy; immune response; inorganic diphosphate transport; intracellular phosphate ion homeostasis; melanocyte differentiation; negative regulation of cell growth, of fat cell differentiation, of glucose import, of glycogen biosynthetic process, of insulin receptor signaling pathway, of protein autophosphorylation; nucleic acid phosphodiester bond hydrolysis; nucleoside triphosphate catabolic process;response to ATP; response to inorganic substance; sequestering of triglyceride	cell surface;extracellular space;plasma membrane	ATP binding;cyclic-GMP-AMP hydrolase activity;dinucleotide phosphatase activity;insulin receptor binding;metal ion binding;nucleic acid binding;activity;phosphatase activity;phosphodiesterase 1 activity; polysaccharide binding;protein homodimerization activity;scavenger receptor activity	−1.74	0.001
A0A8I5NMX7	GPX3	Glutathione peroxidase	hydrogen peroxide catabolic process;response to oxidative stress	extracellular space	glutathione peroxidase activity; identical protein binding; selenium binding	−1.73	0.013
A0A096NRF7	QPCT	Glutaminyl-peptide cyclotransferase	protein metabolism;other metabolic processes		other molecular function	−1.71	0.024
A0A2I3NB39	ITGA8	Integrin subunit alpha 8	cell adhesion;cell organization and biogenesis;developmental processes;signal transduction	plasma membrane;other membranes;ER/Golgi;other cell component		−1.69	0.045
A0A8I5NPT2	CNRIP1	CB1 cannabinoid receptor-interacting protein 1		cytoplasm;plasma membrane	type 1 cannabinoid receptor binding	−1.66	0.006
A0A096NLI0	PPL	Periplakin	cell organization and biogenesis;other biological processes	cytoskeleton;other cell component		−1.65	0.038
A0A096N3B2	ADH4	Alcohol dehydrogenase 4 (class II), pi polypeptide	alcohol metabolic process		oxidoreductase activity; zinc ion binding	−1.64	0.003
A0A096N141	LAMA 3	Laminin subunit alpha 3	cell adhesion;developmental processes;other biological processes	non-structural extracellular; extracellular matrix; other membranes; ER/Golgi	signal transduction activity or receptor binding	−1.64	0.046
A0A096NPO6	EFEMP1	EGF containing fibulin extracellular matrix protein 1	protein metabolism;other metabolic processes;signal transduction	extracellular matrix	kinase activity;other molecular function	−1.59	0.020
A0A096NM33	LOC100997454	TNF alpha induced protein 8 like 3	regulation of apoptotic process			−1.59	0.034
A0A2I3MT23	POSTN	Periostin	cell adhesion			−1.58	0.049
A0A096MTH6	COL6A3	Collagen type VI alpha 3 chain	cell adhesion;other biological processes	non-structural extracellular;extracellular matrix;plasma membrane;other membranes;ER/Golgi;other cell component	enzyme regulator activity	−1.57	0.005
A0A096P654	PLCD3	Phosphoinositide phospholipase C	angiogenesis;intracellular signal transduction;labyrinthine layer blood vessel development;lipid catabolic process.regulation of cell population proliferation	nucleoplasm;plasma membrane	metal ion binding;phosphatidylinositol phospholipase C activity	−1.55	0.011
A0A096MLI9	LRRN4CL	LRRN4 C-terminal like		membrane		−1.54	0.015
A0A8I5N4E7	OLFM1	Olfactomedin like 1		extracellular region		−1.54	0.025
A0A096NZP1	SDK1	Sidekick cell adhesion molecule 1	behavioral response to cocaine;regulation of dendritic spine development	membrane		−1.54	0.027
A0A096PO42	MOXD1	Monooxygenase DBH like 1			copper ion binding;dopamine beta-monooxygenase activity	−1.52	0.001
A0A2I3MWL5	ENTPD1	Ectonucleoside triphosphate diphosphohydrolase 1		membrane	ATP binding;hydrolase activity	−1.52	0.005
A0A2I3N3R2	HROB	Homologous recombination factor with OB-fold	DNA metabolism;other metabolic processes;stress response;other biological processes	other cell component	nucleic acid binding activity	−1.51	0.034
A0A096NBM8	MAP1B	Microtubule associated protein 1B	cell organization and biogenesis; transport; developmental processes; other biological processes	plasma membrane;other membranes; cytosol; cytoskeleton; other cell component	cytoskeletal activity	−1.50	0.043
A0A096NQG4	FBLN2	Fibulin 2	cell organization and biogenesis	non-structural extracellular;extracellular matrix	other molecular function	−1.50	0.046
A0A2I3N6K8	RECK	Reversion inducing cysteine rich protein with kazal motifs	cell-cell signaling; cell organization and biogenesis; developmental processes; signal transduction; other biological processes	plasma membrarne;other membranes;other cell component	enzyme regulator activity;other molecular function	−1.49	0.026
A0A096N2M1	SNTB1	Syntrophin beta 1		anchoring junction; cytoskeleton; protein-containing complex	structural molecule activity	−1.49	0.034
A0A2I3MNX9	WNT2B	Protein Wnt	cell-cell signaling; signal transduction	non-structural extracellular	signal transduction activity or receptor binding	−1.47	0.036
A0A096MVE5	ALB	Albumin	stress response; other biological processes	non-structural extracellular;ER/Golgi;other cell component	nucleic acid binding activity;other molecular function	−1.43	0.046
A0A096NYU9	ANXA9	Annexin	cell adhesion; cell-cell signaling; signal transduction	cytosol;other cell component	other molecular function	−1.41	0.050
A0A2I3N477	ABCC9	ATP binding cassette subfamily C member 9	transport	other membranes	transporter activity;other molecular function	−1.40	0.009
A0A096N6Q7	ARHGAP29	Rho GTPase activating protein 29	signal transduction;other biological processes	other cell component	enzyme regulator activity;other molecular function	−1.40	0.034
A0A2I3M4F5	GRB10	Growth factor receptor bound protein 10	signal transduction;other biological processes		signal transduction activity or receptor binding	−1.39	0.046
A0A096MV66	SCARA5	Scavenger receptor class A member 5	cell organization and biogenesis;stress response;transport;other biological processes	plasma membrane;other membranes;other cell component	other molecular function	−1.37	0.020
A0A2I3MM86	OSBPL10	Oxysterol-binding protein	transport		other molecular function	−1.36	0.004
A0A2I3MXN7	ZFP28	ZFP28 zinc finger protein	regulation of DNA-templated transcription		metal ion binding	−1.35	0.023
A0A096NZP2	EMILIN1	Elastin microfibril interfacer 1	cell adhesion; cell organization and biogenesis; developmental processes; other biological processes	non-structural extracellular; extracellular matrix; plasma membrane; other membranes	signal transduction activity or receptor binding; extracellular structural activity; other molecular function	−1.35	0.027
A0A096NG19	PRKCD	Protein kinase C delta type	cell cycle OR cell proliferation;other biological processes	nucleus;other cell component	kinase activity;other molecular function	−1.34	0.006
A0A2I3MU24	EMCN	Endomucin		membrane		−1.34	0.050
A0A2I3MS76	MFAP4	Microfibril associated protein 4				−1.33	0.018
A0A096MKT1	RHOB	Ras homolog family member B	cell adhesion; cell cycle OR cell proliferation; stress response; transport; developmental processes; signal transduction; other biological processes	plasma membrane;other membranes;nucleus;other cytoplasmic organelle	other molecular function	−1.32	0.029
A0A8I5N5V6	L3HYPDH	trans-L-3-hydroxyproline dehydratase	enzymatic activity			−1.32	0.032
A0A2I3MV94	COL6A3	Collagen type VI alpha 3 chain	cell adhesion	collagen trimer;endoplasmic reticulum lumen	serine-type endopeptidase inhibitor activity	−1.32	0.046
A0A2I3M8Z2	COL6A2	Collagen type VI alpha 2 chain		extracellular matrix;extracellular region;protein-containing complex;sarcolemma	collagen binding	−1.31	0.008
A0A096MQJ9	ABI3BP	ABI family member 3 binding protein	cell organization and biogenesis;other biological processes	extracellular matrix	other molecular function	−1.29	0.011
A0A2I3MSR4	MTURN	Maturin		cytoplasm		−1.28	0.032
A0A096N1G3	HSPB8	Heat shock protein beta-8	stress response;other biological processes	cytosol;nucleus;other cell component	other molecular function	−1.27	0.013
A0A096NJI9	SERPINA6	Serpin family A member 6	glucocorticoid metabolic process	extracellular space	serine-type endopeptidase inhibitor activity;steroid binding	−1.27	0.040
A0A096P6B8	MRC2	Mannose receptor C type 2	collagen catabolic process;endocytosis	membrane	carbohydrate binding;collagen binding	−1.27	0.041
A9L8T6	RHBDF1	Inactive rhomboid protein 1	cell cycle OR cell proliferation;transport;other biological processes	other membranes;ER/Golgi	other molecular function	−1.26	0.028
A0A096N467	CD99	CD99 molecule (Xg blood group)		membrane		−1.26	0.033
A0A096N7S0	THY1	Thy-1 membrane glycoprotein	cell adhesion; cell-cell signaling; developmental processes; signal transduction; other biological processes	plasma membrane;other membranes;ER/Golgi;other cell component	signal transduction activity or receptor binding	−1.26	0.035
A0A8I5NLT2	IGLV3–27	Immunoglobulin lambda variable 3–27				−1.25	0.023
A0A096NIA2	COL6A1	Collagen type VI alpha 1 chain	cell adhesion;other biological processes	non-structural extracellular;extracellular matrix;plasma membrane;other membranes;ER/Golgi;other cell component	enzyme regulator activity	−1.24	0.012
A0A8I5R7S5		lg-like domain-containing protein				−1.24	0.023
A0A8I5NPE0		lg-like domain-containing protein				−1.23	0.018
A0A2I3N9G3	ESYT1	Extended synaptotagmin 1	cell organization and biogenesis;transport	plasma membrane;other membranes;ER/Golgi	other molecular function	−1.23	0.032
A0A8I5NWN1	ITPR1	Inositol 1,4,5-trisphosphate receptor		endoplasmic reticulum membrane	inositol 1,4,5 trisphosphate binding; inositol 1,4,5-trisphosphate-sensitive calcium-release channel activity	−1.22	0.002
A0A8I5NIG6	LOC100997602	Alpha-2-macroglobulin	negative regulation of peptidase activity	extracellular space	serine-type endopeptidase inhibitor activity	−1.22	0.011
A0A096N678	AGT	Angiotensinogen	developmental processes;signal transduction;other biological processes	non-structural extracellular	signal transduction activity or receptor binding;enzyme regulator activity	−1.21	0.005
A0A8I5NDQ3	LOC101019270	Complement C3-like	complement activation, alternative pathway;complement activation, classical pathway;inflammatory response	extracellular region	endopeptidase inhibitor activity	−1.20	0.002
A0A0A0MVT1	MYADM	Myeloid associated differentiation marker	cell organization and biogenesis;other biological processes	plasma membrane; other membranes; cytoskeleton; other cell component		−1.20	0.006
A0A2I3LY52	NEFM	Neurofilament medium polypeptide	cell organization and biogenesis	cytoskeleton;other cell component	other molecular function	−1.20	0.032
A0A2I3MTT2	WLS	Wnt ligand secretion mediator	cell-cell signaling;signal transduction	other membranes;ER/Golgi	other molecular function	−1.20	0.033
A0A2I3LLY4		lg-like domain-containing protein				−1.20	0.042
A0A096NA41	PODN	Podocan				−1.19	0.008
A0A2I3LXZ6		lg-like domain-containing protein				−1.19	0.047
A0A096MR94	SERPINA7	Serpin family A member 7	transport;other biological processes	non-structural extracellular	enzyme regulator activity	−1.18	0.011
A0A2I3NFT2	PBX1	PBX homeobox 1		nucleus	DNA binding;DNA-binding transcription factor activity	−1.18	0.038
A0A096MKK3	OLFML3	Olfactomedin like 3	cell adhesion; protein-protein interactions; intercellular interactions	extracellular matrix glycoprotein		−1.17	0.006
A0A2I3LF86	SLC14A1	Urea transporter	transport	plasma membrane;other membranes	transporter activity	−1.17	0.022
A0A096NTX8	APOH	Beta-2-glycoprotein 1	protein metabolism;other metabolic processes;stress response;other biological processes	non-structural extracellular;other cell component	enzyme regulator activity;other molecular function	−1.17	0.042
A0A0A0MX79	A1BG	Alpha-1-B glycoprotein		plasma glycoprotein		−1.17	0.047
A0A096MRP6	SERPING1	Serpin family G member 1	negative regulation of complement activation, lectin pathway	extracellular space	serine-type endopeptidase inhibitor activity	−1.16	0.003
A0A096N1J7	APOD	Apolipoprotein D (Apo-D)	aging; glucose metabolic process; lipid metabolic process;lipid transport; negative regulation of cytokine production involved in inflammatory response, of focal adhesion assembly, of lipoprotein lipid oxidation;negative regulation of monocyte chemotactic protein-1 production, of platelet-derived growth factor receptor signaling pathway, of protein import into nucleus, of smooth muscle cell proliferation, of smooth muscle cell-matrix adhesion, of T cell migration; response to reactive oxygen species	extracellular space;perinuclear region of cytoplasm	cholesterol binding	−1.16	0.007
A0A8I5NZN5		lg-like domain-containing protein				−1.16	0.047
A0A2I3MN09	AKT3	Non-specific serine/threonine protein kinase	signal transduction		kinase activity;other molecular function	−1.15	0.012
A0A2I3LWC3	DHODH	Haptoglobin	stress response;other biological processes	non-structural extracellular	other molecular function	−1.15	0.048
A0A8I5N3P7		lg-like domain-containing protein				−1.14	0.004
A0A096MX78	ITIH1	Inter-alpha-trypsin inhibitor heavy chain 1	hyaluronan metabolic process;negative regulation of peptidase activity	extracellular region	hyaluronic acid binding; serine-type endopeptidase inhibitor activity	−1.14	0.025
A0A2I3LMN0	CP	Ceruloplasmin	copper ion transport	extracellular space	copper ion binding; oxidoreductase activity; protein-folding chaperone binding	−1.14	0.034
A0A096P228	LOC101024934	Complement C4-A	stress response;innate immune response; inflammatory response; complement activation, classical pathway	non-structural extracellular;other cell component	enzyme regulator activity; endopeptidase inhibitor activity	−1.14	0.036
A0A096N0R9	SERPINC1	Antithrombin-III (Serpin Cl)	blood coagulation;regulation of blood coagulation	extracellular space	heparin binding; identical protein binding; protease binding; serine-type endopeptidase inhibitor activity	−1.14	0.039
A0A096NS45		lg-like domain-containing protein				−1.13	0.044
A0A2I3NBQ7	HSPA12A	Heat shock protein family A (Hsp70) member 12A		extracellular exosome	ATP binding	−1.12	0.019
A0A096MZM0	GNAI1	G protein subunit alpha i1	signal transduction;other biological processes	other cell component	other molecular function	−1.12	0.021
A0A096NRQ5	C8G	Complement C8 gamma chain	complement activation	extracellular region;membrane attack complex		−1.12	0.050
A0A8I5P2L0	PLPP3	Phospholipid phosphatase 3	phospholipid metabolic process	membrane		−1.11	0.027
A0A2I3MRF6	SERPINF2	Serpin family F member 2		extracellular space	serine-type endopeptidase inhibitor activity	−1.10	0.001
A0A096N4Z1	F2	Prothrombin	stress response; signal transduction; other biological processes	non-structural extracellular; plasma membrane; other membranes; ER/Golgi	signal transduction activity or receptor binding;other molecular function	−1.09	0.011
A0A2I3MBR2		lg-like domain-containing protein				−1.09	0.024
A0A8I5N321		lg-like domain-containing protein				−1.08	0.002
A0A2I3MQR3	BAHCC1	BAH domain and coiled-coil containing 1			chromatin binding	−1.08	0.013
A0A8I5MV73		lg-like domain-containing protein				−1.08	0.037
A0A8J8Y2F3		lg-like domain-containing protein				−1.08	0.038
A0A8I5N5Y9	ILK	Integrin-linked protein kinase (59 kDa scrine/threoninc-protein kinase) (Beta-integrin-linked kinase) (ILK-1) (ILK-2) (p59ILK)	branching involved in ureteric bud morphogenesis; cell morphogenesis; cell projection organization; establishment or maintenance of epithelial cell apical/basal polarity; fibroblast migration; integrin-mediated signaling pathway; myelination in peripheral nervous system; negative regulation of neural precursor cell proliferation; nerve development; neural precursor cell proliferation; outflow tract morphogenesis; peptidyl-serine phosphorylation; positive regulation of BMP signaling pathway, of canonical Wnt signaling pathway, of cell proliferation, of DNA-templated transcription, of NIK/NF-kappaB signaling, of osteoblast differentiation, of protein phosphorylation, of substrate adhesion-dependent cell spreading; protein kinase B signaling; substrate adhesion-dependent cell spreading; TNF-mediated signaling pathway	cytosol; focaladhesion; lamellipodium; nucleoplasm; sarcomere	ATP binding;protein kinase binding;protein serine/threonine kinase activity	−1.07	0.026
A0A096N8S0	C8B	Complement component C8 beta chain	stress response; other biological processes	non-structural extracellular; plasma membrane; other membranes		−1.07	0.034
A0A2I3LW92	NEGR1	Neuronal growth regulator 1	cell adhesion;other biological processes	plasma membrane;other membranes		−1.07	0.049
A0A096NXD0	CMA1	Chymase 1	proteolysis		serine-type endopeptidase activity	−1.06	0.002
A0A096N2Y1	C1S	Complement C1s	stress response;innate immune response; complement activation, classical pathway;proteolysis	extracellular region	calcium ion binding;serine-type endopeptidase activity	−1.06	0.004
A0A096NA36	C8A	Complement C8 alpha chain	complement activation, alternative pathway;complement activation, classical pathway;killing of cells of another organism	non-structural extracellular;plasma membrane;membrane attack complex		−1.06	0.030
A0A213M256	IL6ST	Interleukin 6 cytokine family signal transducer	signal transduction	other membranes	other molecular function	−1.06	0.046
A0A096NZY9	PLG	Plasminogen	stress response;other biological processes	non-structural extracellular	other molecular function	−1.05	0.001
A0A096NS83	SERPIND1	Serpin family D member 1	stress response;other biological processes	non-structural extracellular	enzyme regulator activity	−1.05	0.008
A0A096NFH2	OSBPL1A	Oxysterol-binding protein	transport	other cytoplasmic organelle;other cell component	other molecular function	−1.05	0.020
A0A096NDH5	CLIC4	Chloride intracellular channel protein	transport;developmental processes;other biological processes	plasma membrane; other membranes; cytosol; cytoskeleton; mitochondrion; nucleus; other cell component	transporter activity	−1.05	0.026
A0A2I3M4V3	SLC2A1	Solute carrier family 2, facilitated glucose transporter member 1	cell organization and biogenesis;stress response;transport;deve1opmental processes	plasma membrane; other membranes; cytosol; cytoskeleton; nucleus; other cell component	transporter activity;other molecular function	−1.05	0.040
A0A8I5NSE3	LOC116268832	Pregnancy zone protein-like				−1.05	0.043
A0A096MT62	DAG1	Dystroglycan 1	cell organization and biogenesis; protein metabolism; other biological processes	non-structural extracellular; extracellular matrix; plasma membrane; other membranes; cytoskeleton; nucleus; other cell component	cytoskeletal activity;other molecular function	−1.05	0.048
A0A8I5R910		lg-like domain-containing protein				−1.04	0.019
A0A096NQN2	APCS	Pentaxin	stress response;other biological processes	non-structural extracellular	other molecular function	−1.04	0.036
A0A096MSP3	AFM	Afamin	transport;other biological processes	non-structural extracellular	other molecular function	−1.03	0.007
A0A096NBU0	SLC12A4	Solute carrier family 12 member 4	transport	other membranes	transporter activity	−1.03	0.008
A0A096NS36	TRIP6	Thyroid hormone receptor interactor 6	developmental processes;other biological processes	plasma membrane;other membranes;cytosol;cytoskeleton; nucleus;other cell component	signal transduction activity or receptor binding;other molecular function	−1.03	0.027
A0A096P2P5	PLSCR3	Phospholipid scramblase	cell organization and biogenesis; other metabolic processes; transport; other biological processes	plasma membrane;other membranes; cytosol; mitochondrion; nucleus	transporter activity;other molecular function	−1.03	0.030
A0A096NPZ2	ITIH4	Inter-alpha-trypsin inhibitor heavy chain 4	acute-phase response;hyaluronan metabolic process;negative regulation of peptidase activity;response to cytokine	cytoplasm;extrace11ular region;plasma membrane	serine-type endopeptidase inhibitor activity	−1.02	0.005
A9X172	F10	Coagulation factor X	stress response;transport;other biological processes	non-structural extracellular;ER/Golgi	other molecular function	−1.02	0.005
A0A8I5R5F3	IGLV5–52	Immunoglobulin lambda variable 5–52				−1.02	0.007
A0A8I5NAM2	STBD1	Starch binding domain 1	glycophagy; intracellular transport; substrate localization to autophagosome	endoplasmic reticulum;perinuclear region of cytoplasm;T-tubule	cargo receptor activity; enzyme binding; glycogen binding; starch binding	−1.02	0.012
A0A8I5NPY8	F12	Coagulation factor XII	blood coagulation; Factor XII activation; positive regulation of blood coagulation, of fibrinolysis, of plasminogen activation; protein autoprocessing; response to misfolded protein; zymogen activation	extracellular space	calcium ion binding;serine-type endopeptidase activity	−1.02	0.024
A0A8I5NEC6		lg-like domain-containing protein				−1.02	0.030
A0A2I3NGA0	ARHGEF40	Rho guanine nucleotide exchange factor 40		cytosol;plasma membrane	guanyl-nucleotide exchange factor activity	−1.02	0.044
A0A096P5G9	TOP2A	DNA topoisomerase 2	cell cycle OR cell proliferation;cell organization and biogenesis; DNA metabolism; other metabolic processes; stress response; developmental processes; other biological processes	cytoskeleton;nucleus;other cell component	nucleic acid binding activity;other molecular function	−1.01	0.002
A0A8I5NIY0		lg-like domain-containing protein				−1.01	0.007
A0A8I5N962	CAB39L	Calcium binding protein 39 like	intracellular signal transduction	cytosol		−1.01	0.023
A0A2I3LK29	FLOT1	Flotillin 1	cell organization and biogenesis; stress response; transport; developmental processes; signal transduction; other biological processes	plasma membrane; other membranes; cytoskeleton; nucleus; other cytoplasmic organelle; other cell component	other molecular function	−1.00	0.032
A0A2I3LG36	CPNE8	Copine 8			calcium-dependent phospholipid binding	−1.00	0.032
A0A096NBN3	C7	Complement C7	stress response;innate immune response;complement activation, classical pathway;killing of cells of another organism	non-structural extracellular.plasma membrane.membrane attack complex		−1.00	0.033
A0A096NW43	SNTB2	Syntrophin beta 2		anchoring junction; cytoskeleton; Golgi apparatus; nucleoplasm; plasma membrane; protein-containing complex	structural molecule activity	−1.00	0.037
A0A2I3M507	GYG1	Glycogenin 1			UDP-glycosyltransferase activity	−1.00	0.041
A0A8I5NBS6	CD59	CD59 molecule (CD59 blood group)	regulation of complement activation;regulation of complement-dependent cytotoxicity	external side of plasma membrane		−1.00	0.046

Table 1B.

Enterocystoplasty- Ileal grafted proteins < Native proteins

Accession	Gene Symbol	Description	Biological Process	Cellular Component	Molecular Function	Abundance Ratio (log2): Ileal graft/Native	Abundance Ratio P-Value: Ileal graft/Native
A0A096MVY1	GABARAPL2	GABA type A receptor associated protein like 2	autophagy;negative regulation of proteasomal protein catabolic process;protein localization to endoplasmic reticulum	autophagosome membrane;Golgi membrane	ubiquitin protein ligase binding	1.01	0.042
A0A2I3LE60	NDRG1	Protein NDRG1	cell organization and biogenesis; stress response; developmental processes; signal transduction; other biological processes	plasma membrane; other membranes; cytosol; cytoskeleton; nucleus; other cytoplasmic organelle; other cell component	cytoskeletal activity;other molecular function	1.01	0.038
A0A096NLE7	DNAJA3	DnaJ heat shock protein family (Hsp40) member A3	cell organization and biogenesis; DNA metabolism; other metabolic processes; stress response; developmental processes; signal transduction; other biological processes	plasma membrane; other membranes; cytosol; cytoskeleton; mitochondrion; nucleus; other cell component	enzyme regulator activity;other molecular function	1.03	0.045
A0A096NX69	DCAF11	DDB1- and CUL4-assodated factor 11	protein metabolism; other metabolic processes	nucleus; other cell component		1.03	0.007
A0A096NJD3	EWSR1	EWS RNA binding protein 1	regulation of DNA-templated transcription	nucleus	metal ion binding;RNA binding	1.04	0.034
A0A2I3MVU7	KHDC4	KH homology domain-containing protein 4	cell organization and biogenesis; RNA metabolism OR transcription; other metabolic processes	nucleus;other cell component	nucleic acid binding activity	1.05	0.042
A0A2I3MR67	C1QBP	Complement component 1 Q subcomponent-binding protein, mitochondrial	cell organization and biogenesis; signal transduction; other biological processes	plasma membrane; other membranes; cytosol; mitochondrion; nucleus; other cell component	other molecular function	1.05	0.022
A0A8I5NAP1	PARS2	proline--tRNA ligase (Prolyl-tRNA synthetase)	prolyl-tRNA aminoacylation	cytoplasm	ATP binding;proline-tRNA ligase activity	1.05	0.028
A0A8I5MWW1	YME1L1	YME1 like 1 ATPase	cell population proliferation; mitochondrial protein catabolic process; mitochondrial protein processing; negative regulation of apoptotic process; protein hexamerization; protein quality control for misfolded or incompletely synthesized proteins	mitochondrial inner membrane; nuclear body	ATP bitiding;ATP hydrolysis activity;ATP dependent peptidase activity;metalloendopeptidase activity	1.06	0.043
A0A2I3LWY5	TRMU	Mitochondrial tRNA-specific 2-thiouridylase 1	RNA metabolism OR transcription; other metabolic processes	mitochondrion	nucleic acid binding activity; other molecular function	1.06	0.014
A0A096MSH2	MRPS5	Mitochondrial ribosomal protein S5	protein metabolism;other metabolic processes;translation	mitochondrion;translational apparatus	nucleic acid binding activity;other molecular function	1.07	0.002
A0A096P2D4	CLUH	Clustered mitochondria protein homolog	cell organization and biogenesis	other cell component	nucleic acid binding activity	1.08	0.008
A0A2I3N9D9	NASP	Nuclear autoantigenic sperm protein		nucleus		1.09	0.006
A0A8I5NPC3	IMPA2	Inositol-1-monophosphatase	inositol biosynthetic process;phosphatidylinositol phosphate biosynthetic process		inositol monophosphate 1-phosphatase activity;metal ion binding	1.10	0.048
A0A2I3MXH5	GNG2	Guanine nucleotide-binding protein subunit gamma	signal transduction	plasma membrane;other membranes	other molecular function	1.10	0.024
A0A8I5R1Q0	MRPS26	Mitochondrial ribosomal protein S26	protein metabolism;other metabolic processes;translation	mitochondrion;translational apparatus	other molecular function	1.10	0.048
A0A096NMZ1	STARD5	StAR related lipid transfer domain containing 5	cell organization and biogenesis;transport		transporter activity;other molecular function	1.11	0.019
A0A8I5MZ00	MROH7	Maestro heat like repeat family member 7	fatty acid metabolic process		carboxylic ester hydrolase activity;lipid binding;thiolester hydrolase activity	1.11	0.002
A0A096N415	ATP1B3	Sodium/potassium-transporting ATPase subunit beta	transport;other biological processes	plasma membrane;other membranes;cytoskeleton;other cell component	other molecular function	1.11	0.044
A0A096P0I8	ENDOG	Endonuclease	cell organization and biogenesis; DNA metabolism; other metabolic processes; developmental processes; other biological processes	mitochondrion	nucleic acid binding activity; other molecular function	1.12	0.015
A0A2I3MCE5	HSPD1	60 kDa heat shock protein, mitochondrial	protein refolding		ATP binding;ATP-dependent protein folding chaperone	1.13	0.029
A0A2I3M241	TDP2	Tyrosyl-DNA phosphodiesterase 2	phosphodiesterase activity		catalytic activity	1.13	0.034
A0A096P4W5	GDPD1	Glycerophosphodiester phosphodiesterase domain containing 1	N-acylethanolamine metabolic process;phospholipid metabolic process	endoplasmic reticulum; membrane; perinuclear region of cytoplasm	lysophospholipase activity; phosphoric diester hydrolase activity	1.14	0.032
A0A2I3MRI5	NEDD4L	HECT-type E3 ubiquitin transferase	protein metabolism; other metabolic processes		other molecular function	1.14	0.024
A0A096N312	CMPK1 CMPK	UMP-CMP kinase	de novo’ pyrimidine nucleobase biosynthetic process; phosphorylation; pyrimidine nucleotide biosynthetic process	cytoplasm;nucleus	ATP binding; CMP kinase activity; dCMP kinase activity; nucleoside diphosphate kinase activity; UMP kinase activity	1.15	0.034
A0A2I3N7F2	ACY1	N-acyl-aliphatic-L-amino acid amidohydrolase	amino acid metabolic process	cytoplasm;extracellular exosome	aminoacylase activity; identical protein binding	1.15	0.042
A0A096NN39	MRPS11	Mitochondrial ribosomal protein Sll	protein metabolism;other metabolic processes;translation	mitochondrion; translational apparatus	other molecular function	1.16	0.028
A0A096NV76	LGALS3	Galectin	RNA metabolism OR transcription; other metabolic processes; stress response;signal transduction; other biological processes	non-structural extracellular; plasma membrane; other membranes; cytosol; mitochondrion; nucleus	signal transduction activity or receptor binding; other molecular function	1.16	0.011
A0A8I5QZS2	MRPL13	Mitochondrial ribosomal protein L13	protein metabolism;other metabolic processes;translation	ribonucleoprotein complex;ribosome	structural constituent of ribosome	1.16	0.031
A0A096NXB3	DHRS1	Dehydrogenase/reductase 1		endoplasmic reticulum	carbonyl reductase (NADPH) activity	1.16	0.004
A0A8I5N4T1	MRPL51	39S ribosomal protein L51, mitochondrial	protein metabolism;other metabolic processes;translation	mitochondrial large ribosomal subunit	structural constituent of ribosome	1.17	0.001
A0A8I5N8K6	ABCD3	ATP binding cassette subfamily D member 3	long-chain fatty acid import into peroxisome	peroxisomal membrane	ABC-type transporter activity;ATP binding;ATP hydrolysis activity;long-chain fatty acid transporter activity	1.18	0.010
A0A096MUM0	MRPS14	Mitochondrial ribosomal protein S14	protein metabolism;other metabolic processes;translation	Other membranes;mitochondrion;translational apparatus;nucleus	other molecular function	1.18	0.022
A0A096MTU6	GRSF1	G-rich RNA sequence binding factor 1	RNA metabolism OR transcription;other metabolic processes;developmental processes;other biological processes	mitochondrion;other cell component	nucleic acid binding activity	1.18	0.009
A0A096N7R2	H2AX	Histone H2A	DNA metabolism;stress response;signal transduction;other biological processes	cytoskeleton;nucleus;other cell component	nucleic acid binding activity;other molecular function	1.19	0.017
A0A096NUP8	UQCC1	Ubiquinol-cytochrome c reductase complex chaperone, CBP3 homolog	cell organization and biogenesis	other membranes; mitochondrion; other cell component		1.20	0.012
A0A2I3LL81	ASAP2	ArfGAP with SH3 domain, ankyrin repeat and PH domain 2	positive regulation of GTPase activity	cytoplasm	GTPase activator activity;metal ion binding	1.20	0.002
A0A8J8XMP2	CNDP1	Carnosine dipeptidase 1			metal ion binding; metallodipeptidase activity	1.20	0.040
A0A096NSZ8	MRPL38	Mitochondrial ribosomal protein L38	protein metabolism;other metabolic processes;translation	mitochondrial large ribosomal subunit		1.20	0.040
A0A2I3MKQ1	MYOID	Myosin ID		myosin complex	actin binding; ATP binding; cytoskeletal motor activity	1.21	0.026
A0A8I5NAV1	MRPS33	Mitochondrial ribosomal protein S33	protein metabolism;other metabolic processes;translation	mitochondrion;ribonucleoprotein complex; ribosome		1.21	0.014
A0A096MXVZ	MRPS35	Mitochondrial ribosomal protein S35	protein metabolism;other metabolic processes;translation	mitochondrion	other molecular function	1.22	0.018
A0A096MQF1	SLC25A22	Solute carrier family 25 member 22	transport	other membranes; mitochondrion	transporter activity	1.23	0.036
A0A8I5NUR8	BCAT1	Branched-chain-amino-acid aminotransferase	amino acid biosynthetic process; branched-chain amino acid biosynthetic process		branched-chain-amino-acid transaminase activity	1.24	0.001
A0A096N8Q4	HDHD3	Haloacid dehalogenase-like hydrolase domain-containing protein 3		nucleolus		1.24	0.012
A0A2I3MUXO	SLC25A10	Solute carrier family 25 member 10		membrane;mitochondrion; nucleoplasm		1.24	0.011
A0A2I3MYF6	MRPS18C	Mitochondrial ribosomal protein S18C	protein metabolism; other metabolic processes; translation	mitochondrion; translational apparatus	structural constituent of ribosome	1.25	0.029
A0A2I3LIA9	KLC4	Kinesin light chain		cytoplasm; kinesin complex; microtubule		1.25	0.034
A0A096MSY0	HSD17B4	Hydroxysteroid 17-beta dehydrogenase 4	fatty acid beta-oxidation	peroxisome	oxidoreductase activity	1.26	0.024
A0A096NDV6	YBX1	Y-box binding protein 1	transport; developmental processes; other biological processes	non-structural extracellular; plasma membrane; other membranes; cytosol; ER/Golgi;nucleus; other cell component	nucleic acid binding activity; other molecular function	1.26	0.023
A0A096NPN2	UAP1	UDP-N-acetylglucosamine pyrophosphorylase 1		cytosol;nucleoplasm;plasma membrane	identical protein binding; uridylyltransferase activity	1.26	0.019
A0A2I3N9R9	CES1	Carboxylic ester hydrolase			hydrolase activity	1.26	0.036
A0A096NN38	MRPL46	39S ribosomal protein L46, mitochondrial	protein metabolism;other metabolic processes;translation	cell junction;mitochondrial large ribosomal subunit;nucleoplasm	structural constituent of ribosome	1.28	0.001
A0A096NKW9	MRPS34	Mitochondrial ribosomal protein S34	protein metabolism;other metabolic processes;translation	mitochondrion	other molecular function	1.30	0.008
A0A2I3MLC4	LOC101021735	Histone H2A		nucleosome;nucleus	DNA binding;protein heterodimerization activity; structural constituent of chromatin	1.30	0.012
A0A096N3D9	MRPS22	Mitochondrial ribosomal protein S22	protein metabolism;other metabolic processes;translation	mitochondrial small ribosomal subunit	structural constituent of ribosome	1.30	0.018
A0A096N3H8	ZBTB7B	Zinc finger and BTB domain containing 7B	adaptive thermogenesis; lactation; negative regulation of CD8-positive, alpha-beta T cell differentiation; negative regulation of gene expression, of NK T cell proliferation, of T-helper 17 cell differentiation, of transcription by RNA polymerase II; NK T cell differentiation; positive regulation of brown fat cell differentiation, of CD4-positive, alpha-beta T cell differentiation, of cold-induced thermogenesis, of insulin receptor signaling pathway, of interleukin-17 production, of SREBP signaling pathway; response to insulin	nucleoplasm	DNA-binding transcription activator activity, RNA polymerase ll-spedfic; DNA-binding transcription repressor activity; histone deacetylase binding; protein homodimerization activity; RNA polymerase II cis-regulatory region sequence-specific DNA binding	1.31	0.048
A0A2I3NA33	CYP4F3	Cytochrome P450 family 4 subfamily F member 3			heme binding; iron ion binding; monooxygenase activity; oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen	1.32	0.015
A0A096NR61	RTN4IP1	Reticulon 4 interacting protein 1		mitochondrial outer membrane	oxidoreductase activity;zinc ion binding	1.32	0.021
A0A8I5NG73	MGME1	Mitochondrial genome maintenance exonudease 1	mitochondrial DNA repair	mitochondrion	single-stranded DNA exodeoxyribonuclease activity	1.33	0.035
A0A096N4E8	LYRM7	Complex III assembly factor LYRM7	cell organization and biogenesis; other metabolic processes	other membranes;mitochondrion		1.34	0.028
A0A8I5N5Y6	LRPPRC	Leucine rich pentatricopeptide repeat containing	regulation of transcription	mitochondrion		1.34	0.031
A0A096NTK3	PCYT2	ethanolamine-phosphate cytidylyltransfcrase	phosphatidylethanolamine biosynthetic process		ethanolamine-phosphate cytidylyltransferase activity	1.35	0.023
A0A096NC97	MRPS28	Mitochondrial ribosomal protein S28	protein metabolism; other metabolic processes; translation	mitochondrion		1.36	0.011
A0A096MSA0	MRPL1	Mitochondrial ribosomal protein L1	protein metabolism;other metabolic processes;translation	mitochondrion;translationa1 apparatus	nucleic acid binding activity;other molecular function	1.36	0.016
A0A8I5NIV2	MRPL15	39S ribosomal protein L15, mitochondrial	protein metabolism;other metabolic processes;translation	large ribosomal subunit	structural constituent of ribosome	1.36	0.022
A0A8I5NCL6	CBX4	Chromobox 4	negative regulation of transcription by RNA polymerase II;	PcG protein complex		1.37	0.021
A0A2I3MOS9	MRPL42	Mitochondrial ribosomal protein L42	protein metabolism	mitochondrion;ribosome		1.37	0.000
A0A096P4S3	MRPS23	Mitochondrial ribosomal protein S23	protein metabolism;other metabolic processes	other membranes; mitochondrion; translational apparatus;nucleus	other molecular function	1.38	0.022
A0A096NZK5	ITGA6	Integrin subunit alpha 6	cell adhesion;cell organization and biogenesis;signal transduction	plasma membrane;other membranes		1.39	0.005
A9X1A9	MRPL9	39S ribosomal protein L9, mitochondrial	protein metabolism;other metabolic processes;translation	mitochondrion;translational apparatus	other molecular function	1.39	0.017
A0A096MVU3	MRPL3	Mitochondrial ribosomal protein L3	protein metabolism;other metabolic processes;translation	mitochondrion;translational apparatus	other molecular function	1.39	0.016
A0A2I3N5C1	MRPS7	Mitochondrial ribosomal protein S7	protein metabolism;other metabolic processes;translation	mitochondrion;translational apparatus		1.39	0.015
A0A096NLL4	PMM2	Phosphomannomutase	GDP-mannose biosynthetic process	cytosol;neuronal cell body; nucleoplasm	phosphomannomutase activity	1.40	0.018
A0A096MTE6	MRPL22	39S ribosomal protein L22, mitochondrial	protein metabolism;other metabolic processes	translational apparatus	other molecular function	1.40	0.026
A0A8I5R8A8	LOC101016399	Histone H2B		nucleosome;nucleus	DNA binding;protein heterodimerization activity; structural constituent of chromatin	1.40	0.042
A0A2I3NDT4	TM9SF2	Transmembrane 9 superfamily member	ceramide metabolic process; glycosphingolipid biosynthetic process; regulation of heparan sulfate proteoglycan biosynthetic process	Golgi apparatus;membrane		1.41	0.050
A0A2I3MMJ2	RFK	riboflavin kinase	apoptotic process; FMN biosynthetic process; positive regulation of NAD(P)H oxidase activity; reactive oxygen species metabolic process; riboflavin biosynthetic process		ATP binding;riboflavin kinase activity	1.42	0.045
A0A096NCV1	Hl-5	H1.5 linker histone, cluster member	cell organization and biogenesis; other biological processes	nucleus;other cell component	nucleic acid binding activity;other molecular function	1.44	0.034
A0A2I3LF84	MRPL44	Mitochondrial ribosomal protein L44	protein metabolism;other metabolic processes;translation	plasma membrane; other membranes; mitochondrion; translational apparatus; nucleus	nucleic acid binding activity;other molecular function	1.44	0.014
A0A096NV33	GNPNAT1	Glucosamine 6-phosphate N-acetyltransferase	UDP-N-acetylg 1 ucosa m i ne biosynthetic process		glucosamine 6-phosphate N-acetyltransferase activity;identical protein binding	1.44	0.018
A0A096MMX4	NUDT12	NAD capped RNA hydrolase NUDT12		peroxisome	metal ion binding;NAD+ diphosphatase activity;NADH pyrophosphatase activity	1.45	0.022
A0A8I5NZI0	MRPS9	Mitochondrial ribosomal protein S9	protein metabolism;other metabolic processes;translation	ribonucleoprotein complex; ribosome	structural constituent of ribosome	1.46	0.003
A0A096N2Q0	FDX1	Fcrredoxin 1	cellular response to cAMP; cellular response to forskolin; cholesterol metabolic process; hormone biosynthetic process; P450-containing electron transport chain	mitochondrion	2 iron, 2 sulfur cluster binding; electron transfer activity	1.47	0.004
A0A8I5NKC8	SDF4	45 kDa calcium-binding protein (Stromal cell-derived factor 4)		Golgi lumen	calcium ion binding	1.47	0.018
A0A2I3N6P2	ITGB4	Integrin beta	cell adhesion; cell organization and biogenesis; other metabolic processes; stress response; developmental processes; signal transduction; other biological processes	extracellular matrix; plasma membrane; other membranes; nucleus; other cell component	signal transduction activity or receptor binding; other molecular function	1.48	0.005
A0A2I3M1B1	SHTN1	Shootln-1		axon; cytoplasm; cytoskeleton; filopodium; lamellipodium; perikaryon		1.48	0.040
A0A096MLB7	SMC4	Structural maintenance of chromosomes protein	cell cycle OR cell proliferation;cell organization and biogenesis;other biological processes	cytosol;nucleus;other cell component	nucleic acid binding activity;other molecular function	1.49	0.038
A0A096NI92	LSS	Terpene cyclase/mutase family member	cholesterol biosynthetic process; regulation of protein stability; triterpenoid biosynthetic process	endoplasmic reticulum membrane;lipid droplet	lanosterol synthase activity	1.49	0.021
A0A8I5NE09	LOC10109499	Cytochrome c oxidase subunit 7A2	mitochondrial electron transport, cytochrome c to oxygen	mitochondrial respiratory chain complex IV		1.49	0.040
A0A096NTH9	MRPL12	Mitochondrial ribosomal protein L12	protein metabolism;other metabolic processes;translation	mitochondrion;translational apparatus	other molecular function	1.49	0.006
A0A096N123	MRPL49	39S ribosomal protein L49, mitochondrial	protein metabolism;other metabolic processes;translation	mitochondrion;translational apparatus	other molecular function	1.52	0.002
A0A096NCK0	IGF2BP2	Insulin like growth factor 2 mRNA binding protein 2	transport	cytosol;cytoskeleton;nucleus	nucleic acid binding activity	1.52	0.026
A0A8I5NLL6	MRPL39	Mitochondrial ribosomal protein L39	protein metabolism;other metabolic processes;translation		nucleotide binding	1.52	0.032
A0A096NB44	MRPL21	Mitochondrial ribosomal protein L21	protein metabolism;other metabolic processes;translation	mitochondrial large ribosomal subunit		1.52	0.011
A0A2I3NFG5	SLC43A2	Solute carrier family 43 member 2	transport	other membranes	transporter activity	1.53	0.033
A0A096P105	MRPL50	39S ribosomal protein L50, mitochondrial	protein metabolism;other metabolic processes;translation	cytosol;mitochondrial large ribosomal subunit		1.53	0.013
A0A2I3MM22	CRYL1	Crystallin lambda 1	fatty acid metabolic process; glucuronate catabolic process to xylulose 5-phosphate	cytosol	L-gulonate 3-dehydrogenase activity;NAD+ binding; protein homodimerization activity	1.54	0.001
A0A8I5NGD9	PFKFB2	6-phosphofructo-2-kina se/fructose-2,6-biphosphatase 2	fructose 2,6-bisphosphate metabolic process;fructose metabolic process		6-phosphofructo-2-kinase activity; ATP binding; hydrolase activity	1.54	0.026
A0A2I3NOC6	MRPL27	Mitochondrial ribosomal protein L27	protein metabolism;other metabolic processes;translation	mitochondrion;translational apparatus	other molecular function	1.54	0.010
A0A8I5NNA9	MRPL10	Mitochondrial ribosomal protein L10	protein metabolism;other metabolic processes;translation	mitochondrion;translational apparatus		1.54	0.019
A0A2I3LQI7	MRPL54	Mitochondrial ribosomal protein L54	protein metabolism;other metabolic processes;translation	mitochondrial large ribosomal subunit		1.54	0.020
A0A096MR30	HMGB2	High mobility group box 2	cell organization and biogenesis; DNA metabolism; other metabolic processes; stress response; signal transduction; other biological processes	non-structural extracellular; nucleus; other cell component	signal transduction activity or receptor binding;nucleic acid binding activity;other molecular function	1.55	0.021
A0A0A0MWG3	MRPL4	Mitochondrial ribosomal protein L4	protein metabolism;other metabolic processes;translation	translational apparatus;other cell component	other molecular function	1.55	0.026
A0A8I5N952	MRPS12	Mitochondrial ribosomal protein S12	protein metabolism;other metabolic processes;translation	mitochondrial small ribosomal subunit	structural constituent of ribosome	1.55	0.020
A0A8I5N0D7	AKR1C1	Aldo-keto reductase family 1 member Cl			oxidoreductase activity	1.55	0.036
A0A096N6Y2	CASP6	Caspase-6	protein metabolism;other metabolic processes;signal transduction;other biological processes	cytosol; nucleus	other molecular function	1.57	0.008
A0A8I5NXE4	MRPS30	Mitochondrial ribosomal protein S30	protein metabolism;other metabolic processes;translation	mitochondrial large ribosomal subunit	structural constituent of ribosome	1.58	0.049
A0A096P374		Galectin			carbohydrate binding	1.59	0.016
A0A096P4H0	PPIF	Peptidyl-prolyl cis-trans isomerase	cell organization and biogenesis; stress response; transport; other biological processes	other membranes;mitochondrion	other molecular function	1.60	0.008
A0A2I3M4E0	GK	glycerol kinase	glycerol catabolic process; glycerol-3-phosphate biosynthetic process; phosphorylation; triglyceride metabolic process	mitochondrial outer membrane	ATP binding; glycerol kinase activity	1.60	0.031
A0A096P6I7	GSTOl	Glutathione S-transferase omega	glutathione metabolic process	cytoplasm	glutathione dehydrogenase (ascorbate) activity; glutathione transferase activity; methylarsonate reductase activity	1.61	0.042
A0A096ND28	NOS1AP	Nitric oxide synthase 1 adaptor protein	cell organization and biogenesis; other biological processes	plasma membrane; other membranes; cytosol; cytoskeleton; mitochondrion; ER/Golgi; other cell component	other molecular function	1.62	0.005
A0A2I3N1N3	MRPL30	39S ribosomal protein L30, mitochondrial	protein metabolism;other metabolic processes;translation	translational apparatus	other molecular function	1.62	0.025
A0A096NII9	MRPL14	Mitochondrial ribosomal protein L14	protein metabolism;other metabolic processes;translation	translational apparatus	other molecular function	1.62	0.008
A0A096NSA2	SLIRP	SRA stem-loop interacting RNA binding protein			RNA binding	1.63	0.021
A0A096N724	MRPL47	Mitochondrial ribosomal protein L47	protein metabolism;other metabolic processes;translation	mitochondrion;translational apparatus	other molecular function	1.64	0.014
A0A096MYA5	MRPL48	Mitochondrial ribosomal protein L48	protein metabolism;other metabolic processes;translation	mitochondrial large ribosomal subunit		1.64	0.009
A0A096NSQ5	DHX32	DEAH-box helicase 32 (putative)			helicase activity	1.65	0.029
A0A8I5NPG3	MRPL11	Mitochondrial ribosomal protein Lll	protein metabolism;other metabolic processes;translation	mitochondrial large ribosomal subunit	structural constituent of ribosome	1.66	0.038
A0A096MPA6	TKFC	Triokinase/FMN cyclase	carbohydrate phosphorylation; fructose catabolic process to hydroxyacetone phosphate and glycera ldehyde-3-phosphate; glycerol metabolic process; negative regulation of MDA-5 signaling pathway		ATP binding; FAD-AMP lyase (cyclizing) activity; glycerone kinase activity; triokinase activity	1.67	0.029
A0A2I3MBF2	ACSS2	propionate--CoA ligase	lipid biosynthetic process	cytosol	acetate-CoA ligase activity;propionate-CoA ligase activity	1.67	0.003
A0A096N932	MRPL37	Mitochondrial ribosomal protein L37	protein metabolism;other metabolic processes	mitochondrion;translational apparatus	other molecular function	1.67	0.029
A0A8I5NTE3	PECR	Peroxisomal trans-2-enoyl-CoA reductase				1.67	0.019
A0A096NSQ7	SLC9A3R1	Na(+)/H(+) exchange regulatory cofactor NHE-RF	cell organization and biogenesis;transport;developmental processes;signal transduction;other biological processes	non-structural extracellular;plasma membrane;other membranes;cytoskeleton;other cell component	signal transduction activity or receptor binding;other molecular function	1.68	0.002
A0A096NQ11	MRPL24	Mitochondrial ribosomal protein L24	protein metabolism;other metabolic processes	mitochondrion;translational apparatus	nucleic acid binding activity;other molecular function	1.68	0.009
A0A2I3MG31	AKR1C1	Aldo-keto reductase family 1 member Cl			estradiol 17-beta-dehydrogenase [NAD(P)| activity	1.68	0.029
A0A096MNY1	GZMA	Granzyme A	protein metabolism;other metabolic processes;signal transduction;other biological processes	nucleus	other molecular function	1.69	0.038
A0A096MQH2	SDSL	L-serine ammonia-lyase	amino acid metabolic process		identical protein binding;L-serine ammonia-lyase activity;pyridoxal phosphate binding	1.70	0.012
A0A096N523	PAFAH2	Platelet-activating factor acetylhydrolase	lipid catabolic process		l-alkyl-2-acetylglycerophosphocholine esterase activity	1.70	0.022
A0A8I5NB34	PM20D2	Peptidase M20 domain containing 2	proteolysis;regulation of protein metabolic process	nucleoplasm	dipeptidase activity;identical protein binding	1.70	0.008
A0A096P5A5	MINPP1	Multiple inositol polyphosphate phosphatase 1			bisphosphoglycerate 3-phosphatase activity;inositol hexakisphosphate 2-phosphatase activity	1.70	0.017
A0A2I3MGA6	GOLIM4	Golgi integral membrane protein 4		Golgi membrane		1.71	0.030
A0A096N1L0	KIAA0319L	KIAA0319 like		cytoplasmic vesicle;Golgi apparatus;membrane;nucleolus		1.71	0.048
A0A096N129	RBFA	Ribosome binding factor A	RNA metabolism OR transcription;other metabolic processes			1.73	0.009
A0A096N6V2	ACSL5	Long-chain-fatty-acid--CoA ligase		endoplasmic reticulum membrane; mitochondrial outer membrane; nucleolus; nucleoplasm; plasma membrane	ATP binding;long-chain fatty acid-CoA ligase activity	1.73	0.042
A0A2I3LGW7	CAMSAP3	Calmodulin regulated spectrin associated protein family member 3	cell adhesion;cell organization and biogenesis;transport;developmental processes;other biological processes	cytoskeleton;nudeus;other cell component	cytoskeletal activity;other molecular function	1.76	0.041
A0A2I3LML8	RPL36	60S ribosomal protein L36	protein metabolism;other metabolic processes;translation	translational apparatus	other molecular function	1.77	0.034
A0A8I5NLU5	RETSAT	Retinol saturase		endoplasmic reticulum membrane;nudear outer membrane	all-trans-retinol 13,14-reductase activity	1.79	0.017
A0A096NII4	MRPS18A	Mitochondrial ribosomal protein S18A	protein metabolism; other metabolic processes; translation	mitochondrion;translationa1 apparatus	other molecular function	1.80	0.007
A0A8I5MYF8	LOC101021945	Histone H4		nucleoplasm;nucleosome	DNA binding; protein heterodimerization activity; structural constituent of chromatin	1.80	0.006
A0A2I3MM43	ACOT7	Acyl-CoA thioesterase 7	coenzyme A biosynthetic process; long-chain fatty-acyl-CoA catabolic process; medium-chain fatty acid biosynthetic process; medium-chain fatty-acyl-CoA catabolic process; palmitic acid biosynthetic process	cytosol;nudeoplasm	carboxylic ester hydrolase activity; long-chain fatty acyl-CoA binding; palmitoyl-CoA hydrolase activity; protein homodimerization activity	1.82	0.044
A0A096NMH8	DCTPP1	dCTP pyrophosphatase 1	DNA metabolism;other metabolic processes;stress response	cytosol; mitocbondrion; nucleus	other molecular function	1.82	0.010
A0A096NGT6	PUS7	Pscudouridine synthase 7	RNA metabolism OR transcription; other metabolic processes; other biological processes	nucleus	nucleic acid binding activity;other molecular function	1.87	0.032
A0A2I3MAQ3	MRPL34	39S ribosomal protein L34, mitochondrial	protein metabolism;other metabolic processes;translation	translational apparatus	other molecular function	1.88	0.016
A0A096NGH5	SMPDL3A	Acid sphingomyelinase-like phosphodiesterase	nucleoside triphosphate catabolic process;sphingomyelin catabolic process	extracellular space	sphingomyelin phosphodiesterase activity;zinc ion binding	1.90	0.042
A0A2I3MYK2	SLC4A7	Anion exchange protein	transport	plasma membrane; other membranes	transporter activity	1.91	0.010
A0A2I3MX34	SIRT6	Sirtuin 6			NAD+ binding;transferase activity	1.91	0.024
A0A2I3MSI4	ENTPD5	Ectonucleoside triphosphate diphosphohydrolase 5 (inactive)			ATP binding;hydrolase activity	1.91	0.038
A0A096MQ22	CYP27A1	Cytochrome P450 family 27 subfamily A member 1	bile acid biosynthetic process; cholesterol catabolic process		cholestanetriol 26-monooxygenase activity; cholesterol 26-hydroxylase activity; heme binding;iron ion binding; vitamin D3 25-hydroxylase activity	1.92	0.025
A0A2I3LLW9	ACOX1	Acyl-coenzyme A oxidase	fatty acid beta-oxidation using acyl-CoA oxidase	peroxisome	acyl-CoA oxidase activity; FAD binding;fatty acid binding	1.93	0.026
A0A2I3M7X4	MRPL43	Mitochondrial ribosomal protein L43	protein metabolism; other metabolic processes; translation	mitochondrion translationa1 apparatus	other molecular function	1.93	0.021
A0A2I3U68	MRPL53	Mitochondrial ribosomal protein L53	protein metabolism;other metabolic processes;translation	mitochondrial large ribosomal subunit		1.93	0.007
A0A096P4N8	TOM1L1	Target of mybl like 1 membrane trafficking protein	transport; signal transduction;other biological processes	cytosol;other cytoplasmic organelle	enzyme regulator activity;other molecular function	1.94	0.041
A0A096P1S4	ALDH1A1	Aldehyde dehydrogenase 1 family member Al	cellular detoxification of aldehyde; fructosamine catabolic process	cytosol	3-deoxyglucosone dehydrogenase activity	1.96	0.018
A0A2I3LIL0	MFSD14A	Major facilitator superfamily domain containing 14A	transport	other membranes;ER/Golgi	transporter activity	1.97	0.032
A0A096NKS2	TTC38	Tetratricopeptide repeat protein 38		extracellular exosome		1.98	0.009
A0A096NS00	CYP3A5	Cytochrome P450 3A	alkaloid catabolic process; estrogen metabolic process; lipid hydroxylation; oxidative demethylation; retinoic acid metabolic process; xenobiotic catabolic process	endoplasmic reticulum membrane	estrogen 16-alpha-hydroxylase activity; heme binding; iron ion binding;oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen; retinoic acid 4-hydroxylase activity	1.99	0.027
A0A8I5NJY9	LOC101002132	Histone H3		nucleosome;nucleus	DNA binding; protein heterodimerization activity; structural constituent of chromatin	1.99	0.025
A0A2I3N853	AHCYL2	Adenosylhomocysteinase like 2	one-carbon metabolic process	cytosol;intracellular membrane-bounded organelle;neuron projection		2.04	0.023
A0A096NI51	MRPS10	28S ribosomal protein S10, mitochondrial	protein metabolism;other metabolic processes;translation	mitochondrial small ribosomal subunit		2.05	0.011
A0A096NX64	PCX2	phosphoenolpyruvate carboxykinase (GTP)	gluconeogenesis		GTP binding; metal ion binding; phosphoenolpyruvate carboxykinase (GTP) activity	2.06	0.000
A0A2I3LVN6	CPD	Carboxypeptldase D	proteolysis	membrane	metallocarboxypeptidase activity; zinc ion binding	2.07	0.008
A0A2I3NDN8	ATP1A1	Sodium/potassium-transporting ATPase subunit alpha	transport	plasma membrane;other membranes	transporter activity;other molecular function	2.08	0.016
A0A8I5POB2	HPDL	4-hydroxyphenylpyruvate dioxygenase	aromatic amino acid metabolic process	mitochondrion	4-hydroxyphenylpyruvate dioxygenase activity; metal ion binding	2.10	0.008
A0A096N4R1	MRPL16	39S ribosomal protein L16, mitochondrial	protein metabolism;other metabolic processes;translation	mitochondrion translational apparatus	nucleic acid binding activity;other molecular function	2.10	0.006
A0A096MYX7	DGAT1	O-acyltransferase	cell organization and biogenesis; other metabolic processes;other biological processes	other membranes;ER/Golgi	other molecular function	2.11	0.005
A0A8I5N4M8	GMDS	GDP-mannose 4,6-dehydratase	de novo’ GDP-L-fucose biosynthetic process; GDP-mannose metabolic process		GDP-mannose 4,6-dehydratase activity	2.14	0.016
A0A096MYR9	GFPT1	glutamine--fructose-6-phosphate transaminase (isomerizing)	glutamine metabolic process;UDP-N-acetylglucosamine biosynthetic process		carbohydrate derivative binding;glutamine-fructose-6-phosphate transaminase (isomerizing) activity	2.14	0.034
A0A8I5NU25	LOC101009759	Sulfotransferase			sulfotransferase activity	2.14	0.029
A0A096NDY4	MRPL17	39S ribosomal protein L17, mitochondrial	protein metabolism;other metabolic processes;translation	mitochondrion;translationa1 apparatus	other molecular function	2.20	0.020
A0A096N5B0	VNN1	Vanin 1	cell adhesion;other metabolic processes;stress response;other biological processes	other cell component	other molecular function	2.25	0.009
A0A8I5NHT6	MRPL33	39S ribosomal protein L33, mitochondrial	protein metabolism;other metabolic processes;translation	mitochondrial large ribosomal subunit	structural constituent of ribosome	2.27	0.008
A0A096MRZ6	ATP1B1	Sodium/potassium-transporting ATPase subunit beta	cell adhesion; other metabolic processes; transport; signal transduction; other biological processes	plasma membrane;other membranes;cytoskeleton;other cell component	other molecular function	2.37	0.029
A0A096MW86	PIGR	Polymeric immunoglobulin receptor	transport;signal transduction;other biological processes	non-structural extracellular;plasma membrane;other membranes.other cell component	other molecular function	2.39	0.019
A0A8I5NUI9	GALNT3	Polypeptide N-acetylgalactosaminyltransferase	protein O-linked glycosylation	Golgi membrane	carbohydrate binding; glycosyltransferase activity	2.40	0.050
A0A096NT10	ACOX1	Acyl-coenzyme A oxidase	generation of precursor metabolites and energy; hydrogen peroxide biosynthetic process; prostaglandin metabolic process; spermatogenesis; very long-chain fatty acid beta-oxidation	peroxisomal membrane	acyl-CoA oxidase activity;FAD binding;fatty acid binding;PDZ domain binding;protein homodimerization activity	2.45	0.000
A0A096N519	GIPC2	GIPC PDZ domain containing family member 2	protein binding	extracellular exosome		2.47	0.040
A0A096NFC3	HMGCS1	Hydroxymethylglutaryl-CoA synthase (HMG-CoA synthase)	acetyl-CoA metabolic process;cholesterol biosynthetic process;farnesyl diphosphate biosynthetic process, mevalonate pathway		hydroxymethylglutaryl-CoA synthase activity	2.49	0.047
A9X1B6	CKMT1B	creatine kinase	phosphocreatine biosynthetic process;phosphorylation		ATP binding;creatine kinase activity	2.53	0.031
A0A096MWS2	BCL2L15	BCL2 like 15	regulation of apoptotic process	cytosol;nudeus		2.54	0.003
A0A096NJ40	MCM3	DNA replication licensing factor MCM3	cell cycle OR cell proliferation;cell organization and biogenesis;DNA metabolism;other metabolic processes	nucleus.other cell component	nucleic acid binding activity;other molecular function	2.59	0.038
A0A096MLV9	METTL7B	Methyltransferase like 7B			thiol S-methyltransferase activity	2.60	0.007
A0A2I3M0H5	ERAP2	Ami nopeptidase	proteolysis		aminopeptidase activity;metallopeptidase activity;zinc ion binding	2.72	0.031
A0A2I3MYY2	RBM47	RNA-binding protein 47	RNA metabolism OR transcription; other metabolic processes; developmental processes	nucleus	nucleic acid binding activity	2.75	0.006
A0A096NSW2	MYO15B	Myosin XVB		cytoplasm;myosin complex	actin binding;ATP binding;cytoskeletal motor activity	2.75	0.004
A0A8I5R909	HSD11B2	Hydroxysteroid 11-beta dehydrogenase 2	cortisol metabolic process;female pregnancy	intracellular membrane-bounded organelle	11-beta-hydroxysteroid dehydrogenase (NAD+) activity	2.86	0.009
A0A096MS63	HK2	Hexokinasc	cell organization and biogenesis; other metabolic processes; stress response; transport; other biological processes	other membranes; cytosol; cytoskeleton; mitochondrion	kinase activity;other molecular function	2.87	0.023
A0A096NKF0	DECR2	2,4-dienoyl-CoA reductase 2	CTP biosynthetic process;fatty acid catabolic process;GTP biosynthetic process;UTP biosynthetic process		2,4-dienoyl-CoA reductase (NADPH) activity;nucleoside diphosphate kinase activity	2.89	0.007
A0A0A0MUP7	SPC24	Kinetochore protein Spc24	cell cycle OR cell proliferation;other biological processes	nucleus;other cell component		2.90	0.029
A0A096P572	PHYHIPL	Phytanoyl-CoA 2-hydroxylase interacting protein like		cytoplasm		2.90	0.008
A0A2I3MGL1	SLC12A2	Solute carrier family 12 member 2	transport	plasma membrane;other membranes	transporter activity	2.92	0.005
A0A096NRM2	REGIB	Regenerating family member 1 beta			carbohydrate binding	2.92	0.013
A0A2I3LYX4	RBP2	Retinol binding protein 2			lipid binding	2.99	0.005
A0A0A0MUV4	CYP2S1	Cytochrome P450	prostaglandin metabolic process; retinoic acid metabolic process	endoplasmic reticulum membrane	heme binding; hydro-lyase activity; iron ion binding; oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen;thromboxane-A synthase activity	3.00	0.048
A0A8I5NII0	CASP7	Caspasc 7	apoptotic process;proteolysis	protein-containing complex	cysteine-type endopeptidase activity	3.01	0.008
A0A096N4P9	OTC	Ornithine transcarbamylase, mitochondrial (Ornithine carbamoyltransferase, mitochondrial)	ammonium homeostasis;citrulline biosynthetic process;ornithine catabolic process;urea cycle	mitochondrion	amino acid binding;ornithine carbamoyltransferase activity	3.02	0.008
A0A8I5NN65	FBP2	fructose-bisphosphatase	gluconeogenesis	cytosol;plasma membrane	fructose 1,6-bisphosphate 1-phosphatase activity;identical protein binding;metal ion binding	3.06	0.010
A0A096NL20	DUOX2	NAD(P)H oxidase (H2O2-forming)	adenohypophysis morphogenesis; bone mineralization; fertilization; hormone biosynthetic process; hydrogen peroxide catabolic process; inner ear development; multicellular organism growth; response to oxidative stress; thyroid gland development; thyroid hormone generation	apical plasma membrane	calcium ion binding;heme binding;NAD(P)H oxidase H202-forming activity;peroxidase activity	3.09	0.017
A0A0A0MU38	RHPN2	Rhophilin Rho GTPase binding protein 2	signal transduction;other biological processes	plasma membrane;other membranes		3.12	0.004
A0A096NQ50	SCIN	Scinderin	cell organization and biogenesis;other biological processes	cytoskeleton;other cell component	cytoskeletal activity;other molecular function	3.15	0.007
A0A096MX08	HEPH	Hephaestin	transport;other biological processes	plasma membrarte;other membranes	other molecular function	3.15	0.023
A0A2I3M3Q6	EPCAM	Epithelial cell adhesion molecule	developmental processes;signal transduction;other biological processes	plasma membrane;other membranes;other cell component	other molecular function	3.24	0.015
A0A096NMG4	CALML4	Calmodulin like 4			calcium ion binding	3.24	0.037
A0A2I3N7V1	BACE2	Beta-secretase 2	cell-cell signaling; transport;other biological processes	non-structural extracellular		3.27	0.010
A0A096NA44	PLS1	Plastin 1	cell organization and biogenesis; transport; developmental processes; other biological processes	cytoskeleton;other cell component	cytoskeletal activity;other molecular function	3.28	0.015
D2SML9	UGT2B43	UDP-g1ucurortosyltransferase		endoplasmic reticulum membrane	glucuronosyltransferase activity	3.29	0.007
A0A8I5R289	CA13	Carbonic anhydrase		cytosol;intracellular membrane-bounded organelle;myelin sheath	carbonate dehydratase activity;zinc ion binding	3.30	0.031
A0A096NF33	AGR3	Anterior gradient 3, protein disulphide isomerase family member		endoplasmic reticulum	dystroglycan binding	3.31	0.035
A0A096MMJ2	EPS8L3	EPS8 like 3		cellular anatomical entity		3.31	0.003
A0A2I3LRL6	AKR7L	Aldo-keto reductase family 7 like (gene/pseudogene)			reductase activity	3.34	0.003
A0A2I3MW71	MTTP	Microsomal triglyceride transfer protein large subunit	cell organization and biogenesis; protein metabolism; other metabolic processes; transport; other biological processes	cytosol;ER/Golgi;other cell component	transporter activity; other molecular function	3.35	0.009
A0A096MWB0	B3GNT7	Hexosyltransferase	protein metabolism; other metabolic processes	other membranes;ER/Golgi;other cell component	other molecular function	3.36	0.015
A0A2I3MEL2	GALNT5	Polypeptide N-acetylgalactosaminyltransferase	protein O-linked glycosylation	Golgi membrane	carbohydrate binding;polypeptide N-acetylgalactosaminyltransferase activity	3.38	0.011
A0A096MZH7	MYH14	Myosin heavy chain 14		myosin complex		3.40	0.041
AQA8ISQZL5	XRCC5	X-ray repair cross complementing 5	DNA recombinatlon;double-strand break repair via nonhomologous end joining;telomere maintenance	Ku70:Ku80 complex;telomere maintenance;DNA recombination	ATP binding; damaged DNA binding; DNA helicase activity;hydrolase activity;telomeric DNA binding	3.42	0.025
A0A096NLZ8	C7HI5orf48	Normal mucosa of esophagus-specific gene 1 protein				3.44	0.020
A0A096P5J1	KRT20	Keratin 20	cell organization and biogenesis; stress response; other biological processes	cytosol;cytoskeleton	other molecular function	3.48	0.009
A0A096MYA4	MOGAT2	Acyltransferase	diacylglycerol biosynthetic process; intestinal absorption; monoacylglycerol biosynthetic process; triglyceride biosynthetic process	endoplasmic reticulum membrane;perinuclear endoplasmic reticulum membrane	2-acylglycerol O-acyltransferase activity;acetyltransferase activity	3.51	0.011
A0A096NZJ5	GBA3	Glocosylccramidasc beta 3 (gene/pseudogene)	carbohydrate metabolic process		hydrolase activity, hydrolyzing O-glycosyl compounds	3.55	0.014
A0A2I3N061	CLCA1	Chloride channel accessory 1	transport	other membranes;other cell component	transporter activity;other molecular function	3.56	0.000
A0A8I5NKB7	LGALS4	Galectin 4			carbohydrate binding	3.56	0.034
A0A096N416	MEP1B	Meprin A subunit	transport	plasma membrane;other membranes	other molecular function	3.57	0.023
A0A096MX76	FABP2	Fatty acid-binding protein, intestinal	transport	other cell component	other molecular function	3.61	0.019
A0A8I5NGP5	MGST1	glutathione transferase		membrane	transferase activity	3.61	0.020
A0A096NDS2	MUC13	Mucin 13, cell surface associated	maintenance of gastrointestinal epithelium	apical plasma membrane; cytosol; extracellular space	protein homodimerization activity	3.62	0.003
A0A2I3NGG3	CHGA	Chromogranin A	stress response;transport;other biological processes	non-structural extracellular;other cell component		3.65	0.007
A0A096NB01	XDH	Xanthine dehydrogenase/oxidase	cell organization and biogenesis; other metabolic processes; transport; other biological processes	cytosol;ER/Golgi;other cytoplasmic organelle	other molecular function	3.68	0.002
A0A096NJN9	SLC5A1	Solute carrier family 5 member 1	transport;other biological processes	plasma membrane;other membranes;other cytoplasmic organelle;other cell component	transporter activity	3.68	0.020
A0A096MTI8	ELAPOR1	Endosome-lysosome associated apoptosis and autophagy regulator 1	cell organization and biogenesis; other metabolic processes; stress response; other biological processes	plasma membrane;other membranes; ER/Golgi; other cytoplasmic organelle		3.69	0.002
A0A2I3MKQ3	AKR7A2	Aldo-keto reductase family 7 member A2				3.75	0.007
A0A2I3N7K6	LOC100998585	Trypsin-1	proteolysis		serine-type endopeptidase activity	3.78	0.030
A0A2I3MWQ2	ENPEP	Aminopeptidase	cell cycle OR cell proliferation; developmental processes; other biological processes	plasma membrane;other membranes;other cell component	other molecular function	3.86	0.013
A0A096P394	SLC13A2	Solute carrier family 13 member 2	transport	other membranes	transporter activity	3.93	0.022
A0A2I3M2E2	TMIGD1	Transmembrane and immunoglobulin domain containing 1				3.94	0.043
A0A096MQK3	A0C1	Amine oxidase	cellular response to azide; cellular response to copper ion; cellular response to copper ion starvation; cellular response to histamine; putrescine metabolic process; response to antibiotic	extracellular exosome; plasma membrane	calcium ion binding; copper ion binding; diamine oxidase activity; heparin binding; primary amine oxidase activity; protein homodimerization activity; protein-containing complex binding; quinone binding;zinc ion binding	3.97	0.005
A0A096NXS7	LOC101006700	Regenerating family member 3 alpha			carbohydrate binding;identical protein binding	3.97	0.027
A0A2I3MUYO	EMILIN1	Elastin microfibril interfacer 1	fructose metabolic process		ketohexokinase activity	3.99	0.005
A0A8I5NX92	FABP6	Fatty acid binding protein 6			lipid binding	3.99	0.026
A0A0A0MUV2	TJP3	Tight junction protein 3		bicellular tight junction; plasma membrane		4.04	0.013
A0A2I3LPH8	FAB PI	Fatty acid-binding protein, liver	stress response; other biological processes	cytosol; nucleus	other molecular function	4.06	0.004
A0A096N861	SI	Sucrase-isomaltase	sucrose catabolic process	membrane	carbohydrate binding;oligo-l,6-glucosidase activity	4.06	0.007
A0A096N2V6	CPS1	Carbamoyl-phosphate synthase 1	de novo’ pyrimidine nucleobase biosynthetic process; carbamoyl phosphate biosynthetic process; cellular response to ammonium ion; glutamine metabolic process; homocysteine metabolic process; nitric oxide metabolic process; response to lipopolysaccharide; triglyceride catabolic process; vasodilation	mitochondrial nucleoid; nucleolus	ATP binding; carbamoyl-phosphate synthase (ammonia) activity; carbamoyl-phosphate synthase (glutamine-hydrolyzing) activity; metal ion binding; modified amino acid binding	4.08	0.002
A0A096NNI6	PCK1	Phosphoenolpyruvate carboxykinase (GTP)	protein metabolism; other metabolic processes; stress response; other biological processes	cytosol; ER/Golgi	kinase activity; other molecular function	4.08	0.012
A0A8I5MZK3	PRSS2	Serine protease 2	collagen catabolic process; proteolysis	extracellular space	calcium ion binding; serine-type endopeptidase activity	4.09	0.009
A0A096P102	ALDOB	Fructose-bisphosphate aldolase	cell organization and biogenesis; other metabolic processes; other biological processes	cytoskeleton	cytoskeletal activity; other molecular function	4.20	0.006
A0A096MW03	MYO1A	Myosin IA OS=Papio anubis	cell organization and biogenesis; other biological processes	plasma membrane; other membranes; cytoskeleton; other cell component	cytoskeletal activity; other molecular function	4.24	0.018
A0A2I3MVT8	KCTD14	Potassium channel tetramerization domain containing 14	cell organization and biogenesis			4.25	0.013
A0A8I5R102	LOC101005786	Llthostathine-1-alpha isoform X2			carbohydrate binding; growth factor activity; molecular function inhibitor activity	4.28	0.010
A0A096NLE5	SCGN	Sccrctagogin		transport vesicle membrane	calcium ion binding	4.36	0.012
A0A096N2A5	VILI	Villin-l	cell organization and biogenesis; transport; signal transduction; other biological processes	plasma membrane;other membranes;cytoskeleton;other cell component	enzyme regulator activity; cytoskeletal activity; other molecular function	4.37	0.004
A0A2I3NGN1	DDC	Dopa decarboxylase	amino acid metabolic process; catecholamine biosynthetic process		carboxy-lyase activity; pyridoxal phosphate binding	4.43	0.004
A0A096N6M1	NTS	Neurotonsin/neuromedin N	signal transduction; other biological processes	non-structural extracellular; ER/Golgi	signal transduction activity or receptor binding	4.43	0.008
A0A096NIV6	MEP1A	Meprin A subunit	epidermal growth factor receptor ligand maturation	meprin A complex; plasma membrane	metallodipeptidase activity; metalloendopeptidase activity; zinc ion binding	4.46	0.031
A0A096P4Z5	HKDC1	hexokinase	glycolytic process; hexose metabolic process; intracellular glucose homeostasis	mitochondrion; photoreceptor inner segment	ATP binding;glucokinase activity; glucose binding	4.53	0.000
A0A8I5NAJ1	LOC101000241	Peptidase SI domain-containing protein	proteolysis		serine-type endopeptidase activity	4.59	0.017
A0A096N2N1	CDH17	Cadherin 17	cell adhesion; transport; developmental processes; signal transduction	plasma membrane;other membranes;nucleus;other cell component	signal transduction activity or receptor binding; transporter activity; other molecular function	4.62	0.013
AGA2I3MNY7	LGALS2	Galectin 2			carbohydrate binding	4.72	0.009
A0A096NGR8	DEFA6	Defensin alpha 6	stress response; defense response to bacterium, fungus; killing of other organism	non-structural extracellular	protein homodimerization activity	6.64	0.010

Table 1C.

CS-POCO- Regenerated proteins < Native proteins

Accession	Gene Symbol	Description	Biological Process	Cellular Component	Molecular Function	Abundance Ratio (log2): Regenerated/Native	Abundance Ratio P-Value: Regenerated/Native
A0A096NFG4	CHRDL1	Chordin like 1			developmental protein	−1.030	0.038
A0A2I3MNY7	LGALS2	Galectin-2	carbohydrate binding		other molecular function	−1.490	0.025

Table ID.

CS-SIS- Regenerated proteins vs. Native proteins

Accession	Gene Symbol	Description	Biological Process	Cellular Component	Molecular Function	Abundance Ratio (log2): Regenerated/Native	Abundance Ratio P-Value: Regenerated/Native
A0A096P238	ASPN	Asporin	negative regulation of tooth mineralization; negative regulation of transforming growth factor beta receptor signaling pathway; bone mineralization	extracellular region and matrix	calcium ion binding	1.090	0.010
A0A2I3M3L4	HBD	Hemoglobin subunit delta	transport	cytosol; hemoglobin complex	heme binding; metal ion binding; oxygen binding	−1.160	0.021
A0A8I5R8I1	ZNF229	Zinc finger protein 229	regulation of DNA-templated transcription		metal ion binding	−1.190	0.011
A0A096N0J2	FAM180B	Family with sequence similarity 180 member B		extracellular region		−1.100	0.009
A0A096MSH0	N4BP2	NEDD4 binding protein 2	other metabolic processes	cytosol	ubiquitin binding; polydeoxyribonucleotide 5’-hydroxyl-kinase activity;DNA endonuclease activity;ATP binding	−1.020	0.044
A0A2I3MTE7	SMOC1	SPARC related modular calcium binding 1	developmental processes;regulation of osteoblast differentiation	non-structural extracellular	calcium ion binding;extracellular matrix binding	−1.270	0.017

3.3 |. Differentially expressed proteins

For the consideration of differentially expressed protein, the p-value for the protein was lower than 0.05 (a 95% confidence interval). After setting this threshold, we examined all the proteins that were highly expressed in the grafted or regenerated tissue or its matched native tissue by comparing their abundance within those tissues by their log₂ fold ratio. The negative log₂ fold ratio designates a protein with higher expression level in the native tissue when compared to the regenerated grafted tissue. The differentially expressed proteins are shown in Table 1A for E grafted proteins < native proteins, Table 1B for E grafted proteins > native proteins, Table 1C for CS-POCO, and Table 1D for CS-SIS. For each table, the identifying feature of the protein is the UniProt Accession number. Any uncharacterized proteins or proteins with unknown function were updated using information listed on the UniProt and GenBank Gene websites.

As shown in Table 1C, for the CS-POCO graft, only two proteins were significantly differentially expressed. CHRDL1 (chordin like 1) had log2 fold ratio of −1.03 (p-value= 0.03836) while LGALS2 (Galectin-2) had a log₂ fold ratio of −1.49 (p-value= 0.02488). Both proteins had higher expression in the native tissue when compared to the CS-POCO graft regenerated tissue. For Table 1D, protein ASPN (asporin), which had an expression value of log₂ fold ratio of 1.09, while 5 proteins, Hemoglobin, ZNF229 (Zinc finger protein 229), FAM180B (Family with sequence similarity 180 member B), N4BP2 (NEDD4 binding protein 2), SMOC1 (SPARC related modular calcium binding 1), all were expressed at higher expression in the native tissue compared to the CS-SIS tissue at log₂ fold ratio ranging from −1.27 to −1.02.

Tables 1A and 1B showed the differentially expressed proteins for the E group which had the greatest number of proteins that differed significantly between the native tissue and the regenerated tissue. As shown, within the 419 proteins that were differentially expressed, of which 160 proteins were expressed at higher levels in the native tissue compared to regenerated tissue in Table 1A, while 259 proteins with higher expression levels in the entero grafted tissue compared to the native tissue in Table 1B. In Table 1A, the top 10 proteins having the highest abundance ratio in the native tissue compared to entero grafted tissue included Purkinje cell protein 4 (PCP4) (abundance log₂ fold ratio of −4.09, p-value of 0.034), Tripartite motif containing 29 (TRIM29) (abundance log2 fold ratio of −3.55, p-value of 0.039), Type-2 angiotensin II receptor (AGT2) (abundance log2 fold ratio of −3.24, p-value of 0.038), Heparanase 2 (inactive)- HPSE2 (abundance log₂ fold ratio of −3.13, p-value of 0.012), SMB domain-containing protein (SBSPON) (abundance log2 fold ratio of −2.99, p-value of 0.011), LIM zinc-binding domain-containing protein (FHL1) (abundance log2 fold ratio of −2.81, p-value of 0.022), MAGUK p55 scaffold protein 2 (MPP2) (abundance log2 fold ratio of −2.76, p-value of 0.013), uncharacterized protein (abundance log2 fold ratio of −2.68, p-value of. 0.024), EGF like, fibronectin type III and laminin G domains (EGFLAM) (abundance log2 fold ratio of −2.67, p-value of 0.031), and CDK5 regulatory subunit associated protein 1 (CDK5RAP1) (abundance log2 fold ratio of −2.52, p-value of 0.022).

In Table 1B, the top 10 proteins with the highest abundance ratio in the E grafted tissue compared to native tissue included Defensin alpha 6 (DEFA6) (abundance log₂ fold ratio of 6.64, p-value of 0.010), Galectin 2 (LGALS2), (abundance log₂ fold ratio of 4.72, p-value of 0.009), Cadherin 17 (CDH17) (abundance log₂ fold ratio of 4.62, p-value of 0.013), Peptidase S1 domain-containing protein (LOC101000241) (abundance log₂ fold ratio of 4.59, p-value of 0.017), Hexokinase-HKDC1 (abundance log₂ fold ratio of 4.53, p-value of 0.000), Meprin A subunit (MEP1A) (abundance log₂ fold ratio of 4.46, p-value of 0.031), Neurotensin/neuromedin N (NTS) (abundance log₂ fold ratio of 4.43, p-value of 0.008), Dopa decarboxylase (DDC) (abundance log₂ fold ratio of 4.43, p-value of 0.004 ), Villin-1 (VIL1) (abundance log₂ fold ratio of 4.37, p-value of 0.004), and Secretagogin (SCGN) (abundance log₂ fold ratio of 4.37, p-value of 0.004 ).

The biological function of the proteins was categorized as illustrated in Figure 3 with regards to those proteins expressed in E and summarized in Tables 1A and 1B. The gene symbol, protein description, biological function, cellular component, and molecular function were updated when needed to the most up to date UniProt and UniGene databases. As shown in the figure, the distribution of proteins that were highly expressed in the grafted tissue was indicated in the blue color while the native tissue in orange color. The protein categories based on biological process included amino acid metabolism, apoptotic process, ATP binding, calcium binding, carbohydrate binding/process, cell adhesion, cell cycle or cell proliferation, cell-cell signaling, cell organization and biogenesis, cholesterol process, complement activity, DNA binding/chromatin binding/or RNA binding, DNA metabolism, fatty acid metabolism/processing/lipid binding, fructose/mannose/or glycerol process/glucogenesis/glycolytic process, immunoglobulin/Ig-like proteins, metal ion/copper/zinc/or iron ion binding, protein metabolism/proteolysis, serine-type endopeptidase inhibitor activity, signal transduction, stress response, structural activity, transport, and other biological process/cellular component/or nuclear function. Some of the proteins that had overlapping functions were categorized according to the first listed function. In the figure for the 259 proteins that were highly expressed at levels greater than log2-fold ratio in the E-grafted tissue when compared to the native tissue are shown in blue, while the 160 proteins at highly in the native tissue vs. E-grafted tissue are shown in orange. As shown in the distribution graph, in several categories, more proteins were highly expressed in one tissue vs the other tissue, or none in the other tissue. An example of this included the category of amino acid metabolism where the E-grafted tissue had 7 proteins that are highly expressed and none in the native tissue. For proteins that have complement activity, there were 6 proteins that were highly expressed in the native tissue vs. none for the E-grafted tissue. This scenario is similar for the serine-type endopeptidase inhibitor activity category with 9 proteins in the native vs. none in the E-grafted tissue. For the protein metabolism/ proteolysis category, a large number of proteins- 63 were highly expresses in the E-grafted tissue compared to native, while 6 were highly expressed in the native tissue compared to E-grafted tissue.

Figure 3:

Biological Process Distribution for highly differentially expressed proteins in the enterocystoplasty study. The blue color-coded bars indicate the highly expressed proteins in the grafted tissue vs. the native tissue; while the orange coded bars indicate the highly expressed proteins in the native tissue vs. the grafted tissue.

4 |. DISCUSSION

In this study we have reported the proteomic profile of bladder augmented tissue in comparison to its native bladder tissue in a long-term study using a baboon bladder augmentation model. Three augmentation scenarios were employed that included the E-autograph ileal graft, the stem cell seeded polymeric biodegradable scaffold POCO, and the stem cell seeded biological scaffold SIS. The cells used for seeding onto the scaffold included autologous donor-matched bone marrow MSCs and CD34+ HSPCs. Over the course of the study in our baboon model, these cells would regenerate the portion of the cystectomized bladder. In our study, we used the cut off of log2 > 1 (or 2-fold with higher expression in the grafted or regenerated tissue) or log2 < −1 (or 2-fold with higher expression in the native tissue) and the significance level of p-value > 0.05 for the differentially expressed proteins in one tissue vs. the other. Using these criteria, we have found that the gold standard enterocystoplasty procedure that is clinically used yielded the most differentially expressed proteins in the grafted tissue vs the native tissue at a total of 419 proteins, with 259 proteins expressed at higher levels in the grafted tissue vs. native and 160 proteins expressed at higher levels in native vs. grafted tissue. For the proteins with higher expression in the native tissue vs. the grafted tissue included 4 proteins with log2 ratio of −3.13 to −4.09 (for a fold of 8.75 to 17.03), which included PCP4 (Purkinje cell protein 4), TRIM29 (Tripartite motif containing 29), AGTR2 (Type-2 angiotensin II receptor), and HPSE2 (Heparanase 2).

The proteins with the highest fold level of differential protein expression were in the ileal grafted tissue where a total of 65 proteins had a log2 ratio of 3.00 to 6.64 (or a fold of 8 to 99.73). The protein DEFA6, Defensin alpha 6, had the highest differential fold difference of log2 ratio of 6.64 or 99.73. DEFA6 is highly expressed in the secretory Paneth cells that reside in the small intestine [19,20]. DEFA6 protects the intestinal mucosa and defends against invasion of viruses and bacteria by forming fibrils and nanonets that encompasses pathogens [21]. It has however also reported that DEFA6 is also highly expressed in colorectal cancer (CRC) cell lines and patient samples [22]. By knocking down DEFA6 expression via shRNA in cancer cells, Jeong et al observed significantly inhibited cell growth, migration, and invasion in cancer cells in vitro, and inhibited tumorigenesis in vivo compared to control cells. Furthermore, it was determined that high DEFA6 expression to be a strong prognostic indicator for CRC as high expression of DEFA6 was observed in 51.4% (or 181/352) primary colorectal cancer tissue samples with high correlation to poor prognosis [21]. In a study by Husman et al, it was revealed that patients who undergone BAE had an incidence of 5–6% to develop bladder cancer after 50 years old [23]. It is worthy to note that whether or not that DEFA6 was observed to be highly expressed in cancer tissue that investigation into this protein maybe warranted to determine its potential role in tumorigenesis in bladder tissue post BAE.

In our study using the cell seeded graft, CS-POCO or CS-SIS, the proteomic analysis revealed the least number of differentially expressed proteins. CS-SIS had a total of 6 proteins including 1 protein having higher expression level in grafted vs. native at log2 ratio of 1.09 (2.13), and 5 proteins higher expression levels in native vs. grafted tissue and ranged from log2 ratio at −1.160 (fold of 2.23) to −1.270 (2.41). Our in-house CS-POCO graft yielded the least number of differentially expressed proteins with two proteins having higher expression in the native tissue vs. the grafted tissue. These two proteins were CHRDL1 (Chordin like 1) and LGALS2 (Galectin-2) which had log2 ratio at −1.030 (fold of 2.04) and −1.49 (fold of 2.81).

These data suggested that the ileal graft used in enterocystoplasty expressed many proteins that differed in the grafted tissue vs. native tissue, confirming a mismatch in tissue type. Using either CS-POCO or CS-SIS with the least of differentially expressed proteins in the regenerated tissue vs. native indicated better protein compatibility to the native tissue bladder. The data suggested further investigation into the use of CS-POCO or CS-SIS as potential cell seeded graft for the bladder augmentation as they support similar protein expression pattern compared to native bladder tissue.

Significance of Study.

Bladder augmentation enterocystoplasty has been used for decades as the gold-standard surgical procedure to treat severely dysfunctional bladders. Unfortunately, bowel (typically ileum) used for augmentation is an anatomical and physiological mismatch to bladder tissue and results in numerous complications including bladder perforation, secondary and tertiary redo surgeries, metabolic imbalances, excess mucus production, and increased risk of cancer. This is in part due to the intestinal protein expression that serves as a starting point and subsequent foundation in the optimization of pseudo bladder tissue. Within the context of this study, we demonstrate that autologous, bone marrow derived mesenchymal stem cells along with primitive hematopoietic stem/progenitor cells can used to regenerate bladder tissue in a large deficit, non-human primate bladder augmentation model. Data demonstrate that this synergistic cellular combination facilitates the promotion of a protein tissue landscape that is nearly identical to native bladder tissue.

Funding Statement

A.K.S. discloses support for the research of this work from the National Institutes of Health (NIH) [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), R01DK109539; National Institute of Biomedical Imaging and Bioengineering (NIBIB), R01EB026572]. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.