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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: Shock. 2016 May;45(5):540–554. doi: 10.1097/SHK.0000000000000532

Peptidomic Analysis of Rat Plasma: Proteolysis in Hemorrhagic Shock

Federico Aletti 1,2, Elisa Maffioli 3, Armando Negri 3,5, Marco H Santamaria 2, Frank A De Lano 2, Erik B Kistler 4, Geert W Schmid-Schönbein 2, Gabriella Tedeschi 3,5
PMCID: PMC4833562  NIHMSID: NIHMS735276  PMID: 26863123

Abstract

It has been previously shown that intestinal proteases translocate into the circulation during hemorrhagic shock and contribute to proteolysis in distal organs. However, consequences of this phenomenon have not previously been investigated using high-throughput approaches. Here, a shotgun label-free quantitative proteomic approach was utilized to compare the peptidome of plasma samples from healthy and hemorrhagic shock rats to verify the possible role of uncontrolled proteolytic activity in shock. Plasma was collected from rats after hemorrhagic shock (HS) consisting of two-hour hypovolemia followed by two-hour reperfusion, and from healthy control (CTRL) rats. A new two-step enrichment method was applied to selectively extract peptides and low molecular weight proteins from plasma, and directly analyze these samples by tandem mass spectrometry. 126 circulating peptides were identified in CTRL and 295 in HS animals. 96 peptides were present in both conditions; of these, 57 increased and 30 decreased in shock. In total, 256 peptides were increased or present only in HS confirming a general increase in proteolytic activity in shock. Analysis of the proteases that potentially generated the identified peptides suggests that the larger relative contribution of to the proteolytic activity in shock is due to chymotryptic-like proteases. These results provide quantitative confirmation that extensive, system-wide proteolysis is part of the complex pathologic phenomena occurring in hemorrhagic shock.

Keywords: Hemorrhagic shock, Proteolysis, Serine-Proteases, Matrix Metalloproteases, Peptidomics, Mass spectrometry

INTRODUCTION

Circulatory shock is the leading cause of mortality in the intensive care unit [1], and despite decades of basic and clinical research, morbidity and mortality rates remain high. An incomplete understanding of the pathological mechanisms involved in tissue damage, organ dysfunction and failure limits available treatments for shock to source control (e.g. antibiotic therapy) and supportive therapy such as fluids and pressor agents.

The pathways activated in response to an initial insult, for example global ischemia in hemorrhagic shock (HS), suggest that shock involves several common pathologic mechanisms. One specific organ, long suspected as fundamental to the pathogenesis of shock is the intestine, whose failure triggers a widespread inflammatory state, which can ultimately lead to multiple organ failure (MOF) and death. One of the hypotheses that implicate the intestine in the progression of shock is centered around the role of the enteral digestive enzymes [2]. The intestinal mucosal barrier may become severely damaged due to hypoperfusion in hemorrhage and sepsis. As a consequence, its function as a barrier between the intestinal lumen and the interstitial tissue and vasculature may become compromised, and digestive enzymes, such as proteases and lipases may exit the intestinal lumen and reach the systemic circulation, resulting in global cellular and organ dysfunction [25]. Digestive proteases contribute to malfunction of important transmembrane receptors, such as the insulin receptor, possibly through cleavage [4], but they are also known to activate matrix metalloproteinases (MMPs) in vivo, and could therefore act as mediators of secondary cell and tissue injury. Both serine proteases and MMPs have the potential to cause uncontrolled pathological proteolysis, and this phenomenon may play an important role in the evolution of shock-induced organ failure.

We test here the hypothesis that hemorrhagic shock causes an increase in systemic proteolysis of proteins in the presence of increased enzymatic activity levels. We used high-throughput quantitative mass spectroscopy-based proteomic techniques to analyze the peptidome, in order to precisely identify and quantify the peptides in plasma.

Proteomic approaches have been introduced recently in shock research [6], but interpretation of the data is still limited by the lack of a comprehensive model of the disease. Here we report the results of the analysis of the peptidome of rat plasma collected after hemorrhagic shock (HS) in order to quantify the relative changes in the concentration of circulating peptides relative to their physiologic concentration in the plasma of healthy control rats. A systematic analysis of the circulating peptides, both in terms of their origin and possible generation by proteolytic cleavage, was carried out to obtain information on the proteolytic activity of several serine proteases and MMPs present in the bloodstream. The objective of the study was to garner direct evidence in support of one of the possible mechanistic processes underlying shock-induced organ failure and death, i.e. diffuse proteolysis. These results may open future directions to identify possible biomarkers in circulatory shock, by analysis of peptides and proteins that appear in the plasma starting from the experimental protocol set up in the present report.

MATERIALS AND METHODS

A. Experimental Protocol

The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego and conforms to the Guide for the Care and Use of Laboratory Animals, 8th edition, by the National Institutes of Health (2011).

Six male Wistar rats (300–450 g, Harlan Laboratories, Inc., Indianapolis, IN) were randomly assigned to either a control (CTRL, n=3) or a hemorrhagic shock (HS, n=3) group. The animals were housed in the vivarium of the Department of Bioengineering at the University of California San Diego and experiments took place during the morning. The temperature (~ 20–22 °C) and humidity (~ 70–75%) in the vivarium are controlled and monitored regularly, and the light/dark cycles (6 hours light/6 hours dark) are standardized according to the facility procedures. Each cage houses at most two rats. Food and water are given at libitum and the health of animals is confirmed by regular veterinary inspection. The well-being of the animals is ensured according to Federal, State and University regulation.

After general anesthesia (ketamine 75 mg/kg + xylazine, 4 mg/kg; i.m.) the right femoral vein was cannulated for blood withdrawal and intravenous supplemental anesthesia (xylazine, 4 mg/kg; ketamine 7.5 mg/kg i.v.) and the right femoral artery for continuous monitoring of arterial blood pressure. Body temperature was measured continuously rectally, and the animals were placed on a heated stage (at 37°C) and covered with a heat-retaining blanket in order to limit the temperature drop occurring during the hypovolemic phase of the experiment. Both HS and CTRL animals were heparinized (porcine heparin from Sagent Pharmaceuticals, Schamburg, IL) following an initial stabilization after the induction of anesthesia (1 unit heparin/cc total blood volume, estimated at 6% of the body weight). Hemorrhage was induced by blood withdrawal from the femoral vein (0.5 cc/min) to a target mean arterial pressure (MAP) of 35 mmHg. Hypovolemia was maintained for two hours, at which point the shed blood was reinfused (0.5 cc/min). The shed blood was maintained at room temperature (~ 22°C) and gently warmed to 37°C before reinfusion to minimize the temperature gradient.

The animals were monitored for an additional two hours, and euthanized at the end of the observation period, with Beuthanasia-D (120 mg/kg, Merck Animal Health). CTRL animals were monitored under anesthesia and without any further intervention for the same duration of the shock experiment until euthanasia. Death was confirmed by verification of cardiac arrest after thoracotomy to expose the chest cavity.

B. Plasma collection

Just before euthanasia 1 mL of venous blood was collected in a BD Vacutainer ® Plus Plastic K2EDTA (Becton, Dickinson and Company, Franklin Lakes, NJ, U.S.A.) vial after the addition of a protease inhibitor solution (a tablet of cOmplete™ Protease Inhibitor Cocktail, Roche, diluted in water to obtain a 10X solution with the plasma sample), and centrifugation at 1300 rpm for 10 min to separate plasma. The supernatant was collected, stored at +4°C and shipped for analysis by mass spectroscopy.

C. Shotgun analysis and label free quantitation

C.1 Sample preparation

500 μL of each plasma sample were diluted with an equal volume of 32% (v/v) acetic acid, transferred to an Amicon Ultra-0.5 mL centrifugal filter (MWCO 10K), and centrifuged at 5000×g at 4 °C for 2 hour to deplete the high molecular weight proteins [7]. After the first centrifugation, 500 μL of 32% (v/v) acetic acid were added to the Amicon Ultra filter device and centrifuged at 5,000 × g for 2 hours. The filtrate was precipitated with two volumes of cold acetonitrile (ACN) containing 0.1% of trifluoroacetic acid (TFA) (stored at 4 °C for at least 1 hours) and centrifuged at 13,200 rpm for 30 minutes [8] to remove residual proteins. The supernatant containing peptides and low molecular weight proteins was collected, dried (Speed Vacuum), dissolved in 1% (v/v) formic acid and desalted (Zip-Tip C18, Millipore) before mass spectrometric (MS) analysis.

C.2 Liquid chromatography electrospray–tandem MS/MS analysis

LC-ESI-MS/MS analysis was performed on a Dionex UltiMate 3000 HPLC System with a PicoFrit ProteoPrep C18 column (200 mm, internal diameter of 75 μm) (New Objective, USA). Gradient: 1% ACN in 0.1 % formic acid for 10 min, 1–4 % ACN in 0.1% formic acid for 6 min, 4–30% ACN in 0.1% formic acid for 147 min and 30–50 % ACN in 0.1% formic for 3 min at a flow rate of 0.3 μl/min. The eluate was electrosprayed into an LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) through a Proxeon nanoelectrospray ion source (Thermo Fisher Scientific). The LTQ-Orbitrap was operated in positive mode in data-dependent acquisition mode to automatically alternate between a full scan (m/z 350–2000) in the Orbitrap (at resolution 60000, AGC target 1000000) and subsequent collision-induced dissociation (CID) MS/MS in the linear ion trap of the 20 most intense peaks from full scan (normalized collision energy of 35%, 10 ms activation). Isolation window: 3 Da, unassigned charge states: rejected, charge state 1: rejected, charge states 2+, 3+, 4+: not rejected; dynamic exclusion enabled (60 s, exclusion list size: 200). Three technical replicate analyses of each sample were performed. Data acquisition was controlled by Xcalibur 2.0 and Tune 2.4 software (Thermo Fisher Scientific).

C.3 Data processing and statistical analysis

Mass spectra were analyzed using MaxQuant software (version 1.3.0.5). The initial maximum allowed mass deviation was set to 15 ppm for monoisotopic precursor ions and 0.5 Da for MS/MS peaks. Enzyme specificity was set as unspecific. N-terminal acetylation, methionine oxidation, and asparagine/glutamine deamidation were set as variable modifications. The spectra were searched by the Andromeda search engine against the rat Uniprot sequence database (release 03.12.2014). Quantification in MaxQuant was performed using the built in XIC-based label free quantification (LFQ) algorithm [9] using fast LFQ. The required false positive rate was set to 5% at the peptide and 5% at the protein level, and the minimum required peptide length was set to 6 amino acids. Statistical analyses were performed using the Perseus software (version 1.4.0.6, www.biochem.mpg.de/mann/tools/).

Three biological replicates (3 healthy vs. 3 hemorrhagic shock animals) were analyzed. Three technical replicates were carried out for each biological sample. Only peptides present in at least 2 out of 3 technical replicates were considered as positively identified and quantified in each biological replicate. Then, biological replicates were compared, leading to three groups of peptides: only-HS peptides (199 peptides), i.e. peptides that were found in HS samples in at least 2 out of 3 biological replicates; only-CTRL peptides (30), i.e. peptides that were found in CTRL samples in at least 2 out of 3 biological replicates; and peptides present in both HS and CTRL (96), i.e. identified and quantified in at least 2 out of 3 biological replicates both in HS and CTRL samples).

Peptides were considered increased or decreased if they were present only in either the healthy (CTRL) or hemorrhagic (HS) group or, in the case of the 96 peptides found in both groups, if they showed 50 % fold change differences (> 1.5-fold increase or < 0.67-fold decrease) in HS compared to CTRL, as calculated from the ratio of mean XIC-based label free quantification of the respective biological replicates [10].

Proteins were classified as secreted or non-secreted, and the presence of transmembrane topology was searched by combining the results of five different computer programs: Perseus Annotation, SecretomeP [11], SignalP [12], Phobius [13], TMHMM [14].

C.4 Plasma protease activity

The role played in the proteolysis observed following shock by some of the most important digestive enzymes (i.e., trypsin, chymotrypsin, elastase), and by several MMPs known for their proteolytic function in pathologic conditions [15, 16] (i.e. MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-14) was determined by matching the C-terminus of the sequences of the peptides identified in the MS-based analysis with the cleavage site specificities of the proteases.

The relative contribution of a specific protease to the overall proteolytic activity was estimated: i) in the case of HS, as the ratio between the number of peptides generated by the protease and found to be present only in HS or in a higher concentration vs. CTRL, and the total number of peptides present in the HS sample; ii) in the case of CTRL, as the ratio between the number of peptides generated by the protease and found to be present only in CTRL or in a higher concentration vs. HS, and the total number of peptides present in the CTRL sample. The data were also analyzed by a Fisher’s exact test to detect statistically significant enrichment (increase and/or decrease) in the fraction of peptides potentially generated by a specific protease in HS or CTRL as compared to all peptides detected and quantified.

To cross-validate the estimates of protease activity derived from the peptidomics based approach described above, serine protease activity in the plasma was determined by gelatin zymography, as performed by SDS-PAGE gels containing 80 μg/mL gelatin. Gels were renatured by four 15 min washes with 2.5% Triton X-100 and incubated overnight at 37°C in developing buffer (0.05 M Tris base, 0.2 M NaCl, 4 μM ZnCl2, 5 mM CaCl2·2H2O). Gels were subsequently stained (50% methanol, 10% acetic acid, 40% water, and 0.25% Coomassie blue solution) for one hour before de-staining in buffer (10% methanol, 10% acetic acid, 80% water). A standard protein ladder (Invitrogen) was used to estimate the molecular weights of the proteases. Gels were digitized and bands were analyzed in ImageJ (http://imagej.nih.gov/ij/).

RESULTS

A. Peptidomics of plasma in shock

Shotgun label-free quantitative proteomic analysis is widely used to examine differences in global protein/peptide expression. This approach was utilized to compare the peptidome of plasma samples from healthy and hemorrhagic shock rats with the aim to identify and quantify the peptides present in plasma. These data serve to elucidate the possible role of uncontrolled proteolytic activity in shock. Figure 1 provides an overview of the workflow.

Figure 1. Peptidome sample preparation workflow for mass spectrometric analysis.

Figure 1

A new two-step enrichment method to selectively extract peptides and LMW components in plasma samples from healthy and hemorrhagic-shock rats was applied. The process consists of an ultrafiltration step with a 10kDa cut-off filter followed by a precipitation step conducted with a dissociating solution before LC-ESI MS/MS analysis

Due to the high complexity and wide dynamic range of plasma proteins, we applied a new two-step enrichment method to selectively extract peptides and low molecular weight (LMW) components, which are low abundant in plasma. The process consists of an ultrafiltration step with a 10kDa cut-off filter followed by a precipitation step conducted with a dissociating solution to increase the recovery of LMW molecules. To optimize reproducibility and quantification, each sample was then analyzed directly by LC-ESI MS/MS without any further separation step of the peptides mixture using the so-called shotgun approach. Analyses were performed on three biological replicates (3 CTRL vs 3 HS rats), each one analyzed three times (three technical replicates).

126 and 295 peptides were identified in the CTRL and HS groups, respectively (Figure 2A); among the 96 peptides present in both CTRL and HS, 57 increased (>1.5 fold-change) and 30 decreased (<0.67 fold change) in concentration in HS. Overall, the analysis identified 256 peptides, which were increased or present only in HS while 60 peptides were decreased or present only in the healthy group (CTRL). These peptides are listed in Tables 1 and 2 respectively, clearly showing a significantly increased number of peptides derived from an intrinsic proteolytic activity in HS samples in comparison with CTRL.

Figure 2.

Figure 2

A) Venn diagram of the peptides identified in hemorrhagic (HS) and healthy (CTRL) rats. B) Venn diagram of the proteins identified in hemorrhagic (HS) and healthy (CTRL) rats

Table 1. List of the peptides increased or present only in HS.

Peptides were considered increased if they were present only in either the healthy (CTRL) or hemorrhagic (HS) group or if they showed significant fold change differences between the two groups (> 1.5-fold increase or < 0.67-fold decrease in HS compared to CTRL) (ratio mean LFQ HS/mean LFQ CTRL). The specificity of proteases responsible for the generation of the peptides data set is indicated in the table (column: Selected Proteolytic Enzymes).

Sequence ratio HS/CTRL Proteins Genes Selected Proteolytic enzymes
LGEQHFKGLVL 80.28 P02770 Alb chymotrypsin-like
IQKTPQIQVY 28.79 P07151 B2m chymotrypsin-like
SLEIPGSSDPNVIPDGDFSSF 28.62 M0RB00 C4 chymotrypsin-like
LTTNPQGDTLDVSF 25.83 Q6IRS6 Fetub chymotrypsin-like
ASTVRPSFSLGNETLKVPLAL 24.55 Q5I0D7 Pepd chymotrypsin-like
ATGKPRYVVLVPSELY 23.63 Q63041 A1m chymotrypsin-like
EVTSDQVANVMWDY 19.5 P02651 Apoa4 chymotrypsin-like
LNGNSKYMVL 18.79 Q03626 Mug1 chymotrypsin-like
EAHKSEIAHRFKDLGEQHFKGLVL 17.84 P02770 Alb chymotrypsin-like
AAPRPPPAISVSVSAPAF 16.83 D4A7U1 Zyx chymotrypsin-like
LQLTGGHEAF 15.61 B2GVB9 Fermt3 chymotrypsin-like
MSKQAFVFPGVSATAY 15.36 P48199 Crp chymotrypsin-like
DAGLTPNNLKPVAAEF 15.34 Q7TMC7 Tf chymotrypsin-like
QKASLEHMVSMQY 12.56 D4ACQ4 RGD15608 chymotrypsin-like
DEPQSQWDRVKDFATVY 11.55 P04639 Apoa1 chymotrypsin-like
LLFGNSKY 11.35 Q6IE52 Mug2 chymotrypsin-like
KIILDPSGSMNIY 11.14 Q7TP05 Cfb chymotrypsin-like
MDLPGQQPVSEQAQQKLPPLAL 10.72 Q68FT8 Serpinf2 chymotrypsin-like
HEDMSKQAFVFPGVSATAY 7.88 P48199 Crp chymotrypsin-like
HPSSPPVVDTTKGKVLGKY 7.37 P10959 Ces1c chymotrypsin-like
SFSYKPRAPSAEVEMTAYVL 7.23 Q63041 A1m chymotrypsin-like
DVQMTQSPSY 7.14 P01681 chymotrypsin-like
QAAETDVQTLFSQY 7.04 P04638 Apoa2 chymotrypsin-like
PVTSVDAAFRGPDSVF 7.03 P20059 Hpx chymotrypsin-like
PNHFRPEGLPEKY 6.32 F7EHL9 Hps5 chymotrypsin-like
SDKPDMAEIEKFDKSKL 5.03 P62329 Tmsb4x chymotrypsin-like
EDVPAADLSDQVPDTDSETRIL 4.71 M0RBF1 C3 chymotrypsin-like
LNGNSKYMVLVPSQL 3.42 Q03626 Mug1 chymotrypsin-like
DLPGQQPVSEQAQQKLPPLAL 2.54 Q68FT8 Serpinf2 chymotrypsin-like
SDKPDMAEIEKF 1.94 P62329 Tmsb4x chymotrypsin-like
KESTLHLVL 1.61 F1LML2 Ubc chymotrypsin-like
AADISQWAGPLSL P31044 Pebp1 chymotrypsin-like
AALQERLDNVSHTPSSY Q5U2Z3 Nap1l4 chymotrypsin-like
AAPAKGENLSL P27867 Sord chymotrypsin-like
AAVVLENGVLSRKLSDFGQETSY P04176 Pah chymotrypsin-like
AEFYGSLEHPQTHY Q7TMC7 Tf chymotrypsin-like
AETDVQTLFSQY P04638 Apoa2 chymotrypsin-like
AEVTGLSPGVTYLFKVF P04937 Fn1 chymotrypsin-like
AGQAFRKFLPLF P26772 Hspe1 chymotrypsin-like
AGQAFRKFLPLFDRVL P26772 Hspe1 chymotrypsin-like
AGVLSRDAPDIESIL O89000 Dpyd chymotrypsin-like
AHKSEIAHRFKDLGEQHFKGLVL P02770 Alb chymotrypsin-like
AKLLGLTL P55159 Pon1 chymotrypsin-like
ALKNVPFRSEVLAWNPDNLADY Q920L0 Lcp2 chymotrypsin-like
ALKPLAPLLRGYHVVL D4AA35 Asmtl chymotrypsin-like
ALSMPLNGLKEEDKEPLIEL G3V8C4 Clic4 chymotrypsin-like
APPSFFAQVPQAPPVLVFKL P13221 Got1 chymotrypsin-like
ARGSVSDEEMMELREAF Q5XI38 Lcp1 chymotrypsin-like
ASINTDFTLSL P05545 Serpina3k chymotrypsin-like
ASSDIQVKELEKRASGQAFEL P13668 Stmn1 chymotrypsin-like
ASTVRPSFSLGNETLKVPLALF Q5I0D7 Pepd chymotrypsin-like
ASVLTAQPRLMEPIY P05197 Eef2 chymotrypsin-like
ATGKPRYVVLVPSEL Q63041 A1m chymotrypsin-like
ATTFKQDSPGQSSGFVY P18757 Cth chymotrypsin-like
ATTVSTQRGPVY A2RUW1 Tollip chymotrypsin-like
AVYSLSKSY Q03626 Mug1 chymotrypsin-like
DEPQSQWDRVKDF P04639 Apoa1 chymotrypsin-like
DGILGRDTLPHEDQGKGRQLHSLTL P05545 Serpina3k chymotrypsin-like
DIISNILHNF D3ZY96 Ngp chymotrypsin-like
DIVLTQSPVL F1LYU4 chymotrypsin-like
EAHKSEIAHRFKDLGEQHFKGL P02770 Alb chymotrypsin-like
ELVEAYQEQAKGLLDGGVDILL G3V8A4 Mtr chymotrypsin-like
EMLKGMIMSGMNVAHL D3ZH80 LOC689343 chymotrypsin-like
EMQQQELAQMRQRDANL M0R4D8 Taf4a chymotrypsin-like
ETLKDDTEKLKQLNTEQNIL Q5FVG2 Epb41l5 chymotrypsin-like
FAQVPQAPPVLVFKL P13221 Got1 chymotrypsin-like
FGSPLGKDLLFKDSAFGL Q7TMC7 Tf chymotrypsin-like
FIGGDAGDAFDGYDFGDDPSDKF P02680 Fgg chymotrypsin-like
FILKHTGPGILSMANAGPNTNGSQF M0RCZ9 Ppia chymotrypsin-like
FQVAEKPTEVDGGVWSIL Q5EBC0 Itih4 chymotrypsin-like
FRVGPESDKYRLTY P02680 Fgg chymotrypsin-like
FRVGPESDKYRLTYAY P02680 Fgg chymotrypsin-like
FTKTPKFFKPAMPFDL M0RBF1 C3 chymotrypsin-like
FTNIHGRGGGALLGDRWIL D4A1T6 C1r chymotrypsin-like
FVLSPEQINAVY P48199 Crp chymotrypsin-like
FYARGNFEAQQRGSGGVW F7EHL9 Hps5 chymotrypsin-like
GFGDLKTPAGLQVLNDYLADKSY Q7TPK5 Eef1b2l chymotrypsin-like
HKSEIAHRFKDLGEQHFKGLVL P02770 Alb chymotrypsin-like
IAHRFKDLGEQHFKGLVL P02770 Alb chymotrypsin-like
IAVDYLNKHLLQGFRQIL P24090 Ahsg chymotrypsin-like
IIGGRNAELGLFPWQAL Q8CHN8-2 Masp1 chymotrypsin-like
IVEGWDAEKGIAPWQVML G3V843 F2 chymotrypsin-like
KPRLLLFSPSVVNLGTPLSVGVQL M0RB00 C4 chymotrypsin-like
LDTKSYWKALGISPFHEY P02767 Ttr chymotrypsin-like
LELLDHVL G3V852 Tln1 chymotrypsin-like
LGAPQEADASEEGVQRALDF P14841 Cst3 chymotrypsin-like
LQIGATTQQAQKLKGEEVAF P10959 Ces1c chymotrypsin-like
LQKPEAELSPSL P14046 A1i3 chymotrypsin-like
LQKPEAELSPSLIYDLPGMQDSNF P14046 A1i3 chymotrypsin-like
LRSEPLDIKFNKPFILLL P31211 Serpina6 chymotrypsin-like
MEGPAGYLRRASVAQLTQELGTAF P12928-2 Pklr chymotrypsin-like
MEMDKRIY P49911 Anp32a chymotrypsin-like
MEVKPKLY P14942 Gsta4 chymotrypsin-like
MFRNQYDNDVTVWSPQGRIHQIEY P18420 Psma1 chymotrypsin-like
MIVESETQSPLF P17475 Serpina1 chymotrypsin-like
MMDQARSAFSNLF G3V679 Tfrc chymotrypsin-like
MNAAAEAEFNIL Q80Z29 Nampt chymotrypsin-like
MQKDASSSGFLPSFQHF P18757 Cth chymotrypsin-like
PKPDSEAGTAFIQTQQLHAAMADTF P11980 Pkm2 chymotrypsin-like
PYEIKKVF Q5RKI0 Wdr1 chymotrypsin-like
RKLQPNLYVVAELFTGSEDL D4AEH9 Agl chymotrypsin-like
RLLWESGSLL M0RBF1 C3 chymotrypsin-like
RVELDTKSYWKALGISPFHEY P02767 Ttr chymotrypsin-like
SEIAHRFKDLGEQHFKGLVL P02770 Alb chymotrypsin-like
SEKKQPVDLGLLEEDDEF D3ZHW9 Shfm1 chymotrypsin-like
SFSYKPRAPSAEVEMTAY Q63041 A1m chymotrypsin-like
SKPDNPGEDAPAEDMARY P07808 Npy chymotrypsin-like
SLLDEFYKL Q5M9G3 Caprin1 chymotrypsin-like
SLLFGNSKY Q6IE52 Mug2 chymotrypsin-like
SLPEGVVDGIEIY Q63416 Itih3 chymotrypsin-like
SMTDLLSAEDIKKAIGAF P02625 Pvalb chymotrypsin-like
SPMYSIITPNVLRLESEETF M0RBF1 C3 chymotrypsin-like
SPTVFRLLWESGSL M0RBF1 C3 chymotrypsin-like
SPTVFRLLWESGSLL M0RBF1 C3 chymotrypsin-like
SQADFDKAAEEVKRLKTQPTDEEML P11030 Dbi chymotrypsin-like
SSGNAKIGHPAPSF Q63716 Prdx1 chymotrypsin-like
STNESSNSHRGLAPTNVDFAFNL P31211 Serpina6 chymotrypsin-like
SYDRAITVFSPDGHLFQVEY F1LSQ6 Psma7 chymotrypsin-like
TDVANYLDWIQEHTAF D3ZTE0 F12 chymotrypsin-like
TVFRLLWESGSLL M0RBF1 C3 chymotrypsin-like
VDIFEPQGISRLDAQASF B2RYM3 Itih1 chymotrypsin-like
VDLPGGLHQLSFPLSVEPALGIY Q63041 A1m chymotrypsin-like
VFRLLWESGSLL M0RBF1 C3 chymotrypsin-like
VSLEGFTQPVAVF P10959 Ces1c chymotrypsin-like
VTGLSPGVTYLFKVF P04937 Fn1 chymotrypsin-like
VVFTANDSGHRHYTIAALLSPYSY P02767 Ttr chymotrypsin-like
VVLSAPAVESELSPRGGEF Q03626 Mug1 chymotrypsin-like
VYDAGLTPNNLKPVAAEF Q7TMC7 Tf chymotrypsin-like
WEFWQQDEPQSQWDRVKDF P04639 Apoa1 chymotrypsin-like
WTKTLPQKIQELKGSQSKHAEL D3ZE31 Ces2i chymotrypsin-like
YASVLTAQPRLMEPIY P05197 Eef2 chymotrypsin-like
YAVGGRSHKPLDMSKVF M0RB00 C4 chymotrypsin-like
YSIITPNVLRLESEETFIL M0RBF1 C3 chymotrypsin-like
YSLAPQIKVIAPWRMPEF P09034 Ass1 chymotrypsin-like
YVRPGGGFVPNFQL P23764 Gpx3 chymotrypsin-like
YVGRPLVSQYNV 13.72 Q9WUW3 Cfi elastase-like
FDWISYYVGRPLVSQYNV Q9WUW3 Cfi
FRLLGNMIV P02091 Hbb
MATPVVTKTAWKLQEIV Q4VFZ4 Katnb1
FAIEEYSAPFSSDSEQGNA 6.87 Q63041 A1m elastase-like/MMPs
YPSKPDNPGEDAPAEDMA 4.16 P07808 Npy elastase-like/MMPs
FLSRLMSPEEKPAPAA 1.92 P04638 Apoa2 elastase-like/MMPs
AAGTLYTYPENWRAFKALIA Q68FR6 Eef1g elastase-like/MMPs
AEVVFTANDSGHRHYTIA P02767 Ttr elastase-like/MMPs
AGQAFRKFLPLFDRVLVERSA P26772 Hspe1 elastase-like/MMPs
ASDPILYRPVA P11980 Pkm2 elastase-like/MMPs
DAGLTPNNLKPVA Q7TMC7 Tf elastase-like/MMPs
FDKFTWSSLMMSQVVNPA P31211 Serpina6 elastase-like/MMPs
INYYDMNAANVGWNGSTFA D3ZE63 LOC686548 elastase-like/MMPs
MDFLSRLMSPEEKPAPAA P04638 Apoa2 elastase-like/MMPs
MPLFFRKRKPSEEARKRLEYQMCLA D3ZI42 Lrsam1 elastase-like/MMPs
PSKPDNPGEDAPAEDMA P07808 Npy elastase-like/MMPs
SETAPAETTAPAPVEKSPA D3ZBN0 Hist1h1b elastase-like/MMPs
SFSYKPRAPSAEVEMTA Q63041 A1m elastase-like/MMPs
VVFTANDSGHRHYTIA P02767 Ttr elastase-like/MMPs
VVYPWTQRYFDSFGDLSSA P02091 Hbb elastase-like/MMPs
YETDEFAIEEYSAPFSSDSEQGNA Q63041 A1m elastase-like/MMPs
KPRLLLFSPSVVNLGTPLSVG 34.99 M0RB00 C4 elastase-like/MMPs
EAHKSEIAHRFKDLGEQHFKG P02770 Alb elastase-like/MMPs
FGIDKDAIVQAVKGLVTKG G3V826 Tkt elastase-like/MMPs
HPSSPPVVDTTKGKVLGKYVSLEG P10959 Ces1c elastase-like/MMPs
LLLFSPSVVNLGTPLSVG M0RB00 C4 elastase-like/MMPs
LVQKDTVVKPVIVEPEG Q63041 A1m elastase-like/MMPs
VPETGRKDTVVKVLIVEPEG Q03626 Mug1 elastase-like/MMPs
VQKDTVVKPVIVEPEG Q63041 A1m elastase-like/MMPs
SDKPDMAEIEKFDKS 34.7 P62329 Tmsb4x MMPs
VQEQVQEQVQPKPLES 18.24 P02651 Apoa4 MMPs
NPLPSKETIEQEKQAGES 1.83 P62329 Tmsb4x MMPs
AAVSKIAWHVIRNS Q9EQV9 Cpb2 MMPs
EALAAVSKIAWHVIRNS Q9EQV9 Cpb2 MMPs
EKNPLPSKETIEQEKQAGES P62329 Tmsb4x MMPs
EQVQEQVQPKPLES P02651 Apoa4 MMPs
ETQEKNPLPSKETIEQEKQAGES P62329 Tmsb4x MMPs
KNPLPSKETIEQEKQAGES P62329 Tmsb4x MMPs
QVQEQVQEQVQPKPLES P02651 Apoa4 MMPs
TVFRLLWES M0RBF1 C3 MMPs
ALLSPYSYSTTAVVSNPQN 1.77 P02767 Ttr MMPs
FYIMPVMNVDGYDYTWKKNRMWRKN Q9EQV9 Cpb2 MMPs
LNETGDEPFQYKN P09606 Glul MMPs
SFFNLTMKEMVKDVLRGQN B1WC01 Kif20a MMPs
SFSYKPRAPSAEVE 5.16 Q63041 A1m MMPs
AALKDQLIVNLLKE P04642 Ldha MMPs
AMQKIFAREILDSRGNPTVE P15429 Eno3 MMPs
ASKRALVILAKGAEEME O88767 Park7 MMPs
DFESEFVYMLNQQCFKFLQMKRETE F1LW73 MMPs
DGLFSFFKE F7EHL9 Hps5 MMPs
DVGSYQEKVDVVLGPIQLQSPSKE Q8CIZ5 Dmbt1 MMPs
EHICMSEHMCMSEHMCLSEHMCMSE M0R8P2 MMPs
MWASCCNWFCLDGQPE Q8CIN9 Rffl MMPs
QSLLNQQMMGKKQTLQRPTME F1M3B2 Maml2 MMPs
RVELDTKSYWKALGISPFHEYAE P02767 Ttr MMPs
SDKPDMAEIEKFDKSKLKKTE P62329 Tmsb4x MMPs
SDKPDMAEIEKFDKSKLKKTETQE P62329 Tmsb4x MMPs
TSQIRQNYSTEVE Q7TP54 Fam65b MMPs
VDYSVWDHIE Q63692 Cdc37 MMPs
VQKDTVVKPVIVEPE Q63041 A1m MMPs
SETQSPLFVGKVIDPTR 20.81 P17475 Serpina1 trypsin-like
SDKPDMAEIEKFDKSK 3.33 P62329 Tmsb4x trypsin-like
MIVESETQSPLFVGKVIDPTR 3.29 P17475 Serpina1 trypsin-like
DSEVTSHSSQDPLVVQEGSR 3.15 Q6P734 Serping1 trypsin-like
EDVPAADLSDQVPDTDSETR 1.94 M0RBF1 C3 trypsin-like
SFSQVTPAQMDLVFQR 1.91 Q64602 Aadat trypsin-like
SETAPAAPAAPAPAEKTPIK 1.51 P15865 Hist1h1c trypsin-like
DLEAMRMQTEMELRMFRQNEFMYHK D4A3D2 Smyd1 trypsin-like
IISDTETAIAPFLAKIFNPK P09006 Serpina3n trypsin-like
LASVSTVLTSKYR P01946 Hba1 trypsin-like
LTSVPRYSMWFNQIMK G3V8K8 Proz trypsin-like
SDKPDMAEIEKFDKSKLK P62329 Tmsb4x trypsin-like
SETAPAAPAAPAPVEKTPVK M0R7B4 LOC684828 trypsin-like
SPTVFRLLWESGSLLR M0RBF1 C3 trypsin-like
SSPTVFRLLWESGSLLR M0RBF1 C3 trypsin-like
TVFRLLWESGSLLR M0RBF1 C3 trypsin-like
VFRLLWESGSLLR M0RBF1 C3 trypsin-like
VLLAYLTSASSRPTR Q63041 A1m trypsin-like
YPSKPDNPGEDAPAEDMAR P07808 Npy trypsin-like
FVKRQFMNKSLSGPGQ 31.94 B2GV73 Arpc3
VITDNNGQSVFFMGKVTNPM 14.32 P05545 Serpina3k
WTELLAKNPPQTEHTEHT 12.73 P10959 Ces1c
MIVESETQSPLFVGKVID 7.35 P17475 Serpina1
VLLAYLTSASSRPT 6.74 Q63041 A1m
SDKPDMAEIEKFDKSKLKKT 6.56 P62329 Tmsb4x
YQPVTMVTSQGQVVTQAIPQ 6.14 A0A096MJK LOC100364346
LAYLTSASSRPT 3.51 Q63041 A1m
GEDAAQAEKFQHPNTD 2.79 Q66H98 Sdpr
AQASLGEYLFERLTLKHD Q7TP54 Fam65b
ASGVAVSDGVIKVFND M0R6D6 Cfl1
DEPQSQWDRVKDFATVYVD P04639 Apoa1
EEVVSESFASGP P02783 Svs4
EQRLNRRRQNEIMAKIQAAIIQI Q9R095-2 Spef2
FAQVPQAPPVLVFKLIAD P13221 Got1
FLFRDGDILGKYVD P26772 Hspe1
GEDAAQAEKFQHPNT Q66H98 Sdpr
GGGEMNPFETKVKTRIT P04812 Svs5
GGWVLVRDLLIPSNNMLYIVANGMI Q2TGI4 Zdhhc25
GMLNQQLTPVAGMMGGYP D3ZT52 Pbrm1
INDFHRAI F1LSG1 Kcnt1
IQEVTINQSLLTPMNLQIDPTIQ Q4FZU2 Krt6a
LAFQMQYQMAKYQAP M0RAT6 Tmed8
LGRFLESANMFFMVNQKVII E9PTA3 LOC502618
LVITDNNGQSVFFMGKVTNPM P05545 Serpina3k
MDAGLTFKTNQGLQTDQRED M0RBF1 C3
MIVESETQSPLFVGKVIDPT P17475 Serpina1
MKLTSEKLPKNPFSLSQYAAKQ G3V9B8 Lrp2bp
MRDKNRKPVVMKIKPDEIQQNGQT D3ZZW3 Entpd4
MTAYVLLAYLTSASSRPT Q63041 A1m
SDKPDMAEIEKFDKSKLKKTET P62329 Tmsb4x
SDKPDMAEIEKFDKSKLKKTETQ P62329 Tmsb4x
SPMYSIITPNVLRLESEET M0RBF1 C3
TQLKRKENEIELSLLQLREQQAT D3Z8G3 Rpgrip1l
TRAGMERWRDRLALVT G3V978 Dhrs11
VAQASLGEYLFERLTLKHD Q7TP54 Fam65b
VKVLDAVRGSPAVD P02767 Ttr
VVLARLTAQPAPSPED Q03626 Mug1
WKQQVMTTVQNMQHESAQLQEELH D4ABD7 Trip11
YPSKPDNPGEDAPAED P07808 Npy
YVLLAYLTSASSRPT Q63041 A1m

Table 2. List of the peptides decreased or present only in CTRL.

Peptides were considered increased if they were present only in either the healthy (CTRL) or hemorrhagic (HS) group or if they showed significant fold change differences between the two groups (> 1.5-fold increase or < 0.67-fold decrease in HS compared to CTRL) (ratio mean LFQ HS/mean LFQ CTRL). The specificity of proteases responsible for the generation of the peptides data set is indicated in the table (column: Selected Proteolytic Enzymes).

sequence ratio HS/CTRL Proteins Genes Selected Proteolytic enzymes
SGNFIDQTRVLNLGPITRQGVQA 0.66 P27590 Umod elastase-like/MMPs
TVIDQNRDGIIDKEDLRDT 0.66 P04466 Mylpf
ELEAMSRYTSPVNPAV 0.55 D3ZS52 LOC10036304 elastase-like
TAPKIPEGEKVDFDDIQK 0.45 P09739-3; Tnnt3 trypsin-like
MEKEEETTREL 0.35 M0R9D5 Ahnak chymotrypsin-like
GTTSEFIEAGGDIR 0.31 P06399;Q Fga trypsin-like
PRLLLLLLLLPSLAWG 0.26 B0BN16 Igfbpl1 elastase-like/MMPs
DISNADRLGSSEVEQVQ 0.25 P00564 Ckm
GQGLEWIGRIGPGSGDTNY 0.24 M0RBX3 chymotrypsin-like
WESGPEDQLTTPTLEP 0.24 P06759 Apoc3
ELEEAEERADIAESQVN 0.22 F1LRV9;G Myh4;Myh8;M elastase-like/MMPs
HELEEAEERADIAESQVN 0.22 F1LRV9;G Myh4;Myh8;M elastase-like/MMPs
TTALQPWHGAESKTDDSAL 0.21 Q99MH3 Hamp chymotrypsin-like
TTALQPWHGAESKTDDSA 0.21 Q99MH3 Hamp elastase-like/MMPs
TTALQPWHGAES 0.19 Q99MH3 Hamp elastase-like/MMPs
FEVTNLNMKSGMSLKIKGKIHN 0.19 Q9Z144 Lgals2 elastase-like/MMPs
MFRLLTACWSRQKATEGKQ 0.19 Q6AXT3 Arl13a
ATTDSDKVDLSIAR 0.16 P14480 Fgb trypsin-like
TGTTSEFIEAGGDIR 0.16 P06399;Q Fga trypsin-like
SIASTTVPLENQ 0.16 P02650 Apoe
PTIETTEFSDFDLLDALS 0.15 P08934;M Kng1 elastase-like/MMPs
MLRLGALRLRGLALRS 0.15 Q5RKL4;Q Dmgdh elastase-like/MMPs
ETDAIQRTEELEEA 0.14 G3V885;P Myh6;Myh7;M elastase-like/MMPs
ADTGTTSEFIEAGGDIRGPRIVE 0.14 P06399;Q Fga elastase-like/MMPs
GFMGVMNNMRKQRTLCDVI 0.11 Q5XHZ6 Klhl7
ADTGTTSEFIEAGGD 0.1 P06399;Q Fga
ADTGTTSEFIEAGGDIR 0.09 P06399;Q Fga trypsin-like
DTGTTSEFIEAGGDIR 0.08 P06399;Q Fga trypsin-like
TRQTTALQPWHGAES 0.06 Q99MH3 Hamp elastase-like/MMPs
MGTAVLRKLQLVLLL 0.05 B2RYM1 Angptl6 chymotrypsin-like
DIENPSSHVPEF P06399;Q Fga chymotrypsin-like
EEAEEQSNVNLAKF F1LRV9;F1 Myh4;Myh8 chymotrypsin-like
EQIQYRNNGVSSKQLMVF P32232-2 Cbs chymotrypsin-like
KENTIHLTL Q921A3 Ubd chymotrypsin-like
MMNFLRRRLSDSSFIANLPNGY Q63537;Q Syn2 chymotrypsin-like
MSPASLTFFCIGL M0R8A3; Gp6 chymotrypsin-like
TMMGKQEESQAGIIPQL O88658;O Kif1b chymotrypsin-like
VGPIQSLQM D3ZTA8 Rbm33 chymotrypsin-like
ARNKMLTGVVGLVL M0RDH5 chymotrypsin-like
TKYETDAIQRTEELEEA G3V885;P Myh6;Myh7;M elastase-like/MMPs
ADTGTTSEFIEAGGDIRG P06399;Q Fga elastase-like/MMPs
SQDAIMDAIAGQAQAQG A0A096M Ldb3 elastase-like/MMPs
EGELEVTDQLPGQS P02650 Apoe elastase-like/MMPs
AGSVADSDAVVKLDDGHLN B4F7A3 Lgalsl elastase-like/MMPs
KNKMTKEQYIKMNRGIN D4A631;Q Arfgef1;Arfgef elastase-like/MMPs
LEEAEERADIAESQVN F1LRV9;G Myh4;Myh8;M elastase-like/MMPs
VIDLFGNEHDNFTKNLEN D4A3H4 RGD1307621 elastase-like/MMPs
SQMPVLGKSSLRNVE E9PSU2 Donson elastase-like/MMPs
DAGAGIALNDNFVK M0R6I3;M RGD1560797; trypsin-like
DIDPEDEELELGPR P31394;Q Proc trypsin-like
DLEEATLQHEATAATLR F1LRV9;G Myh4;Myh13 trypsin-like
TDSDKVDLSIAR P14480 Fgb trypsin-like
DGPEGPKGRGGPNGDPGPL Q9JI03;G3 Col5a1
EHVQYMAEVIERLSNEPL D3ZWJ0 Gmnn
MEHTGQYLHLVFLMT Q5HZW9 Evi2a
QQQQQQQMQQLQQQ F1LXK8 LOC100362634
SRPSSKTSHRQQEEVKAPQMSQ D3ZK09 Scyl3
TAPKIPEGEKVDFDDIQ P09739-3; Tnnt3
TEEDDPGSSALLDTVQEH G3V8D4 Apoc2
VELGNDATDIEDD P06866-2; Hp

Computational identification of secreted and transmembrane proteins by means of prediction tools including Secreome P, SignalP, Phobius in combination with TMHMM allowed determination of the proteins from which the peptides originated (Figure 2B and Tables 3 and 4). The Tables report also the analysis carried out using the reference Plasma Proteome Database (http://www.plasmaproteomedatabase.org/), which contains a list of the plasma and serum proteins from healthy individuals. Among the peptides which increased or were present only in HS samples, 73% originated from proteins described as secreted or transmembrane while 23% (60 peptides out of 256) originated from 51 proteins out of 124 (41%) that are classified as non secreted. Moreover, 72% of the proteins have been previously reported in plasma according to the Plasma Proteome Database.

Table 3. Proteins increased or present only in HS from which the peptides in Table 1 originate.

List of the proteins predicted to be secreted/transmembrane or non-secreted by Perseus Annotation, Secreome P, SignalP, Phobius and TMHMM software or previously reported in plasma according to the Plasma Proteome Database.

Protein names Protein IDs Genes transmembrane/secreted non secreted plasma proteome DB
Alpha-1-inhibitor 3 P14046 A1i3 x
Alpha-1-macroglobulin Q63041 A1m x
Amylo-1, 6-glucosidase D4AEH9 Agl x x
Alpha-2-HS-glycoprotein P24090 Ahsg x x
Serum albumin P02770 Alb x x
Acidic leucine-rich nuclear phosphoprotein 32 family P49911 Anp32a x x
Apolipoprotein A-I P04639 Apoa1 x x
Apolipoprotein A-II P04638 Apoa2 x x
Apolipoprotein A-IV P02651 Apoa4 x x
Actin-related protein 2/3 complex subunit 3 B2GV73 Arpc3 x x
Protein Asmtl D4AA35 Asmtl x x
Argininosuccinate synthase P09034 Ass1 x x
Beta-2-microglobulin P07151 B2m x x
Protein C1r D4A1T6 C1r x x
Complement C3 M0RBF1 C3 x x
Complement C4 M0RB00 C4 x x
Caprin-1 Q5M9G3 Caprin1 x
Hsp90 co-chaperone Cdc37 Q63692 Cdc37 x x
Carboxylesterase 1C P10959 Ces1c x
Protein Ces2a D3ZE31 Ces2i x
Da1-24 Q7TP05 Cfb x x
Complement factor I Q9WUW3 Cfi x x
Cofilin-1 M0R6D6 Cfl1 x x
Chloride intracellular channel protein 4 G3V8C4 Clic4 x x
Carboxypeptidase B2 Q9EQV9 Cpb2 x x
C-reactive protein P48199 Crp x x
Cystatin-C P14841 Cst3 x x
Cystathionine gamma-lyase P18757 Cth x x
Protein Dhrs11 G3V978 Dhrs11 x
Deleted in malignant brain tumors 1 protein Q8CIZ5 Dmbt1 x x
Dihydropyrimidine dehydrogenase [NADP(+)] O89000 Dpyd x x
Ac2-067 Q7TPK5 Eef1b2l x x
Elongation factor 1-gamma Q68FR6 Eef1g x x
Elongation factor 2 P05197 Eef2 x x
Beta-enolase P15429 Eno3 x x
Ectonucleoside triphosphate diphosphohydrolase 4 D3ZZW3 Entpd4 x x
Band 4.1-like protein 5 Q5FVG2 Epb41l5 x x
Coagulation factor XII D3ZTE0 F12 x x
Prothrombin G3V843 F2 x x
Ferritin Q7TP54 Fam65b x
Fermt3 protein B2GVB9 Fermt3 x x
Fetuin-B Q6IRS6 Fetub x x
Fibrinogen gamma chain P02680 Fgg x x
Fibronectin;Anastellin P04937 Fn1 x x
Aspartate aminotransferase P13221 Got1 x x
Glutathione peroxidase 3 P23764 Gpx3 x x
Glutathione S-transf. alpha-4 P14942 Gsta4 x
Hemoglobin subunit alpha-1/2 P01946 Hba1 x x
Hemoglobin subunit beta-1 P02091 Hbb x x
Histone H1.5 D3ZBN0 Hist1h1b x x
Serum amyloid A protein F7EHL9 Hps5 x x
Hemopexin P20059 Hpx x x
10 kDa heat shock protein P26772 Hspe1 x x
Itih1 B2RYM3 Itih1 x x
Inter-alpha-trypsin inhibitor heavy chain H3 Q63416 Itih3 x x
Inter alpha-trypsin inhibitor, heavy chain 4 Q5EBC0 Itih4 x x
Katanin p80 WD40 repeat-containing subunit B1 Q4VFZ4 Katnb1 x x
Potassium channel subfamily T member 1 F1LSG1 Kcnt1 x x
Kinesin-like protein B1WC01 Kif20a x x
Keratin, type II cytoskeletal 6A Q4FZU2 Krt6a x x
Lymphocyte cytosolic protein 1 Q5XI38 Lcp1 x x
Lymphocyte cytosolic protein 2 Q920L0 Lcp2 x
Protein Pknox2 A0A096MJK1 LOC100364346 x
Protein LOC502618 E9PTA3 LOC502618 x
Protein LOC684828 M0R7B4 LOC684828 x
Macrophage migration inhibitory factor D3ZE63 LOC686548 x x
Pyruvate kinase D3ZH80 LOC689343 x
LRP2-binding protein G3V9B8 Lrp2bp x
Protein Lrsam1 D3ZI42 Lrsam1 x x
Protein Maml2 F1M3B2 Maml2 x
Mannan-binding lectin serine protease 1 Q8CHN8-2 Masp1 x x
Methionine synthase G3V8A4 Mtr x x
Murinoglobulin-1 Q03626 Mug1 x
Murinoglobulin-2 Q6IE52 Mug2 x
Nicotinamide phosphoribosyltransferase Q80Z29 Nampt x x
Nucleosome assembly protein 1-like 4 Q5U2Z3 Nap1l4 x x
Neutrophilic granule protein (P D3ZY96 Ngp x
Pro-neuropeptide Y P07808 Npy x x
Phenylalanine-4-hydroxylase P04176 Pah x x
Protein DJ-1 O88767 Park7 x x
Protein Pbrm1 D3ZT52 Pbrm1 x x
Phosphatidylethanolamine-binding protein 1 P31044 Pebp1 x x
Xaa-Pro dipeptidase Q5I0D7 Pepd x x
Pyruvate kinase PKLR P12928-2 Pklr x x
Pyruvate kinase M1/M2 P11980 Pkm2 x
Serumparaoxonase/arylestera se1 P55159 Pon1 x x
Pept.-prolyl cis-trans isomA P10111 Ppia x x
Peroxiredoxin-1 Q63716 Prdx1 x x
Protein Proz G3V8K8 Proz x x
Proteas. Sub. alpha type-1 P18420 Psma1 x x
Proteas. sub. alpha type F1LSQ6 Psma7 x x
Parvalbumin alpha P02625 Pvalb x
E3 ubiq.-prot. ligase rififylin Q8CIN9 Rffl x
Protein Zc3h12b D4ACQ4 RGD1560891 x
Protein Rpgrip1l D3Z8G3 Rpgrip1l x
Alpha-1-antiproteinase P17475 Serpina1 x x
Ser. Prot. inhibitor A3K P05545 Serpina3k x
Ser. Prot. inhibitor A3N P09006 Serpina3n x
Corticosteroid-binding globulin P31211 Serpina6 x x
Protein Serpinf2 Q68FT8 Serpinf2 x x
Plasma protease C1 inhib. Q6P734 Serping1 x x
Protein Shfm1 D3ZHW9 Shfm1 x
Protein Smyd1 D4A3D2 Smyd1 x x
Sorbitol dehydrogenase P27867 Sord x x
Q9R095-2 Spef2 x
Stathmin P13668 Stmn1 x x
Sem. ves. Secr. protein 4 P02783 Svs4 x
Sem. ves. Secr. prot. 5 P04812 Svs5 x
Serotransferrin Q7TMC7 Tf x x
Transferrin rec. protein 1 G3V679 Tfrc x x
Transketolase G3V826 Tkt x x
Protein Tln1 G3V852 Tln1 x x
Protein Tmed8 M0RAT6 Tmed8 x
Thymosin beta-4 P62329 Tmsb4x x x
Toll-interacting protein A2RUW1 Tollip x x
Protein Trip11 D4ABD7 Trip11 x x
Transthyretin P02767 Ttr x x
WD repeat-cont. protein 1 Q5RKI0 Wdr1 x x
Palmitoyltransferase Q2TGI4 Zdhhc25 x
Protein Zyx D4A7U1 Zyx x x
Uncharacterized protein F1LW73 x
Uncharacterized protein F1LYU4 x
Uncharacterized protein M0R4D8 Taf4a x
Ig kappa chain V region S211 P01681 x

Table 4. Proteins decreased or present only in CTRL from which the peptides in Table 2 originate.

List of the proteins predicted to be secreted/transmembrane or non-secreted by Perseus Annotation, Secreome P, SignalP, Phobius and TMHMM software or previously reported in plasma according to the Plasma Proteome Database.

Protein names Protein IDs Genes transmembrane/secreted non secreted plasma proteome DB
Protein Ahnak M0R9D5 Ahnak x x
Angiopoietin-like 6 B2RYM1 Angptl6 x x
Apolipoprotein C-II G3V8D4 Apoc2 x x
Apolipoprotein C-III P06759 Apoc3 x x
Apolipoprotein E P02650 Apoe x x
Brefeldin A-inhibited guanine nucleotide-exchange protein 1 D4A631 Arfgef1 x x
ADP-ribosylation factor-like protei Q6AXT3 Arl13a x
Cystathionine beta-synthase P32232-2 Cbs x x
Creatine kinase M-type P00564 Ckm x x
Collagen alpha-1(V) chain Q9JI03 Col5a1 x x
Dimethylglycine dehydrogenase Q5RKL4 Dmgdh x x
Protein Donson E9PSU2 Donson x x
Ecotropic viral integration site 2A Q5HZW9 Evi2a x x
Fibrinogen alpha chain P06399 Fga x x
Fibrinogen beta chain P14480 Fgb x x
Geminin (Predicted) D3ZWJ0 Gmnn x x
Protein Gp6 M0R8A3 Gp6 x
Hepcidin Q99MH3 Hamp x
Haptoglobin P06866-2 Hp x x
Insulin-like growth factor binding protein-like 1 B0BN16 Igfbpl1 x
Kinesin-like protein KIF1B O88658 Kif1b x x
Kelch-like protein 7 Q5XHZ6 Klhl7 x
Kininogen-1 P08934 Kng1 x x
Protein Ldb3 A0A096MKD Ldb3 x x
Galectin-2 Q9Z144 Lgals2 x
Galectin B4F7A3 Lgalsl x x
Histone-lysine N-methyltransferas F1LXK8 OC100362634 x
Myosin-4 F1LRV9 Myh4 x x
Myosin regulatory light chain 2 P04466 Mylpf x x
Vitamin K-dependent protein C P31394 Proc x
Protein Rbm33 D3ZTA8 Rbm33 x
Uncharacterized protein D4A3H4 RGD1307621 x
Protein Scyl3 D3ZK09 Scyl3 x
Synapsin-2 Q63537 Syn2 x
Troponin T P09739-3 Tnnt3 x
Ubiquitin D Q921A3 Ubd x
Uromodulin P27590
M0RDH5
Umod x
x
x
Uncharacterized protein M0RBX3 x

B. Proteolytic activity

A higher protease activity was found in plasma samples following hemorrhagic shock. In particular, the analyses took into account, as possible effector enzymes for the previously described proteolysis, the main intestinal serine proteases (trypsin-like, chymotrypsin-like and elastase-like enzymes) and the most common MMPS (i.e. MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-14). Both those peptides, which were increased or present only in the HS group, and peptides which were increased or present only in the CTRL group were examined, taking into account the C-terminus of each peptide and the cleavage site specificities of the proteases. In this way, it was possible to elaborate on the possible families of enzymes responsible for the cleavage of the proteins listed in the Tables and the subsequent generation of the related peptides. This analysis showed a huge increase in peptides generated by all proteolytic enzymes examined, in particular a significant increase of peptides possibly generated by chymotryptic-like enzymes: 53% in post-shock plasma compared to 22% in control, (Fisher’s exact test p value of 8.06E-06) (Figure 3 and Table 5).

Figure 3. Pie chart showing the number and the % of peptides in plasma samples of CTRL and HS.

Figure 3

The analysis was carried out both on peptides increased and present only in HS group (Table 1) and peptides decreased in HS and present only in healthy group (Table 2) focusing on the cleavage site specificities of the main serine proteases potentially derived from the intestine (trypsin-like, chymotrypsin-like and elastase-like enzymes) and on the most common MMPS: (i.e. MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-14)

Table 5. Plasma protease activity.

The contribution of a specific protease was calculated as the relative proportion between the number of peptides increased and present only in HS and on peptides decreased in HS and present only in CTRL and the total number of peptides present in the HS or CTRL sample.

HS
Digestive protease Cleavage site C-term peptide count (tot 256) % Total %
Trypsin-like K (Lys) 7 2.7 7
R (Arg) 12 4.7
Chymotrypsin-like W (Trp) not before P 1 0.4 53
Y (Tyr) not before P 39 15.2
F (Phe) not before P 37 14.5
L (Leu) not before P 58 22.7
M (Met) not before P /
Elastase-like and Metalloprotease (MMPs) A (Ala) 18 7.0 24
G (Gly)   8 3.1
V (Val)   4 1.6
S (Ser) 11 4.3
N (Asn)   4 1.6
E (Glu) 16 6.3
CTRL
Digestive protease Cleavage site C-term peptide count (tot 60) % Total %
Trypsin-like K (Lys) 2 3.3 17
R (Arg) 8 13.3
Chymotrypsin-like W (Trp) not before P / 22
Y (Tyr) not before P 2 3.3
F (Phe) not before P 3 5.0
L (Leu) not before P 7 11.7
M (Met) not before P 1 1.7
Elastase-like and Metalloprotease (MMPs) A (Ala) 4 6.7 36
G (Gly) 3 5.0
V (Val) 1 1.7
S (Ser) 5 8.3
N (Asn) 7 11.7
E (Glu) 2 3.3

Consistent with the results from the peptidomic analysis, zymography demonstrated a significant increase in serine-protease activity in HS compared to CTRL plasma, with increased activity at a molecular weight compatible with that expected for chymotrypsin (Figure 4).

Figure 4. Plasma serine protease activity by gelatin zymography (n = 3 rats/group).

Figure 4

(A) Serine protease activity band (~ 20–30 kDa) and relevant statistics (B) **p < 0.01 CTRL vs. HS.

DISCUSSION

The main finding of this work is that plasma displays an increase in peptides possibly generated by serine proteases after hemorrhagic shock, linking proteases to the larger presence of circulating peptides. Our data support and quantitatively confirm for the first time the hypothesis of extensive, system-wide proteolytic cleavage as part of the pathologic phenomena occurring in shock.

The importance of proteomics as a tool to shed light on the mechanisms underlying cardiovascular disease, and to propose biomarkers for early diagnosis and monitoring of the responsiveness to therapy has already been recognized [6, 17, 18] but, to our knowledge, no systematic study has utilized a peptidomic approach to investigate systemic proteolysis in the general and highly complex context of the possible pathological mechanisms taking place in shock. Therefore, a high-throughput mass spectroscopy-based approach as the peptidomic one described in this work appeared ideal to quantitatively investigate the problem of enzymatic proteolysis in hemorrhagic shock.

Several studies have examined lymph proteins in hemorrhagic shock [1922]. The assumption in these studies is that the mesenteric lymph is a vehicle for injurious factors produced or released in the intestine, which are mediators of severe injury to organs distal other than the intestine. However, analysis of the lymph, despite its importance for understanding the generation and early transport of cytotoxic mediators, has the disadvantage of a more limited translational potential, due to the difficulty of collecting gut lymph samples from shock patients. In contrast, further investigations, initially conducted using this mass spectrometry based approach with plasma (a very easily available and abundant source) and involving a suitable larger number of animals and conditions, may lead to the detection of potential biomarkers which could then be routinely quantified using widespread and much less demanding detection techniques, such as antibody-based methods.

A common finding in previous reports is that several proteins are upregulated in shock lymph, especially proteins with inflammatory and pro-coagulation properties. D’Alessandro and coworkers [22] reported that the upregulation of such proteins is accompanied by an impaired homeostasis of the balance between proteases and anti-proteases, especially serine proteases and MMPs, and that this could entail an activation of neutrophils following extracellular matrix metalloproteinase involvement. These observations are consistent with the evidence that digestive enzymes can promote “autodigestion” [25]. Hence, we took the next step in this investigation, the analysis of peptides in plasma, with the goal of looking for evidence of proteolytic processes in shock, which could prove impactful in future clinical studies.

Several categories of proteins were affected by the proteolysis detected in our study:

  1. heat shock proteins related to mitochondrial function, which are known to be impaired in shock [23, 24];

  2. serine protease inhibitors (Inter alpha-trypsin inhibitors, serine protease inhibitor A3N and A3K, plasma protease C1 inhibitor, alpha macroglobulins, etc.), indicating a possible response of the anti-proteases to the increased concentrations of circulating serine proteases in shock, and possibly suggesting that the increased protease activity produces extensive proteolysis because of a failure of the physiologic self-defense against proteases;

  3. several cytoplasmic (e.g. aspartate aminotransferase) and membrane proteins (e.g. β2 microglobulin), suggesting that the degradation process also occurs inside cells or that cell contents leak into the bloodstream and become substrates for circulating proteases;

  4. proteins involved in the coagulation cascade, typically involved in the response to hemorrhage, such as fibrinogen, coagulation factor XII, etc.;

  5. proteins involved in the inflammatory response initiated by hemorrhage, and specifically in the innate immune response, such as C-reactive protein, complement proteins, macrophage migration inhibitory factors, thymosin β4, etc.

Many of the proteins from which the newly found fragments in shock are derived, besides being involved in a system-wide inflammatory and pro-coagulant response, are also typically associated with organ dysfunction, especially in the liver and the heart, and their presence in the plasma of HS patients, if verified, could serve as markers of organ failure and shock progression. The possible relationship between proteolysis and organ failure should be further investigated also on the basis of the result showing that the large majority of the cleaved proteins, from which the peptides increased in HS were generated, are cytoplasmic and membrane proteins. This could indicate cellular damage, which may play a role in organ dysfunction.

The enhanced activity of chymotrypsin-like proteases, estimated from the number of peptides generated by these enzymes with respect to the total number of peptides generated by proteolysis in shock, represents an important finding, which integrates previous data on protease activity in plasma and vital organs [25]. Low-throughput, semi-quantitative techniques such as gel or in situ zymography are unable to discriminate among serine proteases. This intrinsic limitation of these approaches was overcome by the quantitative nature of mass spectroscopy.

It is important to observe that, despite the fact that chymotrypsin-like enzymes appear to be responsible for much of the proteolytic generation of peptides in shock, all the proteolytic enzymes analyzed and reported in Table 5, including elastase-like and MMPs, generate a larger number of peptides in shock than in control animals. However, the overwhelming contribution of chymotrypsin-like enzymes (more than 50% of peptides generated in shock) leads to an apparently limited contribution (i.e., percentage value of peptide generation, Table 5) of other proteases.

An important implication of our findings is that they fit and validate previous reports on the possible effects of digestive enzymes which escape the intestinal lumen because of the damage that gut ischemia induces in the mucosal barrier [25] and cause damage in distal organs (“Autodigestion Hypothesis”). The proteolytic activity was not only reported to be increased in plasma, but also in distal organs, and the framework of the Autodigestion Hypothesis first provided a possible explanation for the role of digestive proteases in the organ damage which characterizes circulatory shock. In particular, it was reported that the proteolytic function of these enzymes affect transmembrane receptor integrity, e.g. shock was shown to reduce the density of the extracellular domain of the insulin receptor [25]. The results presented in this manuscript do not include the presence of fragments of the extracellular domain of transmembrane receptors such as the insulin receptor, possibly because of the relative low abundance in comparison to other more abundant plasma proteins. Still, the hypothesis that proteolytic activity takes place and coincides with increased activity of digestive enzymes, such as the chymotrypsin-like enzymes, was quantitatively validated by the findings obtained through the proposed peptidomic approach.

The choice of the experimental hemorrhagic shock model, and the related advantages and possible pitfalls of this model deserve to be discussed as well. This study used the Fixed-Pressure Hemorrhage (Wiggers) model, which is one of the standard models for experimental hypovolemic shock. The predicted mortality rate for this type of model exceeds 75% [4], and animals that do not survive consistently demonstrate multiorgan failure as measured by gross tissue examination, histology, and certain cellular and tissue markers (e.g., Troponin I, lung wet/dry ratios).

As detailed by Fülöp and colleagues [26], this model has the advantage of being highly standardized, controlled and reproducible, and as such is optimal for the study of the pathophysiology and proteomics/peptidomics of shock. In particular: i) the controlled procedure for blood withdrawal and return (executed at the same rate of 0.5 cc/min, as explained in the Materials and Methods) is completely reproducible; ii) the objective control of the hemodynamic state of the animal during hypovolemia is ensured by the maintenance of blood pressure around the target value of 35 mmHg; iii) the monitoring of conditions such as body temperature (decreased following bleeding, constant throughout the hypovolemic phase, and increased again to physiologic levels upon reperfusion) is conveniently repeatable. All these elements are consistently observed in our experience with this HS model [35, 25]. Of particular benefit for proteomics/peptidomics studies is this particular model minimizes trauma to the animals (e.g., a laparotomy was not done), mitigating the potential for confounders to the analysis of hemorrhage per se.

One of the main technical requirements of the Fixed-Pressure Hemorrhage model (and of every experiment where the animal is cannulated) is the use of anticoagulation to prevent the clotting of catheters and ensure the ability to withdraw, add, and store whole blood during the ischemic period. We used systemic heparinization both in the shock and control groups, which prevented any bias between the shock and the control groups. However, there could be a potential limitation of our results, in that heparin may have potentially interfered with proteolytic mechanisms, specifically the clotting cascade. Heparin increases (up to 600-fold [27, 28]) the inhibitory activity of antithrombin, a serpin (serine protease inhibitor) which binds, upon activation by heparin [2730], to thrombin, trypsin and other coagulation factors. Further, proteases other than circulating serine proteases have been shown to be inhibited more effectively in the presence of heparin [30]. This may have led to an underestimate of the amount of proteolytic activity and subsequent peptide generation occurring in shocked animals compared to controls; thus our observations are most likely conservative in nature.

A second limitation of the experimental model is the necessity for storage of the drawn blood during hypovolemia. In order to maintain mean arterial pressure around the target level (i.e., ~ 35 mmHg in this experiment), it may be necessary to draw and/or reinfuse small aliquots of blood during the two-hour hypovolemic phase, and this requires that the shed blood is readily available at body temperature. Previous reports from our laboratory obtained both in hemorrhagic and non- hemorrhagic shock models, which do not entail ex vivo storage of blood for a pre-defined amount of time (e.g., splanchnic arterial occlusion shock or septic (peritonitis) shock or by endotoxin administration) have consistently shown that protease levels and activity are increased in shock independent of the model studied [4, 3133]. Still, the possible effect of the storage of the shed (heparinized) blood at room temperature on excess proteolytic activity should be limited. Zimmerman and coworkers demonstrated that the temperature (including the range of room temperatures) and time of storage do not affect significantly blood proteins, which are broadly stable at the peptide level [34]. Additionally, heparin also has a protective effect on the stability of whole blood proteins at room temperatures, as reported by Henriksen et al. [35].

Conclusions and Perspectives

In summary, the main conclusions of this study are:

  • -

    a quantitative confirmation that massive proteolysis occurs on the organism scale as a fundamental degrading phenomenon induced by shock;

  • -

    serine proteases, whose plasma activity is increased in shock, are largely responsible for the observed proteolysis;

  • -

    the experimental conditions of sample treatment and analysis suitable for the future identification of novel biomarkers of hemorrhagic shock in plasma has been set up.

One of the limitations of our study is the presentation of a “static” picture of the effect of shock at the end of the hypovolemia and reperfusion experiment. However, the possibility of collecting multiple blood samples in the rat is strongly limited by the sensitivity of the animal to blood withdrawal, especially during the hypovolemic phase, and a dynamic description of the phenomena reported in this manuscript during a similar hemorrhagic shock protocol requires the use of larger animals. Another possible limitation concerns the low number of animals included in the study. However, the use of technical replicates, which ensures robustness to the results obtained from each biological replicate, and the detection of a large increase in proteolytically produced peptides in all HS samples compared to CTRL, allows us to conclude that the present data fully support the direct experimental evidence of a general proteolytic activity in shock.

The role of the peptides, which are generated in shock, should be the subject of future analyses, given their potential role in shock. For example, vasoactive intestinal peptides, which are generated in the gut and promote cardiovascular responses (e.g., vasodilation and blood pressure reduction) are an example of peptides with patho-physiological relevance, to be further investigated, particularly in circulatory shock. If peptides present only in plasma from hemorrhagic shock animals, but not in control plasma, have possible vasoactive effects, they could become important pathogenic factors to study in the context of organ dysfunction induced by shock as well as new therapeutic targets.

The results presented in this manuscript serve as a starting point towards validation in large animals and clinical studies, in which an appropriate design should also include the assessment of the “dynamic” effects of shock on the peptidome and proteome (i.e., several blood samples over time from the same subject). Such studies should also propose the test of protease blockade as a cornerstone of an innovative therapeutic approach to protein degradation and peptide formation in circulatory shock.

Acknowledgments

This research was funded by the “ShockOmics” grant #602706 of the European Union, by the “CelSys Shock” Marie Curie International Outgoing Fellowship PIOF-GA-2012-328796 of the European Union in support of the first author, by the NIH grant GM 85072, and by the Career Development Award (CDA2) 1IK2BX001277-01A1 from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development.

Conflicts of Interest and Source of Funding: FAD and GWSS own stock in Inflammagen Inc., a company that develops new shock treatments. Research supported by the “ShockOmics” grant #602706 of the European Union; by the “CelSys Shock” Marie Curie International Outgoing Fellowship PIOF-GA-2012-328796 of the European Union; by the NIH GM 85072; and by Career Development Award (CDA2) 1IK2BX001277-01A1 from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development.

Footnotes

AUTHORS

FA: contributed in the conception of the study and execution of the animal experiments and drafted the manuscript

EM: was involved in the proteomics analysis

AN: was involved in the proteomics analysis

MHS: contributed to the design of the study and to the animal experiments

FAD: contributed to the design of the study and to the animal experiments

EBK: contributed to the conception of the study as a whole, contributed to the manuscript

GWSS: contributed to the conception of the study as a whole, contributed to the manuscript

GT: contributed in the conception of the study, coordinated and executed the proteomics analysis, contributed to the manuscript

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