Proteomics of Dense Core Secretory Vesicles Reveal Distinct Protein Categories for Secretion of Neuroeffectors for Cell-Cell Communication

Abstract

Regulated secretion of neurotransmitters and neurohumoural factors from dense core secretory vesicles provides essential neuroeffectors for cell-cell communication in the nervous and endocrine systems. This study provides comprehensive proteomic characterization of the categories of proteins in chromaffin dense core secretory vesicles that participate in cell-cell communication from the adrenal medulla. Proteomic studies were conducted by nano-HPLC Chip MS/MS tandem mass spectrometry. Results demonstrate that these secretory vesicles contain proteins of distinct functional categories consisting of neuropeptides and neurohumoural factors, protease systems, neurotransmitter enzymes and transporters, receptors, enzymes for biochemical processes, reduction/oxidation regulation, ATPases, protein folding, lipid biochemistry, signal transduction, exocytosis, calcium regulation, as well as structural and cell adhesion proteins. The secretory vesicle proteomic data identified 371 distinct proteins in the soluble fraction and 384 distinct membrane proteins, for a total of 686 distinct secretory vesicle proteins. Notably, these proteomic analyses illustrate the presence of several neurological disease-related proteins in these secretory vesicles, including huntingtin interacting protein, cystatin C, ataxin 7, and prion protein. Overall, these findings demonstrate that multiple protein categories participate in dense core secretory vesicles for production, storage, and secretion of bioactive neuroeffectors for cell-cell communication in health and disease.

Introduction

The nervous system utilizes dense core secretory vesicles for regulated secretion of chemical neurotransmitters and neurohumoural factors that are represented by neuropeptides, catecholamines, and related neuroeffector molecules for cell-cell communication (1–5). These secretory vesicles represent the primary subcellular site for the biosynthesis, storage, and secretion of neurotransmitters and hormones utilized for cell-cell communication in the nervous and endocrine systems for health and disease.

The dense core secretory vesicles of chromaffin cells of the peripheral sympathetic nervous system are a representative model for neurochemical enzymes utilized in brain for the biosynthesis of neuroeffectors composed of neuropeptides and catecholamines (dopamine, norepinephrine, and epinephrine) (5–7). The majority of prior studies have studied individual proteins of these dense core secretory vesicles (8–13). However, a more global understanding of secretory vesicle components is essential to gain knowledge of the repertoire of protein systems that function in this organelle. Elucidation of the proteome characteristics of dense core secretory vesicles can provide valuable insight into the functional protein processes for production and secretion of neuroeffectors, the goal of this study.

The high sensitivity of current mass spectrometry (MS) instrumentation, coupled with efficient HPLC (high-pressure liquid chromatography) separation of peptides, allows proteomic investigations to identify hundreds of proteins from small amounts of samples. Furthermore, enrichment of moderate to low abundant proteins in chromaffin secretory vesicles for this study was achieved by removal of the abundant chromogranin A protein. Peptide identifications from mass spectrometry data were obtained using two independent search algorithms for database searching, combined with searches against a shuffled decoy database for estimation of false discovery rate (FDR) for tryptic peptide identifications. The overall proteomic data resulted in identification of 371 soluble and 384 membrane proteins from dense core secretory vesicles, for a total of 686 distinct secretory vesicle proteins.

Significantly, proteomic data illustrated distinct biochemical functions in dense core secretory vesicles composed of proteins for neuropeptides and neurohumoural factors, protease systems, neurotransmitter enzymes, receptors, biochemical enzymes, regulation of redox status, protein folding, ATPases, lipid and carbohydrate functions, signal transduction and GTP-binding proteins, and proteins for exocytosis. Interestingly, several proteins known to participate in neurological diseases were indicated consisting of the amyloid precursor protein (APP), huntingtin-interacting protein, ataxin 7, and prion protein that represent key elements involved in the mechanisms of Alzheimer’s disease (14–18), Huntington’s disease (19–22), spinocerebellar ataxia (23–25), and prion disease (26–28). These secretory vesicles also contain the CLN8 protein involved in neurodegeneration and mental retardation of EPMR (epilepsy and mental retardation) (29–32), and the P20-CGGBP protein involved in the fragile X syndrome of mental retardation (33). Furthermore, these vesicles also contain regulatory factor X4 involved in bipolar disorder (34), and KIAA0319 that is involved in dyslexia (35).

Overall, proteomic investigation of dense core secretory vesicles revealed functionally distinct categories of protein systems in this organelle, with several involved in neurological disease. These proteomic data illustrate a view of the secretory vesicle ‘system’ for secretion of neuroeffectors mediating neuronal and endocrine cell-cell communication in health and disease.

Materials and Methods

Purification of chromaffin secretory vesicles from bovine adrenal medulla and preparation of soluble and membrane components

Dense core secretory vesicles, represented by chromaffin secretory vesicles (also known as chromaffin granules), were purified from fresh bovine adrenal medulla by differential sucrose density gradient centrifugation, as described previously (37,38), involving extensive wash steps to obtain purified chromaffin granules. We have documented the high purity of this preparation of isolated secretory vesicles by electron microscopy (Fig. 1) and biochemical markers (36–38). Sucrose gradient purification results in a preparation of purified, intact chromaffin secretory vesicles that lack biochemical markers for the subcellular organelles of lysosomes (acid phosphatase marker) (38), cytoplasm (lactate dehydrogenase marker) (37), mitochondria (fumarase and glutamate dehydrogenase markers) (36,37), and endoplasmic reticulum (glucose-6-phosphatase marker) (37). Enzyme markers have been measured in the purified chromaffin secretory vesicle preparation as 1% or less of total homogenate markers, which, thus, indicate the high purity of these isolated secretory vesicles (36–38).

Figure 1. Purity of isolated chromaffin granules (secretory vesicles) evaluated with (Met)enkephalin as a marker for chromaffin granules and with acid phosphatase as a marker for lysosomes.

(a) Preparation of purified chromaffin granules by differential density centrifugation. The flow chart illustrates the purification scheme for chromaffin granules from bovine adrenal medulla homogenate, achieved by differential centrifugation. The homogenate (in 0.32 M sucrose buffer) is centrifuged at 365 × g to remove nuclei (P1, pellet 1) from the supernatant (S1, soluble fraction 1) that represents a crude fraction of chromaffin granules. The Granules of pelleted by centrifugation at 12,000 × g and washed three times in 0.32 M sucrose buffer to obtain enriched chromaffin granules (P5 fraction) that undergoes purification on a 1.6/0.32 M sucrose gradient subjected to ultracentrifugation (120,000 × g) to obtained a pellet of purified chromaffin granules.

(b) Analyses of crude fraction of chromaffin granules on multi-step sucrose gradient. The crude chromaffin granule fraction (P2) was analyzed on a multi-step sucrose gradient of 2.2 M to 1.2 M sucrose as described in the methods. Gradient fractions were assayed for (Met)enkephalin (●) that is present in chromaffin granules, and for the lysosomal enzyme marker acid phosphatase (○). The crude P2 fraction of chromaffin granules contains enkephalin and acid phosphatase.

(c) Analyses of purified chromaffin granules on multi-step sucrose gradient. The purified chromaffin granules were analyzed on the multi-step sucrose gradient of 2.2 M to 1.2 M sucrose as described in the methods. Gradient fractions were assayed for (Met)enkephalin and acid phosphatase. The presence of the purified chromaffin granules is indicated by the peak of (Met)enkephalin. The multi-step gradient showed no peak of acid phosphatase, indicating effective removal of lysosomes. These data document the purity of the chromaffin granule preparation.

(It is noted that cytosolic proteins may possibly associate with the outside of the granule membrane during homogenization, and after freeze-thawing, such cytosolic proteins may become present in the soluble fraction giving the interpretation that they might be luminal proteins of the granules. Nonetheless, cytosolic proteins are likely to have importance because cellular function of the chromaffin granule must involve cytoplasmic proteins for regulated movement to achieve exocytosis.)

In addition, this study further assessed the removal of the lysosomal enzyme marker acid phosphatase from the purified preparation of chromaffin granules compared to unpurified sample of chromaffin granules obtained at an early step in the purification procedure (illustrated in figure 1a). Purified and unpurified granules were analyzed on a multi-step sucrose gradient of 2.2 to 1.2 M sucrose (2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, and 1.2 M sucrose steps each consisting of 2.5 ml) by ultracentrifugation at 120,000 × g in a SW28 rotor (25,000 rpm) at 4° C for 100 min. Gradient fractions of 0.5 ml were collected from the bottom of the tube (2.2 M sucrose), and fractions were assayed for (Met)enkephalin by RIA as previously described (5) as a marker for chromaffin granules, and acid phosphatase activity as a marker for lysosomes as described previously (38). Results show that the purified chromaffin granules lack acid phosphatase activity, indicating effective removal of lysosomes of density near that of chromaffin granules (explained in fig. 1 of results). These new data and established purity in the literature (36–38) document the purity of these chromaffin secretory vesicles for this study.

Soluble and membrane components of the purified chromaffin granules were prepared by lysing (by freeze-thawing) purified chromaffin granules in isotonic buffer conditions consisting of 150 mM NaCl in 50 mM Na-acetate, pH 6.0, with a cocktail of protease inhibitors (10 μM pepstatin A, 10 μM leupeptin, 10 μM chymostatin, 10 μM E64c, and 1 mM AEBSF). The lysed granules were centrifuged at 100,000 × g (SW60 rotor) at 4° C for 30 minutes. The resultant supernatant was collected as the soluble fraction. The pellet was collected as the membrane fraction, and washed two times by re-suspending in the lysis buffer and centrifugation (100,000 × g, 30 min). The final pellet was resuspended in the lysis buffer and designated as the membrane fraction.

The soluble and membrane fractions were each subjected to removal of the abundant chromogranin A (CgA) protein, by its binding to calmodulin-Sepharose (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ) (39). The soluble fraction and membrane fraction (solubilized in 50 mM CHAPS) were each incubated with a slurry of calmodulin-Sepharose at 4° C overnight in equilibration buffer (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 2 mM CaCl₂, and protease inhibitors consisting of 5 μM E64c, 5 μM leupeptin, 5 μM chymostatin, 5 μM pepstatin A, 5 μM bestatin, 1 μM GEMSA, and 50 mM PMSF). The mixture was centrifuged and the supernatant collected as the soluble fraction without CgA. This step removed approximately 90–95% of CgA, based on assessment by anti-CgA western blots.

Proteins in the membrane fraction were concentrated by chloroform-methanol precipitation. To the membrane fraction (400 μg in 300 μl) was added MeOH (400 μl), chloroform (100 μl), and deionized water (300 μl) with mixing between each step, followed by centrifugation (14,000 × g for 1 min). The top aqueous layer was removed, while retaining the protein precipitate at the top of the chloroform layer; after addition of MeOH (400 μl), mixing, and centrifugation (14,000 × g for 2 min), the pelleted protein was collected for trypsin digestion (40).

Reduction/alkylation and trypsin digestion of samples

Reduction and alkylation were performed using Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) (Pierce, Rockford, IL) reduction followed by cysteine alkylation with iodoacetamide (Sigma-Aldrich, St. Louis, MO). Briefly, a sample of the CG soluble fraction (8.6 μg) was lyophilized, redissolved in 12 μl 20% acetonitrile, followed by addition of 4 μl 100 mM TCEP in 20% acetonitrile (28.7 mg/ml TCEP) and reduction at 55°C for 20 minutes. The reduced sample was cooled to room temperature, 4 μl of 100 mM iodoacetamide in 20% acetonitrile was added and incubated for 30 minutes at room temperature (in the dark) for alkylation of free cysteine residues. For proteins of the membrane fraction (400 μg), the proteins precipitated by chloroform-methanol (described above) were subjected to the same procedure for reduction and alkylation using 20 μl of TCEP solution and 5 μl of iodoacetamide solution.

The reduced and alkylated proteins of the chromaffin granule soluble (CGS) and membrane (CGM) fractions were subjected to trypsin digestion. The CGS protein sample (8.67 μg in approximately 1 μl) was diluted by addition of 20 μl 25 mM ammonium bicarbonate (pH 7.0) and 1 μl 100 mM CaCl₂, and 10 μl trypsin stock solution was added (200 ng total trypsin, using stock solution consisting of 20 ng/μl sequencing grade trypsin in 25 mM ammonium bicarbonate, pH 7.0, trypsin was from Promega #V5111). Final trypsin digestion conditions for CGS (31.5 μl total volume) were 0.275 μg/ml CGS protein, 6.5 ng/μl trypsin, 25 mM ammonium bicarbonate, pH 7.0, and 3.2 mM CaCl₂. For the CGM sample (100 μg protein in ~10 μl), it was prepared by addition of 90 μl trypsin (1800 ng total trypsin, using stock solution consisting of 20 ng/μl trypsin in 25 mM ammonium biocarbonate, pH 7.0), 95 μl ammonium bicarbonate, pH 7.0, and 5 μl 100 mM CaCl₂. The trypsin digestion conditions for CGM (200 μl total volume) were 0.5 μg/μl protein, 9.0 ng/μl trypsin, 25 mM ammonium bicarbonate, pH 7.0, and 2.5 mM CaCl₂. Trypsin digestion of CGS and CGM samples was conducted by incubation at 37° C for 18 hours.

Nano-liquid chromatography tandem mass spectrometry (nano-HPLC Chip MS/MS)

Soluble and membrane chromaffin granule sample digests were subjected to nano-LC-MS/MS, loaded at 2.9 μg and 1.0 μg total protein for each LC-MS/MS analysis. All LC-MS/MS analyses were performed in triplicate on an Agilent XCT Ultra ion trap mass spectrometer coupled to an Agilent 1100 nano-HPLC system fitted with a HPLC-Chip system. The LC separation was performed on an Agilent C18 analytical HPLC chip (Agilent Zorbax C18 Chip, 150 mm × 75 μm, 40 nl trap) and utilized a gradient of solvent B (acetonitrile with 0.25% formic acid) in solvent A (water with 0.25% formic acid). The gradient progressed from 3% B to 45% B in 40 minutes followed by an increase to 75% B in 10 minutes. The mass spectrometer was set for data dependant scanning in MS/MS mode on the three most abundant ions present in the MS scan. The exclusion time was set to 0.1 minutes, isolation window set to 4 amu, and voltages set to −1850V (capillary), −500V (counterelectrode) and 1.30V (fragmentation). Smart ion target was set to 500,000 to correct for background ions. The maximum injection time was set to 100 ms. All other default settings were used and left unaltered in all experiments.

Analyses of MS/MS data and database search parameters using Spectrum Mill bioinformatics platform (Agilent Technologies, Santa Clara, CA)

The Spectrum Mill database search platform used 1.4 amu for precursor mass tolerance and 0.8 amu fragment mass tolerance with all other default parameters retained. The search was unrestricted and included non-tryptic peptide identifications to prevent forced identifications for non-tryptic peptides. Protein identifications resulted from searches against a bovine protein database (extracted from the NCBI-nr protein sequence database consisting of 38,197 protein entries). We employed a 1.0 amu precursor mass tolerance and 0.3 amu fragment mass tolerance against the IPI bovine database. The search was restricted to tryptic and semi-tryptic peptides. In all searches, carbamidomethylation was included as a variable modification to compensate for the possibility of incomplete alkylation.

Validation for MS/MS identifications

Protein identifications were evaluated using analysis against proteins known to be present in these samples and by Spectrum Mill database search against a scrambled decoy database (41). Based on these analyses, protein identifications were evaluated by implementing a two-tiered system for peptide identifications. The first tier consisted of proteins with high confidence, peptide scores of ≥10 and Scored Peak Intensity Percent (%SPI) ≥70. When implemented, the False Discovery Rates (FDR) for these protein identifications using tryptic peptides were ~1%, even when single peptides were considered. Protein identification with single tryptic peptides can be confidently achieved as illustrated by previous reports on confident protein identifications based on single peptides (42). The second tier targeted proteins showed confidence levels corresponding to peptide score ≥8 and %SPI >60, combined with the requirement that at least two tryptic peptides of the parent protein were utilized for identification. The levels of these scores were considered to represent identification of proteins based on prior biochemical studies documenting the presence of low abundance proteins in the chromaffin secretory vesicles, which include endopin (43), cathepsin D (44), cathepsin B (45), and prohormone convertase (46). The FDR for this second tier was estimated at 1–2% by decoy database analysis. It is important to note that the FDR determined by decoy database analysis is an estimate and is dependent on the quality of spectra and the randomized database (41). Therefore, we manually evaluated our scoring thresholds in a manner similar to reported by Wang, et al. (41).

Protein organization and clustering

Tryptic peptides shared between several proteins are only counted for the protein that has overall the most matching, unique peptides. Batch Entrez (http://ncbi.nlm.nih.gov/entrez/batchentrez.cgi?db=Protein) was used to generate FASTA formatted protein sequence databases for each GenInfo Identifier (GI) number for proteins identified by the MS experiment. BLASTCLUST was used to perform pairwise comparisons followed by single-linkage clustering of the statistically significant matches (>95% sequence similarity over 90% of the sequence length) (http://www.ncbi.nlm.nih.gov/blast/). The protein list is thus the smallest set of proteins explaining the identified proteins present. Following this analysis, an annotated, non-redundant table of soluble and membrane proteins was compiled (Supplemental Tables A and B).

The functional categories of identified proteins were defined by the gene ontology (GO) resource (http://www.geneontology.org). Further information on the function of proteins was obtained through KEGG and Interact pathway databases, as well as through the MEROPS database to provide additional information relevant to proteases. A series of GO terms in each category was acquired though text searching of specific keywords relating to function and localization. In addition to gene ontologies, both identified and unidentified protein sequences were queried against the the InterPro (http://www.ebi.ac.uk/interpro/) database, SignalP resource (http://www.cbs.dtu.dk/services/SignalP/) and TMHMM resource (http://www.cbs.dtu.dk/services/TMHMM/) in order to assess protein family. All automated searches were enhanced with PubMed searches to assess recent literature where proteins are known to serve multiple functions.

Several peptide sequences were identified that were not functionally annotated in initial database searches. Based on the identified tryptic peptide sequences, predicted mouse and human sequences were aligned back to bovine sequences using the TIGR gene indices (http://tigrblast.tigr.org/tgi/). Proteins demonstrating strong homology to existing bovine sequences were included in the non-redundant assembly of identified chromaffin granule proteins.

Proteomic data (Table 1) combines proteins identified in the sample after the calmodulin affinity step from experiments of this study with proteins identified before the calmodulin affinity step from our previous more limited proteomic study of bovine chromaffin granules [47]. Thus, proteins that may bind to the calmodulin affinity column are included in this complete proteomic data set of proteins identified from this study and our previous, smaller proteomic study of bovine chromaffin granules; the combined proteomic data set is illustrated in Table 1.

Table 1.

Functional Categories of Soluble and Membrane Proteins in Chromaffin Secretory Vesicles.

The functional categories of proteins identified as soluble and membrane components of chromaffin secretory vesicles are illustrated in this table. The main functional categories (large bolded titles) are divided into subcategories. This table combines the extensive group of proteins identified by nano-HPLC Chip MS/MS tandem mass spectrometry in this study with other proteins in these vesicles identified by 1-D gel separation and MS/MS analyses (47).

Protein Function	Genbank	Soluble	Membrane
Production of Neurotransmitters and Neurohumoural Factors
Neuropeptides (Proproteins) and Neurohumoural Factors	Genbank	Soluble	Membrane
Adrenomedullin*	27806927
Amyloid beta A4 precursor#	76613693
Angiopoietin-4 precursor (ANG-4)	76633205
Cathelicidin 1+	27807341
Chromogranin A*	116548
Chromogranin B*	12
Chromogranin C*	27806421
Decorin (bone proteoglycan II)*	54660107
Epithelium-derived growth factor (EGF)	76637576
Glycoprotein II+	1809215
Glycoprotein III (clusterin)*	27806907
Interleukin 27	76655433
Neuroendocrine secretory protein 55 (NESP-55)*	2262205
Osteocrin	76671357
Osteogenin (BMP3)*	76620194
Platelet basic protein precursor (PBP)	76619991
Proenkephalin*	83405428
Pro-Neuropeptide Y*	40022234
Secretogranin III*	76663325
Transforming growth factor-beta binding protein*	135671
Ubiquitin/ribosomal fusion protein	28189665
VEGF (vascularendothelial growth factor)+	27806821
VGF nerve growth factor inducible*	76654056
Protease Systems	Genbank	Soluble	Membrane
ADAM metallopeptidase with thrombospondin type 1	76648708
AFG3-like protein 1	76677962
Alpha-1-antichymotrypsin precursor (ACT)	76694546
Alpha-2-plasmin inhibitor	76643654
Autophagy 4 homolog D (APG4)	76621759
Bleomycin hydrolase	76643627
Carboxypeptidase D*	76643631
Carboxypeptidase E*	76675819
Carboxypeptidase G2	76647796
Cathepsin B+	9955277
Cathepsin D*	76658398
Cathepsin L#	1542853
COP9 constitutive photomorphogenic	74267826
COP9 signalosome	76654132
Cystatin C+	27806675
Cystatin E/M	61097917
Disintegrin and metalloprotease domain 4	76684774
Endopin 2C#	62126072
HECT, UBA and WWE domain containing 1	76659533
IAP, Inhibitor of Apoptosis	76638039
Leukotriene A4 hydrolase	74355010
Meltrin	47564064
Metalloprotease 1	76672659
Neuroendocrine protein 7B2*	88682959
Polyubiquitin	89994036
PP11 serine protease	76618319
Prohormone convertase 1*	61817689
Prohormone convertase 2+	13878928
Proprotein convertase subtilisin/kexin type 4	76622897
Reelin precursor	76677746
Serine (or cysteine) proteinase inhibitor	74268410
Tissue inhibitor of metalloproteinase 1 (TIMP-1)+	27806161
Transmembrane protease, serine 3	76608355
Ubiquitin*	76638698
Ubiquitin A-52 (residue ribosomal protein fusion product 1)	76620757
Ubiquitin associated protein 2	76624820
Ubiquitin protein ligase E3B	76639147
Vpr-binding protein	76649016
XIAP associated factor-1	78045549
YME1-like, metalloprotease	76632200
Neurotransmitter Enzymes/Transporters	Genbank	Soluble	Membrane
4-aminobutyrate aminotransferase precursor	76678944
Bestrophin anion channel	76629024
Calcium channel, voltage-dependent, alpha	76615073
Dopamine beta-monooxygenase*	1083022
Glutamate decarboxylase 1	76609605
Glutaminase	86438072
Neuronal pentraxin I*	76669694
PNMTase (phenylethanolamine N-methyltransferase)+	130374
Protein tyrosine phosphatase, receptor	76614769
Rod photoreceptor cng-channel	1050441
Sodium channel protein type IV alpha subunit	76645224
Sodium/potassium/calcium exchanger 1	76684545
Synaptic vesicle monoamine transporter (VAT2)*	457486
Transient receptor potential cation channel	76625397
Tyrosine 3-monooxygenase (Tyrosine hydroxylase)	27807401
Vesicle amine transport protein 1 (VAT1)	76671278
Vesicular inhibitory amino acid transporter	76646508
Receptors	Genbank	Soluble	Membrane
Adiponectin receptor 2	76679749
Bone morphogenetic protein receptor	76619536
Bradykinin receptor B1	76647810
Cholinergic receptor, nicotinic, alpha polypeptide 3	27807295
EC2-V2R pheromone receptor	76675084
EPH receptor A7	76679477
Estrogen-related receptor gamma	76636898
Fc receptor-like 3 precursor	76670706
Fibroblast growth factor receptor 4 precursor (FGFR-4)	76622759
Gamma-aminobutyric acid receptor	76637789
Glutamate [NMDA] receptor	76631132
Insulin receptor substrate 4	76659092
Interleukin-1 receptor-like 2 precursor (IL-1Rrp2)	76628778
Muscarinic acetylcholine receptor M5	76627274
Olfactory receptor 212	76683959
Olfactory receptor 5A1	76636626
Olfactory receptor 833	76693698
Peripheral-type benzodiazepine receptor-associated protein 1	76639758
Progesterone receptor (PR)	76678665
T-cell receptor alpha chain C region	76626982
Thyroid hormone receptor associated protein 2	76639110
TNF receptor associated protein	81673141
Vascular endothelial growth factor receptor 3 (VEGFR-3)	76663199
Biochemical Processes
Enzymes	Genbank	Soluble	Membrane
1-aminocyclopropane-1-carboxylate synthase	76636325
Ash1 (absent, small, or homeotic)	76612493
Arylacetamide deacetylase (AADAC)	76607894
Aspartate aminotransferase 1	29135295
Bisphosphoglycerate mutase	61839453
C-1-tetrahydrofolate synthase, cytoplasmic	76628072
CG7544-PA	76643676
Cyclophilin B (PPIB)	74268324
Dihydropyrimidinase-related protein 4 (DRP-4)	61878819
Enolase 2	88682888
Enolase 3	88954201
Folate receptor 1 precursor	76635818
Glycerol-3-phosphate acyltransferase	76628542
Malate dehydrogenase (MADH2)*	81674781
Metallothionein-like 5 (testis-specific, tesmin)	76658427
Methionine adenosyltransferase II, beta	81673843
NAD synthetase 1	73587273
Paraoxonase 2	61888862
Phosphoribosylglycinamide formyltransferase	61966468
Reticulon 4 interacting protein 1	76649171
Ribonucleoside-diphosphate reductase	76630263
Splicing factor, arginine/serine-rich 15	76645770
Thioredoxin domain containing protein 5	76676581
Carbohydrate Functions	Genbank	Soluble	Membrane
Alpha-(1,3)-fucosyltransferase (Galactoside 3-L-fucosyltransferase)	76631232
Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase	61864843
Amylo-1,6-glucosidase, 4-alpha-glucanotransferase	76613348
Bactericidal/permeability-increasing protein-like 3	76633471
Beta 1,4-N-acetylgalactosaminyltransferase-transferase-3	76663169
Beta-1,3-N-acetylglucosaminyltransferase bGnT-6	76645514
Beta-1,4-galactosyltransferase 2	76614325
Chondroitin sulfate glucuronyltransferase	76616133
Galactose-1-phosphate uridylyltransferase isoform 1	61555177
Galactose-3-O-sulfotransferase 3 isoform 1	76658175
Glucosidase II*	76657688
Golgi sialoglycoprotein MG-160+	76639850
Maltase-glucoamylase	76678173
Mannosidase*	76624022
N-acetylglucosaminyltransferase V+	76610006
Ribophorin II	76638094
UDP-glucose:glycoprotein glucosyltransferase 1	76609241
Lipid Functions	Genbank	Soluble	Membrane
3,2-trans-enoyl-CoA isomerase, mitochondrial precursor	76652799
Acetyl-Coenzyme A carboxylase beta	76639133
Acyl-Coenzyme A dehydrogenase	27806205
Acyl-Coenzyme A synthetase	76676487
Aminophospholipid transporter (APLT)+	27807317
Arachidonate 12-lipoxygenase	76643357
Carnitine O-palmitoyltransferase I	76658339
Cerebroside sulfate activator (SAP-1)+	115502446
Diacylglycerol kinase, beta	76681581
Fatty acid binding protein 11	89994084
Glucocerebrosidase precursor	76612148
High density lipoprotein-binding protein	76634040
Lipin-2	76651852
Low-density lipoprotein receptor-related protein	76609709
N-acylsphingosine amidohydrolase+	76655702
Patatin-like phospholipase domain containing 2	76658479
Phospholipase A1 member A (Pla1a)	83405374
Transport-secretion protein	76658477
Internal Conditions of Secretory Vesicles
Reduction-Oxidation	Genbank	Soluble	Membrane
Biliverdin reductase A precursor	76615466
Catalase	78369302
Cytochrome b561*	27807323
Cytochrome C oxidase+	117102
Cytochrome P450*	76669152
Dimethylaniline monooxygenase [N-oxide-forming] 2	76637349
Endoplasmic reticulum oxidoreductin 1-Lbeta	76656155
Glutathione peroxidase 3*	585223
Myeloperoxidase precursor (MPO)	76642663
NAD(P) transhydrogenase, mitochondrial precursor	128400
Ubiquinol-cytochrome C reductase complex+	136691
ATPases and Nucleotide Metabolism	Genbank	Soluble	Membrane
ADP-ribosylation factor-like 10C	76648606
ANT 1 (adenine nucleotide translocator 1)+	32189340
ATP H+ transporting VI+	4502315
ATP Synthase (gamma chain)+	2493093
ATP synthase alpha chain, mitochondrial precursor	76652040
ATP6IP1 protein	28461231
ATPase type 13A2	74267862
ATPase, aminophospholipid transporter (APLT), Class I, type 8A*	27807317
ATPase, H+ transporting*	102
ATPase, H+ transporting, lysosomal, V1 subunit C	28603816
ATPase, H+ transporting, subunit A	27807453
ATPase, H+ transporting, V1 subunit B*	28603772
ATP-binding cassette sub-family D	61863306
ATP-binding cassette, sub-family A	76652817
Concentrative Na+-nucleoside cotransporter	76661614
Ectonucleoside triphosphate diphosphohydrolase 1 protein	76654745
HT028 (ATPase)*	61823467
MSTP042+	75832069
Proton-associated sugar transporter A	76671385
TER ATPase (transitional endoplasmic reticulum)+	73586667
V-ATPase (vacuolar ATPase accessory subunit B)+	549205
V-ATPase (vacuolar ATPase accessory subunit D)	62460538
V-ATPase (vacuolar ATPase accessory subunit E1)	27807375
V-ATPase (vacuolar ATPase accessory subunit F1)*	94574271
V-ATPase (vacuolar ATPase accessory subunit SFD alpha isoform)	2895578
V-ATPase (vacuolar ATPase polypeptide IV)	89602
V-ATPase (vacuolar ATPase synthase subunit H)+	12643366
Protein Folding	Genbank	Soluble	Membrane
C1GALT1-specific chaperone 1	74356336
Chaperonin 10+	1167
Cysteine string protein (CSP)*	1232163
Glucose-regulated protein precursor (GRP 78)	76630569
Heat Shock Protein 27*	71037405
Heat Shock Protein 40*	76619510
Heat shock protein 60	76648520
Heat shock protein 70	73586960
Peptidylprolyl isomerase B+	27806469
Prion protein	21666990
Wiskott-Aldrich syndrome protein interacting protein (WIP)	76609571
Transporters (solute)	Genbank	Soluble	Membrane
Organic anion transporter 3	42538740
P87	259174
Sodium-dependent glucose transporter SGLT-I	14486596 76661712
Solute carrier family 2
Solute carrier family 22, anion/cation transporter	76657700
Solute carrier family 25, mitochondrial carrier glutamate	27807185
Solute carrier family 26, anion transporter	76668427
Solute carrier family 38, carrier protein	94966787
Solute carrier family 39, zinc transporter	76651620
Solute carrier family 4, sodum borate transporter	30794360
Solute carrier family 6	76693476
Regulated Secretion Mechanisms
Signal Transduction and GTP-Binding Proteins	Genbank	Soluble	Membrane
1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase	76648298
1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase-like 4	76637596
Activator of S phase kinase	76671561
Adenylate cyclase-inhibiting G alpha protein	27805887
A-kinase anchor protein 4	41386786
AHNAK-related protein*	76657680
Ankyrin repeat domain 37	76668139
AXL receptor tyrosine kinase	76684583
Breast cancer membrane protein 11	76614919
Calcium/calmodulin-dependent protein kinase IIA	76623502
Calcium/calmodulin-dependent protein kinase IV	76679349
CAMP-dependent protein kinase inhibitor beta	76625537
Catenin	76656278
CDK5 and ABL1 enzyme substrate 2 (Interactor with CDK3 2)	76632977
Centaurin-gamma 1 GTPase	76618822
Cyclic nucleotide-gated channel beta subunit 1e	3309626
DIRAS family, GTP-binding RAS	73587391
Doublecortin kinase 2	76668033
Dual specificity protein phosphatase 2	76628632
FK506 binding protein+	25066280
Frizzled 9 precursor (Frizzled-9)	76653703
FYVE, RhoGEF and PH domain containing 2	76650063
FYVE, RhoGEF and PH domain containing protein 5	76661294
Guanine nucleotide binding protein (G protein), alpha*	30794332
Guanine nucleotide binding protein (G protein), beta*	1085447
Guanine nucleotide binding protein (G protein), type B	399711
Guanine nucleotide binding protein (G protein), gamma*	27807509
Guanine nucleotide exchange factor	77362757
Guanylate kinase-associated protein	76647874
Heart alpha-kinase	76662373
Immunity-related GTPase family, Q1	76641801
Inositol polyphosphate-4-phosphatase, type II	76660578
Intestinal cell kinase	76650734
IP3 receptor associated cGMP kinase	7341097
Leucine-rich repeat kinase 1	76646793
Lin-7	76641963
Mitogen-activated protein kinase 1+	28461209
Mitogen-activated protein kinase 5	76660373
Mitogen-activated protein kinase 8	76656852
Mitogen-interacting protein kinase I*	77736562
Myomegalin	76670843
N-ethylmaleimide-sensitive factor	76645272
Phosphatase and tensin homolog	76655292
Phosphatidylinositol 4-kinase beta	38372429
Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase PTEN	76671072
Phosphoinositide-3-kinase adaptor protein 1	76608515
PKCI-1-related HIT protein+	14211923
Plexin B2 precursor (MM1)	76617505
Polo-like kinase 2	76660620
Preferentially expressed antigen in melanoma like 5	92096623
Protein kinase C, epsilon type (nPKC-epsilon)	76629287
Protein kinase, DNA-activated, catalytic polypeptide	76634333
Protein phosphatase 1, regulatory (inhibitor)	76645130
Protein phosphatase 1J (PP2C domain containing)	76613182
PYRIN-containing APAF1-like protein 7	76614247
Rab-1	76629651
Rab-12	76651878
Rab-14*	76668547
Rab-15	76628095
Rab-2	76686605
Rab-21+	2500067
Rab-27A	61875226
Rab-27B	24459171
Rab-2B	76627105
Rab-33B	76638452
Rab-34	73587147
Rab-35+	76639295
Rab-37	76645964
Rab-39A	76611110
Rab-3A	27806127
Rab-3B	27806113
Rab-3C+	86438380
Rab-4B	76641702
Rab-5B	76618738
Rab-6B	76608256
Rab-7+	74354082
Rab-8B	76627752
Receptor-type tyrosine-protein phosphatase N2	76676438
Regulator of G-protein signalling 2, 24kDa	76636875
Retinitis pigmentosa	21303187
Rho guanine nucleotide exchange factor 12	76635393
Rho guanine nucleotide exchange factor 4	76609133
Serine/threonine-protein kinase 38	76650116
Serine/threonine-protein kinase PLK2	76660624
SET binding factor 1	76617331
Signal-induced proliferation-associated 1	76628293
Son of sevenless homolog 1	76629071
Src homology 3 domain-containing guanine nucleotide exchange factor	76676576
Sterile alpha and TIR motif containing 1	76643315
Synaptojanin 2 binding protein	74354054
Testis-specific serine kinase 6	76621013
Tousled-like kinase 1	76609603
Transducin protein 4	76666047
Tuberin (Tuberous sclerosis 2 homolog protein)	76652568
Tyrosine-protein kinase JAK2	76661504
Tyrosine-protein phosphatase-like N precursor (R-PTP-N)*	76610663
Very large G-protein coupled receptor 1	76660074
Virus-induced signaling protein	86438388
WD-repeat protein	76612957
Wingless-type MMTV integration site family, member 10B	76618188
WNK lysine deficient protein kinase 2	76664151
Wnt inhibitory factor 1 precursor (WIF-1)	76618509
Vesicular Trafficking and Exocytosis	Genbank	Soluble	Membrane
Clathrin+	27806689
Coatomer alpha subunit (Alpha-coat protein)	76615759
Dynamin 2	76660288
Epsin-2	76644169
Formin binding protein 4	76636519
Golgi autoantigen*	76641568
Huntingtin interacting protein 1	76649404
Islet cell autoantigen 512	5305476
Kinesin	76648026
Piccolo (presynaptic cytomatrix protein)	76615069
Pleckstrin	76608906
SEC31-like 2	76654762
Sec5 protein	76661880
Sorting nexin 4	76640294
Synapsin Ia	108935
Synaptophysin*	33112658
Synaptotagmin 1*	27806387
Synaptotagmin II	76687864
Synaptotagmin VI	76613150
Synaptotagmin VII*	76657575
Synaptotagmin-4	61809317
Syntaxin-1A	417841
THUMP domain containing 1	76653091
Tomosyn	76626187
Unc-18 protein	631583
VAMP 3 (cellubrevin)*	61845787
Vesicular membrane protein p24	76651226
Calcium Regulation	Genbank	Soluble	Membrane
Annexin A1	74
Annexin A2*	27807289
Annexin A4*	1063258
Annexin A6*	76623595
Annexin A7	76656523
Annexin A11+	113969
Bestrophin isoform 1	61842255
Calcium binding protein P22	76688289
Calnuc (Nucleobindin)+	189308
Mucolipin 3	81673761
SPARC-like 1	74354032
Voltage-dependent T-type calcium channel alpha-1	76674163
Morphological Functions of Secretory Vesicles
Structural Proteins	Genbank	Soluble	Membrane
Bone proteoglycan II	28189579
Brevican	88682949
Cartilage acidic protein 1	76654689
Centromere protein I (CENP1)	86821813
Centromeric protein E (CENP-E)	76676943
CG15021-PA	76648468
CG2843-PA	76644813
Collagen, type I	76686475
Collagen, type VI	76667061
Collagen, type XVIII	76608417
Collagen, type XXVII	76625290
Collogen cyanogen bromide, type II	5354051
Colonic and hepatic tumor over-expressed protein	76636503
Cytokinesis 8	76624391
Desmoplakin	76651410
Diaphanous 3	76661030
Drebrin 1	76622581
Dynein	76648966
Echinoderm microtubule associated protein like 5	76673417
Elastin microfibril interfacer 1*	61845535
Fibrinogen	75812954
Fibronectin type III	76613259
Galectin-related inter-fiber protein	76654374
Gap junction protein, (connexin 31.9)	76644686
Hydrocephalus inducing	76673899
KIAA1914 protein	76655117
Microtubule associated serine/threonine kinase 2	76614276
Microtubule-associated protein 4	27806553
Myosin, heavy polypeptide 9+	27807325
Myosin, light polypeptide kinase	76609105
Nebulin	76609853
Nesprin-2	76628088
Obscurin	76620689
Pericentrin 2	76607770
Periphilin 1	76618432
Proline arginine rich coiled coil 1	76614586
Proteoglycan 3	76705880
Proteolipid protein 1 (PLP1)	74354814
Radial spokehead-like 3	76625577
Spectrin domain with coiled-coils 1	76644035
Sperm associated antigen 4-like	76633467
Symplekin	76641008
Talin 1+	76627770
Testican 2+	76656478
Tubulin*	76628240
Cell Adhesion/Cell-Cell Interactions	Genbank	Soluble	Membrane
Ankyrin 3	76656250
Cadherin*	76640658
CD18 antigen	2407809
CD63 antigen*	45439308
CD81 antigen (target of antiproliferative antibody 1)	73586978
Cell adhesion molecule JCAM	76608323
Contactin 1+	1060861
Dishevelled-associated activator of morphogenesis 1	76665330
Dystonin	76649774
Epithelial V-like antigen 1	62751654
Integrin alpha-8 precursor	76632354
KIAA0319 (dyslexia)	76674660
Laminin beta-1 chain precursor	76615137
Leucine rich repeat and fibronectin type III domain containing	76658000
Nidogen-2 precursor (NID-2) (Osteonidogen)	76627657
Swan isoform 4 (neurexin)	76634905
Thrombospondin-3 precursor	76612130
UCC1+	76664807
Vinexin (SH3-containing adapter molecule-1)	76624656
Other Protein Categories
Cell growth and development	Genbank	Soluble	Membrane
Gametogenetin	76641302
Mitotic-specific cyclin B1	76615016
Tectonic	76639025
Immune	Genbank	Soluble	Membrane
Anti-testosterone antibody+	440
Cardiotrophin-1 (CT-1)	76687585
Collectin-43	50355694
Complement component 6	88954145
Complement component 7	88954301
Ig gamma-1 chain C region, membrane-bound form	76678688
Ig heavy chain*	1575493
Ig lambda chain V-I region BL2 precursor	76690231
IgG Fc receptor FcRN	7339746
Immunoglobulin superfamily, member 2	76612971
Killer cell immunoglobulin-like receptor 3DL1	76687295
Large proline-rich protein (BAT2)	76650909
Major histocompatibility complex, class I-related	76660744
Myeloid/lymphoid or mixed-lineage leukemia	76644552
Stromal cell derived factor 4	73586919
TOLL-like receptor 7	76151015
UL16 binding protein 3	76693242
Transcription and Translation	Genbank	Soluble	Membrane
Ataxin 7	76613103
BCOR protein (BCL-6 corepressor)	76670256
Bromodomain and PHD finger containing, 1 isoform 6	76648396
CAMP responsive element binding protein 5	76615443
Cbp/p300-interacting transactivator	76668278
CCAAT/enhancer binding protein zeta	76629167
Cell division cycle 16	76634504
Cell division cycle 42	76611597
Centrosomal protein 2	76633389
C-Fos	32481980
Chromodomain helicase DNA binding protein 7	76634422
Chromosome condensation (RCC1) and BTB (POZ) domain	76667931
Cleavage and polyadenylation specific factor 1	27807297
Clock isoform 4	76619910
Core-binding factor, beta subunit	76640732
CPEB3 (cytoplasmic polyadenylation element binding protein 3)	76661464
DEAD (Asp-Glu-Ala-Asp) box polypeptide 48	76645558
DNA cross-link repair 1A protein	76655155
DNA methyltransferase 1 associated protein 1	89994078
DNA replication complex GINS protein PSF2	76639980
DNA-binding protein SATB1	76608636
DRE1 protein	76636127
Eukaryotic translation initiation factor 2	76629540
Eukaryotic translation initiation factor 3	74353982
Exosome component 10	76637212
Fidipidine	76610089
Forkhead box K1	76654354
Fusilli	76634878
General transcription factor II H	76650520
High mobility group protein 4	61878473
High-mobility group protein 3	76658822
Histone acetyltransferase GCN5	76644862
Histone H2B 291B	76625998
HIV TAT specific factor 1	76658704
HP1-BP74	76611630
Hypothetical zinc finger protein KIAA1196	76632891
Hypoxia-inducible factor-3 alpha	76642167
Ladybird homeobox homolog 1	76654810
Luc7	76652552
Methyl-CpG binding domain protein 1	86437962
Myoneurin	77567823
Neuro-oncological ventral antigen	76667375
Nibrin+	76634945
Nuclear autoantigen Sp-100	76610804
Nuclear RNA export factor 3	76659002
Nucleolar complex associated 3	76640610
Nucleoporin	76646535
P20-CGGBP	76608705
PGC-1 related co-activator	76654860
PHD finger protein 22	76684907
Pinin, desmosome associated protein	27807293
Polyhomeotic 1	76616376
Pre-B-cell leukemia transcription factor interacting protein 1	76612171
Regulatory factor X4 isoform c	76619030
Replication initiator 1	76616173
Retinoic acid induced 16	76624642
Ribosomal protein L23a	76613395
Ribosomal protein L29	74268009
Ribosomal protein L4	62460480
Ribosomal protein S27a	76620759
Ribosomal protein S6 kinase polypeptide 3	76659718
RNA-binding protein 11	94966923
RNA-binding protein 28	76615708
RNA-binding protein EWS 8	76639325
RPS27A protein	73586974
Schlafen 10	76642925
Serologically defined colon cancer antigen 33	76651519
Sex comb on midleg-like protein 2	76676174
SFRS5 splicing factor	76662260
Shugoshin-like 2	76610144
Sp4 transcription factor	76673507
Spermatogenic leucine zipper 1	94966881
Target of myb1, Tom-1 protein	78042494
TATA box binding protein like 2	76660220
Transcription elongation factor B polypeptide 3 binding protein 1	76622840
Transcription factor PU.1	76636497
Transcription factor RAM2	76614939
Transcription factor Sp1	76617820
Transcriptional intermediary factor 1-gamma (TIF1-gamma)	76613148
Treacle protein	76683158
Woc	76672108
Valosin-containing protein (VCP)	77735541
Zinc finger CCCH-type containing 12A	76614519
Zinc finger protein 111	76616182
Zinc finger protein 262	76614570
Zinc finger protein 385	76617575
Zinc finger protein 398	76616180
Zinc finger protein 469	76640087
Zinc finger protein 608	76667970
Zinc finger protein 623	76634062
Zinc finger protein 644	76613525
Zinc finger protein 694	76610151
Zinc finger protein 8	76685031
Zinc finger protein 84	76642204
Zinc finger protein 85	76641574
Miscellaneous
Miscellaneous	Genbank	Soluble	Membrane
Absent in melanoma 1 protein	76608722
Albumin	162648
Apical-like protein (APXL protein)	76659839
Apoptotic chromatin condensation inducer	76626863
BCL2-like 14	82571795
Beta2-Microglobulin*	41386683
Breast cancer antiestrogen resistance 3	66792756
Bromodomain and WD repeat domain containing 2	76655253
C9orf55	76659997
Cas-Br-M ecotropic retroviral transforming sequence-like 1	76615140
CG7593-PA	76657521
Chromosome 17 open reading frame 27	76670292
Chromosome 9 open reading frame 19	61812939
Desmocollin 2	76651700
ELG protein	76643730
ESCO2 (establishment of cohesion)	76624097
F33H2.2	76638347
Golgi phosphoprotein 3	76650110
HBx-Hepatitis B virus X interacting+	74268261
Hemoglobin*	12655818
Kelch protein 3	76623241
KIAA1862	76616193
KIAA1900	76663072
Lysosome membrane protein II (LIMP II)	76620042
Membrane-associated ring finger 3 (C3HC4)	76660388
Membrane glycoprotein 1 precursor (LAMP-1)	1683365
MGC133986 protein	86437964
MICAL-2	76676434
Mitochondrial carrier homolog 1	76650069
Mitochondrial import inner membrane translocase subunit Tim17 A	76680711
Odd Oz/ten-m homolog 2	76623795
Otolin-1	76607840
Partitioning defective-6 homolog alpha (PAR-6 alpha)	76633889
PC4 and SFRS1 interacting protein 1	76624197
Phosphorylated CTD interacting factor 1	76633669
PRBP (plasma retinol-binding protein)+	132403
Pyrin domain containing 4	82571578
R119.5	76665822
RAD52	66792838
Ran-binding protein 3 (RanBP3) isoform 2	76622053
Ring finger protein 111	76627819
Ryanodine receptor 1, skeletal muscle	76641296
SCD6 homolog A	77736251
SID1 transmembrane family member 1 precursor	76684359
Spastin isoform 1	76682638
Spermatogenesis associated 13	76631579
Spermatogenesis associated 5	76687571
Storkhead box 2	76655606
Tankyrase 1	76655843
Tetraspanin 7*	76665086
Transcobalamin II+	27806385
Transmembrane protein 16B	76673511
Transmembrane protein 63C	76628460
Transmembrane protein 79	59858019
Tripartite motif protein 7	76622789
Tumor necrosis factor, alpha-induced protein 3	76626126
WW domain binding protein 11	76633774
Vacuolar protein sorting factor 4B	76652249
Y37D8A.2	76669076
Unknown	Genbank	Soluble	Membrane
CG11617-PA	76632406
CG6379-PA	76650147
CG13957-PA	76609707
CG17569-PB	76665263
CG32045-PC	76631513
CG3338-PA	76643561
CG4751-PA	76614067
CG5987-PA	76644502
CG7709-PA	76642510
FLJ44048 protein	76668361
H43E16.1	76680309
Hypothetical gene supported by AK075558; BC021286	76637226
Hypothetical protein FLJ13868	61554841
Hypothetical protein LOC539970	74267976
Hypothetical protein XP_583091	76659995
Hypothetical protein XP_584302	61823940
Hypothetical protein XP_585938	76658228
Hypothetical protein XP_590236	76616022
Hypothetical protein XP_592499	76672076
Hypothetical protein XP_594162	76637663
Hypothetical protein XP_598213	76619718
Hypothetical protein XP_600405	76690587
Hypothetical protein XP_600782	76678727
Hypothetical protein XP_868808	76649193
Hypothetical protein XP_869427	76635764
Hypothetical protein XP_873356	76769235
Hypothetical protein XP_580444	76634839
Hypothetical protein XP_596183	76638981
Hypothetical protein XP_608396	76678883
Hypothetical protein XP_876844	76638096
Hypothetical protein XP_876914	76638098
Hypothetical protein XP_881202	76639161
Hypothetical protein XP_882313	76632294
Hypothetical protein XP_882414	76662668
Hypothetical protein XP_882587	76630059
Hypothetical protein XP_883381	76631223
Hypothetical protein XP_883390	76640533
LPR protein	619
Neuroblastoma-amplified protein	76666100
Protein for IMAGE:8054235	83406129
Protein for MGC:140076	92098401
RIKEN cDNA 0610040D20	61870202
RIKEN cDNA 9130210N20	76634018
Sushi repeat-containing protein	94966791
TAG-278	76685297
THAP domain containing 4	83759162
Transmembrane 9 superfamily protein member 4	76633198
Unknown (protein for MGC:127406)	73587279
Unknown (protein for MGC:140139)	92096913
YEATS domain containing 2	76607509
ZK1067.4	76650437
ZK742.2	76620462

Proteins indicated by ‘+’ were found in the Wegrzyn et al.; 2007 study (47) but not in this study. Proteins indicated by ‘*’ are proteins identified in both the Wegryzn et al.; 2007 study (47) and this study. In addition, protein indicated by ‘#’ were identified by previous focused studies on the amyloid precursor protein (55, 143), cathepsin L (8), and endopin 2C (56). Thus, this table represents the overall proteomic characterization of chromaffin secretory vesicle proteins.

Analyses of selected chromaffin granule proteins by western blots and immunofluorescence confocal microscopy

Western blot analyses of chromaffin granules were utilized to assess the presence of several proteins related to neurological diseases. Western blots of cystatin C, huntingtin interacting protein, ataxin 7, and prion protein were conducted using SDS-PAGE gel electrophoresis and western blots methods as we have described previously (48–51). Cystatin C in western blots was detected with anti-cystatin C (at 1:1000 dilution, from US Biological, Swampscott, MA). Huntingtin interacting protein was analyzed in western blots of chromaffin granules with anti-SET2 antisera by immunoprecipitation prior to western blots (at 1:1000 dilution, from Chemicon, Temecula, CA) as described previously (51, 52). Ataxin 7 was detected by anti-ataxin 7 generated by the La Spada laboratory (antisera K (49) utilized at 1:2000, after immunoprecipitation of ataxin 7 performed as we have described previously (51, 52). Detection of prion in western blots utilized monoclonal antibody SAF-84 (1 μl/ml, from Cayman Chemical, Ann Arbor, MI).

The presence of a neurological disease protein, cystatin C, as an example, in chromaffin cells was assessed by immunofluorescence confocal microscopy to confirm its localization in secretory vesicles. Chromaffin cells in primary culture were prepared from fresh bovine adrenal medulla tissue as previously described (53). Cells were subjected to colocalization studies of enkephalin-containing chromaffin secretory vesicles for the presence of cystatin C (anti-cystatin C rabbit, 1:50 dilution, from US Biological, Swampscott, MA) in enkephalin-containing secretory vesicles (detected by anti-(Met)enkephalin mouse, from Abcam company, Cambridge, MA or from Chemicon-Millipore company, Billerica, MA) by immunofluorescence confocal microscopy, conducted as we have previously described (8, 50). Cystatin C was detected with anti-rabbit IgG-Alexa Fluor 568 (goat) (1:50 dilution, red fluorescence, Molecular Probes, Eugene, Oregon) with comparison to localization of (Met)enkephalin (ME) in secretory vesicles detected with anti-mouse IgG Alexa Fluor 488 (goat) (1:50 dilution, green fluorescence). Immunofluorescent images were obtained with the Delta Vision Spectris Image Deconvolution Systems on an Olympus IX70 confocal microscope using the software Softwrox Explorer from Applied Precision.

Results and Discussion

Identification of an extensive number of chromaffin granules (CG, also known as secretory vesicles) proteins by nano-HPLC Chip MS/MS tandem mass spectrometry

Differential centrifugation was utilized to obtain purified chromaffin granules (CG) from bovine adrenal medulla homogenate, illustrated in figure 1a. This purification scheme is well-established in the field (36–38). Organelles are removed from the CG fraction by a series of centrifugation steps that remove nuclei (P1 fraction), microsomes (S2 fraction), and mitochondria and lysosomes (S3, S4, and S5 fractions, not shown in Fig. 1a). The enriched fraction of CG (P5 fraction) is purified by a 0.32/1.6 M sucrose gradient, resulting in a pellet of purified CG. Comparison on a multi-step sucrose gradient of 2.2 M to 1.2 M sucrose shows that both the crude and purified CG samples contain peaks of (Met)enkephalin at about 1.7–2.0 M sucrose. While the crude CG contains a peak of acid phosphatase (Fig. 1b), a marker for lysosomes, such a peak for lysosomes is absent in the purified CG (Fig. 1c). The purity of these CG have been confirmed by electron microscopy (47) that shows the homogeneity of the preparation. These data and those from other studies (36–38) establish the purity of chromaffin granule obtained by density gradient.

The soluble and membrane fractions of these purified secretory vesicles were separated to provide functional predictions of identified proteins in the soluble secreted pool, or as membrane-related proteins that participate in maintaining integrity of the organelle. Soluble and membrane fractions were obtained by lysis and centrifugation of chromaffin granules in isotonic salt conditions in buffer of pH 6.0 that represents the internal vesicle environment (54).

Furthermore, to enhance nano-LC-MS/MS analyses of moderate to lower abundance proteins, the highly abundant chromogranin A (CgA) protein was removed by affinity chromatography (as described in methods), which removes the major 66–70kDa CgA protein band (Fig. 2). Each of the soluble and membrane fractions were subjected to trypsin digestion and nano-HPLC Chip MS/MS tandem mass spectrometry with the XCT Ultra ion trap mass spectrometer (Agilent) for sensitive analyses of peptides (estimated down to the attomole range). Evaluation of MS/MS spectra by the Spectrum Mill search program yielded identification of proteins in soluble and membrane fractions with ~1% FDR (determined by shuffled decoy database analysis). Subsequently the entire dataset was also searched using an alternate search algorithm, OMSSA, to confirm and validate peptide identifications.

Figure 2. Removal of abundant chromogranin A protein from soluble and membrane fractions of purified chromaffin granules.

Chromaffin granule soluble and membrane fractions were each subjected to calmodulin affinity chromatography to remove the most abundant protein consisting of full-length CgA. The soluble fraction of these chromaffin granules is illustrated (lane 1), showing full-length CgA of ~66–70 kDa that has been identified by mass spectrometry in previous studies (10, 137). After affinity chromatography on calmodulin-Sepharose conducted two times, removal of the full-length CgA is illustrated (lane 2). Equal relative volumes (5 μl) of soluble chromaffin granule sample was applied to lanes 1 and 2 (corresponding to ~2 μg and ~6.5 μg protein, respectively). The CgA depletion step recovered ~30–35% of the original proteins of the soluble chromaffin granule sample. CgA also exists as cleaved proteolytic fragments in the chromaffin granules which presumably are largely removed by the calmodulin-Sepharose affinity step. After the affinity step, the overall pattern of protein bands (lane 2) resembles that of the soluble granule sample before the affinity step, with the exception of removal of CgA protein(s).

Proteins found in the soluble and membrane fractions of the secretory vesicles by the nano-HPLC Chip MS/MS approach are illustrated in Supplemental Tables A and B, respectively. Nano-HPLC Chip MS/MS identified more than 600 proteins in both the soluble and membrane fractions. These results demonstrate the high efficiency of the nano-HPLC Chip MS/MS tandem mass spectrometry system to identify hundreds of proteins from several micrograms of sample per analysis.

Total proteome of chromaffin secretory vesicles

To obtain an overall proteomic view of the purified adrenal medullary secretory vesicles (chromaffin granules) the extensive nano-HPLC Chip MS/MS identification of more than 600 proteins obtained in this study was combined with our prior data of proteins from these secretory vesicles subjected to gel electrophoresis separation prior to MS/MS analyses, (47) and several purified proteins (8, 55–57). The present study identified proteins in chromaffin secretory vesicles after affinity removal of the most abundant chromogranin A (CgA) protein, to allow identification of other proteins of more modest abundance. The prior study of chromaffin secretory vesicles with CgA identified ~100 proteins via one-dimensional gel electrophoresis (47). Thus, the overall proteomic analysis of the chromaffin secretory vesicles of this extensive proteomic study for identification of more than 600 proteins, combined with our prior smaller proteomic study (47), has identified 371 distinct soluble proteins and 384 distinct membrane proteins (Fig. 3, Venn diagram), with 69 proteins present in both soluble and membrane fractions. These data illustrate the presence of a total of 686 distinct proteins identified in the soluble and membrane fractions of chromaffin secretory vesicles.

Figure 3. Venn diagram of common and different proteins in soluble and membrane fractions of chromaffin granules.

This Venn diagram illustrates the the majority of the chromaffin secretory vesicle proteins identified in this study using nano-HPLC Chip MS/MS, combined with several proteins identified in in earlier proteomic studies using gel electrophoresis for protein enrichment [43]. The soluble fraction contained 371 distinct proteins and the membrane fraction contained 384 distinct proteins. Proteins common to both soluble and membrane fractions are illustrated as the intersecting area of the Venn diagram, indicating 69 proteins that were present in both soluble and membrane compartments of these secretory vesicles. The soluble and membrane fractions contained a total of 686 unique proteins in chromaffin secretory vesicles.

Distinct functional categories of proteins in soluble and membrane components of chromaffin secretory vesicles

The categorization of the chromaffin secretory vesicle proteins indicates the presence of distinct functional categories of biochemical systems (Fig. 4, and Table 1). Protein categories were identified for neurotransmitter and neurohumoural mechanisms, as well as diverse biochemical processes that include maintenance of the internal environment of these vesicles. A large portion of proteins participate in regulated secretion via signal transduction and exocytosis. These functions are combined with vesicular trafficking that involves structural proteins. Protein components within these categories are described in detail below.

Figure 4. Comparison of soluble and membrane proteins by pie charts.

The relative portion of proteins in each functional category are compared for the soluble (panel A) and membrane (panel B) fractions of chromaffin secretory vesicles. Each functional category of the pie chart is shown as a distinct color.

Production of neurotransmitters and neurohumoural factors: neuropeptides and neurohumoural factors, protease systems, neurotransmitter enzymes/transporters, and receptors

Proteins involved in secretory vesicle-mediated cell-cell communication were identified as proneuropeptides and processing proteases, neurohumoural agents, enzymes and transporters for classical small molecule neurotransmitters, and receptors. Numerous proneuropeptide and prohormone precursors were identified such as proenkephalin, proNPY, chromogranins, and others that undergo proteolytic processing (5, 8–10, 46, 50, 58–63) to generate active neuropeptides that function as neurotransmitters and hormones. These vesicles also contain several neurohumoural factors such as VGF nerve growth factor and vascular endothelial growth factor (64–66).

Numerous proteases of the serine, aspartyl, cysteine, and metalloprotease classes were identified. Several subtilisin-like prohormone convertases (PC1/3, PC2, PACE 4) were identified, which participate in proneuropeptide processing (5, 67–69). The cysteine protease cathepsin L has been identified by MS/MS tandem mass spectrometry utilizing enrichment prior to MS/MS (5, 8); cathepsin L participates in secretory vesicles for processing proneuropeptides for the production of enkephalin (5, 8), NPY (62), and POMC-derived peptide hormones consisting of β-endorphin, ACTH, and α-MSH (46). The cysteine protease cathepsin B was identified, which was recently discovered to participate in the production of neurotoxic beta-amyloid related to Alzheimer’s disease (45, 70). The aspartyl protease cathepsin D was also present [38]. Numerous metalloproteases were found including the ADAM metallopeptidase, and carboxypeptidases (D, E, and G2) (71–73). Components of the ubiquitin system for protein degradation were identified (74–76). In addition, endogenous protease inhibitors were indicated that included TIMP-1 (77), inhibitor of PC1/3 (78), and cystatins (79). These results indicate the presence of numerous protease and protease inhibitors in these secretory vesicles.

Proteins that participate in the biosynthesis and metabolism of small molecule neurotransmitters, as well as receptors, were identified. Catecholamine synthesizing enzymes were present that include tyrosine hydroxylase, dopamine beta-monooxygenase, and PNMT (phenylethanolamine N-methyltransferase) (80, 81). In addition, transporters for vesicular localization of catecholamines were identified (82–84). Interestingly, several receptor proteins were identified which may be present in secretory vesicles for transport to the plasma membrane [85].

Biochemical processes: enzymes, carbohydrate and lipid functions, protein folding, transporters

The secretory vesicles contained enzymes for a variety of biochemical reactions. Enzymes for numerous biochemical reactions were identified including aspartate aminotransferase for amino acid modification, cofactor related tetrahydrofolate synthase, and enolase (86–88). In addition, a number of carbohydrate and lipid metabolizing enzymes were identified (89–90). These included carbohydrate transferases, mannosidase, and glucosidase. Lipid-related enzymes included arachidonate lipoxygenase, phospholipase, and acyl-CoA synthetase.

Internal environment of secretory vesicles: reduction oxidation, ATPases and nucleotide metabolism

Homeostatic mechanisms for maintaining the unique internal conditions of the secretory vesicle require reduction-oxidation regulation, ATP/nucleotide related proteins for pH regulation, and protein factors for protein folding. Regulation of reducing and oxidative conditions is evident with the presence of cytochromes, perioxidase, catalase, and related proteins. Numerous membrane-associated ATPase isoforms were present which participate in proton transport that maintains the acidic internal pH (pH 5–6) of these secretory vesicles (54, 91–95). Conditions for appropriate protein folding or protein configuration are represented by chaperone proteins that include heat shock proteins, chaperonin, and isomerase (96–98). These regulators of the internal secretory vesicle environment are utilized for effective production of neurotransmitters, hormones, and neurohumoural factors in this organelle.

Regulated secretion: signal transduction and GTP-binding proteins, vesicular trafficking and exocytosis, calcium regulation

Significant representation of secretory vesicle proteins consisted of functions for regulated secretion involving signal transduction and GTP-binding proteins, proteins for exocytosis and vesicular trafficking, and calcium regulation. An extensive collection of Rab GTP-binding proteins (99–103) was present, which are critical for intracellular trafficking and transport of secretory vesicles and in vesicle exocytosis. Furthermore, numerous proteins involved in signal transduction pathways by protein kinases (104–107) and phosphatases (107–109) were identified. The presence of these proteins suggest regulation of the phosphorylation status of target proteins within secretory vesicles. The process of exocytosis of secretory vesicles for release of vesicle contents to the extracellular environment utilizes synaptotagmin isoforms and synaptophysin related proteins (110–112). Also, the presence of kinesin suggests its utilization by dense core secretory vesicles for trafficking to the plasma membrane via interactions with cellular structural proteins (113, 114). Notably, regulated secretion is calcium-dependent, which utilizes proteins that regulate calcium metabolism in secretory vesicles. These proteins include several isoforms of annexins which function in calcium-dependent phospholipid binding during exocytosis (105).

Morphological functions of secretory vesicles: structural proteins, cell adhesion and cell-cell interactions

Numerous structural proteins were identified including collagen, myosin, spectrin, proteoglycan, and tubulin which may function in morphological features of secretory vesicles (116, 117). Such cytoplasmic proteins are utilized for intracellular movement of the secretory vesicles to the plasma membrane for regulated secretion. For example, microtubule and myosin structural proteins may be involved in intracellular granule movement. Such cytoplasmic proteins may be linked to the chromaffin granule membrane through protein interactions, but would lack signal peptide sequences. In addition, several proteins involved in cell adhesion or cell-cell interactions were found, which included cadherin, integrin, laminin, and related components.

Potentially novel functional proteins of secretory vesicles

It was of interest that several identified proteins included those with functions in the immune system, cell growth and development, and transcription and translation. Several proteins with immune system functions were found including immunoglobulins and immunoglobulin receptor. Several studies have demonstrated nervous system stimulation of immunoglobulin secretion (118), as well as immunoglobulin receptor trafficking through regulated secretory vesicles (119). Recent studies have also indicated transport of chemokines in large dense core vesicles as mechanisms for secretion of cytokines (120, 121). Thus, dense core secretory vesicles may be involved in both the regulated secretion of immunological factors as well as neurohumoural factors, hormones, and neurotransmitters.

Proteins involved in translation of RNAs were identified such as ribosomal proteins and RNA-binding proteins. Recent studies have demonstrated localization and translation of mRNAs in axons (122–124), which occur in the vicinity of secretory vesicles that are transported to axons and nerve terminals. It is possible that components for mRNA translation may reside in subregions of the neuroendocrine cell where secretory vesicles undergo transport and trafficking for regulated secretion. An alternative possibility is that contaminating RNA granules may be present in the chromaffin granule preparation, but that is unlikely since RNA granule markers (FMRP, Pur alpha and beta, RACK1, S6, Staufen2, or Syncrip) (125) were not identified in this chromaffin granule proteomic study. Indeed, the purity of the isolated chromaffin granules has been established in prior studies with additional enzyme marker data in this study to demonstrate that chromaffin granules of high purity were utilized for this proteomic study.

In addition, several proteins representing transcription factors were identified. Thus far, little is known about the roles of such factors in secretory vesicles. Furthermore, several miscellaneous and unknown proteins were indicated from MS/MS data analyses.

Chromaffin granule proteins related to neurological diseases

Several key proteins involved in neurological disease mechanisms were present in these secretory vesicles. These vesicles contain several neurodegenerative disease related proteins consisting of the amyloid precursor protein (APP), huntingtin interacting protein, ataxin 7, CLN8 protein, and prion protein (Table 2). The amyloid precursor protein (APP) undergoes proteolytic processing to generate toxic beta-amyloid peptide, a neurotoxic factor involved in the development of Alzheimer’s disease (14–18, 39, 45, 70). Beta-amyloid peptide and proteases for its production have been demonstrated in chromaffin secretory vesicles (39, 45, 70). The protease inhibitor cystatin C is involved in epilepsy (126, 127). The huntingtin interacting protein (19–22) is known to bind to the mutant huntingtin (htt) protein with polyglutamine expansion of Huntington’s disease (128). The ataxin 7 protein is the product of the SCA7 gene that possesses polyGln expansions, representing a CAG triplet-repeat neurodegenerative disease (23–25). The CLN8 protein is involved in genetic EPMR syndrome for epilepsy and mental retardation as a mutant CLN8 autosomal recessive disorder (29–32). The mutant CLN8 gene represents one of several neuronal ceroid lipofuscinoses (NCLs) neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in various tissues. The prion protein is a key component of prion neurodegenerative diseases based on misfolding of the prion protein (26–28). It is notable that multiple factors known to participate in severe neurodegenerative diseases are present in chromaffin secretory vesicles.

Table 2.

Proteins Identified in Chromaffin Secretory Vesicles with Neurological and Neurodegenerative Disease Functions.

Protein	Neurological Disease
Amyloid precursor protein	Alzheimer’s Disease
Ataxin 7	Spinocerebellar Ataxia type 7
CLN8 protein	Neuronal Lipofuscinosis, EPMR, epilepsy and mental retardation
Cystatin C	Epilepsy
Huntingtin interacting protein 1	Huntington’s Disease
KIAA0319	Dyslexia
Nesprin-2	Muscular Dystrophy
P20-CGGBP	Fragile X Syndrome, mental retardation
Prion protein	Prion Disease
Regulatory factor X4 isoform c	Bipolar Disorder

Proteins were identified from chromaffin secretory vesicles by LC-MS/MS and subjected to bioinformatic analyses as described in the methods. Several proteins were found which are known to participate in several neurological and neurodegenerative disease conditions.

Furthermore, proteins related to several neurological diseases were identified (Table 2) as the P20-CGGBP protein of the fragile X syndrome for mental retardation (33), the regulatory factor X4 involved in bipolar disorder (34), the KIAA0319 protein involved in dyslexia (35), and nesprin-2 related to muscular dystrophy (129–131). Thus, the dense core secretory vesicle organelle contains several key proteins that participate in neurological diseases.

The presence of selected neurological disease proteins in chromaffin secretory vesicles were assessed by western blots of purified vesicles. Western blots (Fig. 5) show the presence of cystatin C of ~14 kDa (132, 133), huntingtin (htt) interacting protein as several bands of 150–250 kDa that interact with fragments of htt present in brain (48), ataxin 7 of ~98–100 kDa (49, 134), and prion protein of a main and of ~30–35 kDa (135, 136) combined with a 55–60 kDa band (a possible multimer form). Cellular immunofluorescence microscopy indicated the localization of cystatin C (Fig. 6), as example, in chromaffin secretory vesicles that contain the enkephalin peptide neurotransmitter (8). These data show that the LC-MS/MS results can appropriately indicate the presence of such proteins in chromaffin secretory vesicles.

Figure 5. Presence of cystatin C, huntingtin interacting protein, ataxin 7, and prion protein in chromaffin secretory vesicles demonstrated by western blots.

Purified chromaffin secretory vesicles were subjected to western blots for analyses of cystatin C (panel a), huntingtin interactin protein (HIP) (panel b), ataxin 7 (panel c), and prion protein (panel d). Cystatin C of ~12–14 kDa in these vesicles (panel a) is similar in MW (molecular weight) to that reported in other studies [132, 133]. Huntingtin-interacting proteins (3 bands) in the area of ~ 150–250 kDa were observed (panel b). Ataxin 7 of about 98–100 kDa (panel c) is similar to that found in prior studies [49]. Prion protein of several apparent molecular weights of ~30 kDa, 36–40 kDa, and 50–60 kDa were observed (lane d).

Figure 6. Cellular localization of cystatin C with enkephalin-containing secretory vesicles of chromaffin cells in primary culture.

The localization of cystatin C to secretory vesicles that contain the enkephalin neuropeptide was observed by immunofluorescence confocal microscopy of chromaffin cells primary culture. Colocalization of enkephalin (green fluorescence) and cystatin C (red fluorescence) was demonstrated by the yellow fluorescence of merged images. Examples of secretory vesicle colocalization of cystatin C and enkephalin are indicated by the arrows.

Number of proteins identified in distinct functional categories of chromaffin secretory vesicles

Comparisons of the numbers of proteins in each category showed the varying distributions of components within each category in chromaffin secretory vesicles (Table 3). The functional categories of these proteins represent the diverse biochemical systems utilized for secretory vesicle exocytosis of active molecules for cell-cell communication.

Table 3.

Total Number of Proteins Identified in the Soluble and Membrane Secretory Vesicle Compartments in Designated Functional Categories.

Protein Category	Soluble	Membrane
Neuropeptides/Neurohumoural	18	19
Protease Systems	26	21
Neurotransmitter Enzymes/Transporters	9	11
Receptors	14	11
Enzymes	13	11
Carbohydrate Functions	11	9
Lipid Functions	10	9
Reduction-Oxidation	7	7
ATPases/Nucleotide Metabolism	8	23
Protein Folding	2	10
Transporters (solute)	4	8
Signal Transduction/GTP-binding	65	76
Vesicular Trafficking and Exocytosis	17	15
Calcium Regulation	4	10
Structural Proteins	24	26
Cell Adhesion/Cell-Cell Interactions	12	10
Cell growth and development	2	1
Immune	11	2
Transcription/Translation	49	47
Miscellaneous	28	37
Unknown	37	15

Numbers of proteins found in each functional category for the soluble and membrane compartments of chromaffin secretory vesicles are indicated.

Conclusion: Proteomic data reveals that secretory vesicles utilize multiple categories of proteins utilized for production and release of neuroeffectors for cell-cell communication

Secretory vesicle biosynthesis, storage, and secretion of neuroeffector molecules

This study illustrates the most comprehensive proteomic data of chromaffin secretory vesicles that provide neuroeffectors mediating cell-cell communication from the adrenal medulla. Proteins of these secretory vesicles function in the biogenesis and maturation of secretory vesicles, synthesis and storage of bioactive molecules consisting of neuropeptides and catecholamines, and secretion of bioactive molecules which involves secretory vesicle transport and docking to the plasma membrane for regulated secretion (Fig. 7). These proteins provide the biochemical basis for regulated secretion of active neurotransmitter and neurohumoural agents for cell-cell communication. These data illustrate that the dense core secretory vesicles function with distinct functional protein categories. Production of peptide neurotransmitters and hormones, as well as catecholamine neuroeffectors, within the chromaffin secretory vesicles utilize proteins that control the intravesicular environment with respect to redox conditions, acidity (acid pH at 5.5–6.0), protein folding, as well as carabohydrate and lipid conditions. Transporters are needed for bringing certain neuroeffectors into the chromaffin granule. Regulated secretion of chromaffin granules requires extensive use of signal transduction and GTP-binding proteins, combined with proteins needed for secretory vesicle trafficking and exocytosis (including structural proteins for this purpose). Furthermore, because regulated secretion is calcium-dependent, calcium binding proteins are present to mediate regulated secretion. The proteomic data indicate that secretory vesicles utilize proteins of multiple functions for production, storage, and regulated secretion of neuroeffectors for cell-cell communication.

Figure 7. Multiple protein categories for biosynthesis, storage, and regulated secretion of neuroeffectors for cell-cell communication in health and disease.

The chromaffin granule proteome consists of distinct functional categories of proteins utilized for secretory vesicle production, storage, and regulated secretion of neuroeffector molecules. The architecture of proteins of the soluble (blue area) and membrane (gray area) function in the initial biogenesis and subsequent maturation of secretory vesicles, which produce and store bioactive chemical molecules for secretion. The secreted neurotransmitters and hormones mediate cell-cell communication among physiological target organs.

Complementary biochemical studies assist in substantiating proteomic data

An important criterion for proteomic studies is the subject of confidence and validation of protein identifications obtained from tandem mass spectrometry data. The unique feature of this proteomic study was consideration of protein biochemistry data from these secretory vesicles to guide and enhance appropriate confidence levels in bioinformatic analyses of MS/MS data for protein identification, including single peptide identifications (137). Protein biochemistry studies of chromaffin secretory vesicles provide information of previously identified proteins in this organelle at moderate and low levels; this information can assist in defining reliable scoring thresholds. For example, the serpin endopin 2C is of moderate abundance since it was isolated from these secretory vesicles with a 500-fold enrichment (43), and was observed in this study at intermediate peptide scoring thresholds for identification. Cathepsin B and cathepsin L are low abundance proteins, demonstrated by their enrichment requiring 2 × 10⁵-fold (45) and 2 × 10⁶ – fold purification (8), respectively; after enrichment of these proteins, they were identified with high confidence scoring MS/MS data (8, 45). Confidence levels of these MS/MS data are consistent with previous bioinformatic analyses of MS/MS data (137, 138). These types of biochemical studies assisted in guiding appropriate confidence levels for identification of proteins from tandem mass spectrometry data, including those identified by single peptide (tryptic) identification. Application of biochemical data for relative protein abundances enhances analyses of proteomic data.

Proteomic data of secretory vesicle proteins from adrenal medulla in this study and from other tissues in related studies

Important secretory mechanisms for the chromaffin secretory vesicles are indicated by the expansive nature of these proteomic studies that have identified 686 distinct proteins in chromaffin secretory vesicles. Other studies of regulated secretory vesicles have been reported for the dense core secretory vesicles (144–149) and synaptic vesicles (150–154) for investigation of their proteomic features. While it is of interest to compare the identified proteins among these studies, their different methodologies for purification of organelles and protein extraction conditions, combined with different mass spectrometry instrumentation and different bioinformatic database searches should be considered in details of such comparisons. Nonetheless, features of some of the prior proteomic studies of various secretory vesicle systems are summarized here. A proteomic study of pancreatic zymogen granule membranes yielded identification of 101 proteins by 2-D gel electrophoresis, in-gel trypsin digestion, followed by LC-MALDI (139). Another proteomic study of insulin secretory granules has identified 130 different proteins by SDS-PAGE, excision of gel slices, trypsin digestion, followed by nano-LC-ESI-MS/MS (140). Proteomic analyses of synaptic vesicles, which undergo regulated secretion, identified 410 proteins (141). Analyses of multiple organelles isolated from abundant rat liver yielded identification of proteins of the rough and smooth microsomes, and Golgi fractions of the rat secretory pathway; this study identified 1400 proteins that was possible with multiple fractions from an abundant tissue source (142). However, these rat liver organelle components represent the constitutive secretory pathway present in liver cells, which lack the regulated secretory pathway. It will be of interest for future joint efforts to compare different secretory vesicle systems in different cell types.

Summary

This proteomic study of chromaffin dense core secretory vesicles has provided new knowledge of the protein architecture of the regulated secretory vesicle system. The application of sensitive high throughput nano-HPLC Chip MS/MS to proteomic studies, combined with biochemical information, of the dense core secretory vesicle has revealed the presence of distinct functional protein categories for secretory vesicle production and secretion of bioactive molecules – neurotransmitters and hormones – that control physiological functions through cell-cell communication. Significantly, several proteins involved in neurodegenerative and neurological diseases were identified in these secretory vesicles, suggesting their involvement with the secretory vesicle protein systems in the regulation of cell-cell communication. Overall, this proteomic study has revealed an extensive group of functional protein systems in regulated dense core secretory vesicles for cell-cell communication in health and disease.

Abbreviations

ACTH

adrenocorticotropin hormone

ADAM

a disintegrin and metalloprotease

AEBSF

4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride

APP

amyloid precursor protein

CG

chromaffin granule

CGS

chromaffin granule soluble fraction

CGM

chromaffin granule membrane fraction

CHAPS

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

EPMR

epilepsy and mental retardation

FDR

false discovery rate

GEMSA

guanidinoethylmercaptosuccinic acid

GTP

guanosine triphosphate

MS

mass spectrometry

MS/MS

tandem mass spectrometry

α-MSH

α-melanocyte stimulating factor

nano-HPLC Chip MS/MS

nano-high pressure liquid chromatography Chip tandem mass spectrometry

NCL

neuronal ceroid lipofuscinoses

NPY

neuropeptide Y

PC

prohormone convertase

PMSF

phenylmethanesulphonylfluoride

POMC

proopiomelanocortin

PNMT

phenylethanolamine N-methyltransferase

TCEP

Tris-(2-carboxyethyl)-phosphine

TEM

transmission electron microscopy