A survey of membrane proteins in human serum.

Serum and membrane proteins are two of the most attractive targets for proteomic analysis. Previous membrane protein studies tend to focus on tissue sample, while membrane protein studies in serum are still limited. In this study, an analysis of membrane proteins in normal human serum was carried out. Nano-liquid chromatography-electrospray ionization mass spectrometry (NanoLC-ESI-MS/MS) and bioinformatics tools were used to identify membrane proteins. Two hundred and seventeen membrane proteins were detected in the human serum, of which 129 membrane proteins have at least one transmembrane domain (TMD). Further characterizations of identified membrane proteins including their subcellular distributions, molecular weights, post translational modifications, transmembrane domains and average of hydrophobicity, were also implemented. Our results showed the potential of membrane proteins in serum for diagnosis and treatment of diseases.

Introduction

As the most easily obtained sample from patients, blood plasma is the primary specimen used to diagnose and monitor many diseases. Because changes in certain components of the plasma are indicative of abnormalities in the body system, many researches have been carried out in search for disease biomarkers in the plasma since the early history of clinical study. After proteomics emerged as a major discipline at the end of the 20th century, plasma study was launched in this new research direction, which could potentially analyze hundreds of proteins at the same time, instead of the one-protein study allowed by traditional genetic/biochemical approach.

The plasma contains thousands of proteins, including those originating from most, if not all kinds of cells and tissues.1 The plasma is considered one of the most promising proteomes for proteomic research, and many proteomic techniques and methods have been utilized and modified to analyze the human plasma.2 In an early study of human plasma, Pieper et al could identify 325 proteins using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS).3 Advancements in the field of proteomics in the past decade have assisted plasma studies in both breadth and depth, exemplified by the forming of a human plasma Peptide Atlas by researchers participating in the Plasma Proteome Project of the Human Proteome Organization (HUPO).4 Comparative studies of the plasma have revealed many potential markers and some insights into the mechanisms of tissue specific diseases, such as cancers,5 Alzheimer’s disease,6 and myocardial infarction.7

Membrane proteomics is a highly focused branch of proteomics. Approximately 30% of proteins encoded by the mammalian genome are transmembrane proteins.8,9 Membrane proteins play an important part in many important cellular processes, including cross-membrane transportation, cell adhesion, signal transduction, immune response, etc.10 Due to their roles as transporters, receptors and structural proteins as well as their impacts on intracellular processes, they are the usual candidates for drug development: about 60% of approved drugs target membrane proteins.11 Many membrane proteins were also found to directly associate with various human diseases, such as Alzheimer’s disease, diabetes, Hodgkin’s disease and liver cirrhosis.12 Thus, researches on membrane proteins also hold promises for developments of diagnosis and disease treatment.

The recent studies on human and mammalian membrane proteins tend to focus on cell/tissue samples.13–15 Using carbonate extraction, trypsin digestion and NanoLC-MS/MS, a multilaboratory project has been found to profile membrane proteins from mouse liver.16,17 Another study based on similar approaches, more than 200 proteins were detected in pancreatic cancer cells.18 Furthermore, a large number (862) of membrane proteins were identified in the brain cortex.19 However, we have not found any independent research that focus only on membrane proteins in serum. As a subset of the plasma, serum contains thousands of proteins, including the proteins of membrane origin, even though their abundance can be low. In this study, some results of profiling and characterization of membrane proteins in human serum by using combination of NanoLC-ESI-MS/MS methods and bioinformatics tools are shown. The present study was approved by the Ethics Committee of the Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST).

Materials and Methods

Materials

Dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate, ammonium acetate, trypsin (proteomics sequencing grade), sodium bicarbonate and Triton X-100 were purchased from Sigma- Aldrich (St. Louis, MO, USA). Formic acid (FA) and triflouracetate (TFA) were obtained from Fluka (Fluka Chemie GmbH, Buchs, Switzerland). Acetonitrile (ACN, chromatogram grade) and other chemicals (analytical grade) were obtained from Barker (Pittsburgh, USA). The Bradford assay kit, Aurum serum protein mini kit, acrylamide, bis-acrylamide, urea, glycine, Tris, CHAPS, and SDS were purchased from Bio-Rad (Hercules, CA, USA). All equipment and standard reagents used directly should be clean as necessary.

Sample preparation

Sera from healthy middle-aged individuals (20–40 years old) were supplied by Bach Mai Hospital, Hanoi, Vietnam, and stored at −80 °C until analysis. After that, albumin and IgG were depleted from the samples using the Aurum serum protein mini kit (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, serum protein column was washed with 1 ml of serum protein binding buffer. For each sample, 60 μl of serum was diluted with 180 μl serum protein binding buffer and then, 200 μl of diluted serum was loaded onto the column. Subsequently, the depleted serum – unbound fraction was collected by centrifugation at 10,000 × g for 20 sec. The removal of albumin and IgG was evaluated by 12.6% SDS-PAGE.

In-solution trypsin digestion

The depleted sera containing approximately 30 μg of total protein were dried by vacuum centrifugation and then dissolved in 50 mM NH4HCO3. The samples were reduced by 10 mM dithiothreitol (DTT) at 56°C for 30 min and alkylated by 5 mM iodoacetamide (IAA) at room temperature for 1 hr. Proteins in the samples were digested using trypsin (Sequencing grade, Sigma-Aldrich, St. Louis, MO, USA) at ratio of 1:50 (w/w) enzyme to protein at 37 °C for 12 hrs. The digestion was stopped by formic acid with final concentration of 0.1%.

Nano two-dimensional chromatography and mass spectrometry

Tryptic peptides were dissolved in 0.1% formic acid and loaded onto Strong Cation Exchange (SCX) Chromatography Column (LC Packing, Dionex, The Netherlands) for separation in the first dimension. The second dimension was performed using a C18 Reversed Phase (RP) Column (GraceVydac, Hesperia, CA, USA) with mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in 85% acetonitrile (B). Peptides were eluted in a linear gradient from 0% to 100% mobile phase B at a flow rate of 0.2 μl/min for 90 min.

Tandem mass spectrometry analysis were performed using an ABI QSTAR®XL hybrid quadrupole/TOF MS/MS instrument (Applied Biosystems/MDS Sciex, Ontario, Canada) equipped with a nanoelectrospray source (Protana XYZ manipulator). Positive mode nanoelectrospray was generated from fused-silica PicoTip emitters with a 10 μm aperture (New Objective, Woburn, MA) at 2.5 kV. MS and MS/MS spectra were recorded and processed in IDA mode (Information Dependent Acquisition) controlled by Analyst QS software. The range of the MS full scan was from 200 to 1500 amu followed by MS/MS fragmentation of the three most intense precursor ions. The dynamic ion selection threshold for MS/MS experiments was set to 45 counts.

Identification of membrane proteins

The obtained MS and MS/MS spectra were searched against the NCBInr and the Swiss-Prot protein sequence database using Mascot™ V1.8 software (Matrix Science Ltd., London, UK). The parameters for searching were set as following: enzymatic digestion with trypsin with one potential missed cleavage; a peptide and fragment mass tolerance of ±0.5; carbamidomethyl (cysteine) as fixed modification; oxidation (methionine) as variable modification. Protein identifications were performed using a Mowse scoring algorithm with a confidence level of 95% and with at least two matched peptides. For further verification, proteins were validated using the open-source software MSQuant v 1.5 (http://msquant.sourceforge.net/).

Membrane proteins were sorted from total identified serum proteins based on UniProt protein database (http://www.uniprot.org). SOSUI prediction algorithm was used to predict transmembrane domains and average values of hydrophobicity of those membrane proteins.20

Results

Identification of membrane proteins in serum

The major aim of this study is to detect and profile membrane proteins from human serum. With the strategy and methods described above, 217 membrane proteins were detected from 2778 matched peptides (see Table 1, Supplementary Data). It is interesting to note that more than 90% of the identifications were based on 3 or more matched peptides. From the total of 217 identified membrane proteins, 129 proteins have at least one transmembrane domain (TMD) based on SOSUI prediction algorithm (see Table 1, Supplementary Data).

Evaluation of membrane protein molecular weight

The distribution of molecular weights of membrane proteins from serum was shown in Figure 1. Membrane proteins in serum have a wide range of molecular weights. Proteins with molecular weights from 100–200 kDa make up the largest group, accounting for 43% the total of 217 identified membrane proteins. The second most abundant is the group of proteins with molecular weights in the range of 200–300 kDa (26%). It is also notable that several membrane proteins (6%) have molecular weights above 500 kDa.

Figure 1

Distribution of molecular weights of membrane proteins found in human serum.

Note: Membrane protein identifications and molecular weights were obtained from MS and MS/MS data using Mascot software.

Transmembrane domains (TMDs) and average of hydrophobicity

TMD is one of the most distinguished features of membrane proteins. In our study, 129 (59.4%) proteins were predicted to have at least one TMD in the total of 217 membrane proteins according to SOSUI prediction algorithm. The majority of these 129 proteins have one TMD (25 proteins) and two TMDs (40 proteins), but a considerable number have more than 8 TMDs, which are often receptors, transporters or ion channels.

In this study, SOSUI prediction algorithm was also used to evaluate the hydrophobicity of membrane proteins from human serum. The average hydrophobicity values of all identified membrane proteins were calculated based on their amino acid sequences. It is notable that almost all proteins have average hydrophobicity values below zero. Only 17 proteins have positive average hydrophobicity values and most of them (16 proteins) are integral membrane proteins or transmembrane proteins. In addition, 7 UniProt annotated membrane proteins could not be analyzed by SOSUI prediction algorithm because their sizes have more than 5000 amino acids.

Subcellular distribution of membrane proteins

The subcellular distribution of 217 identified membrane proteins was determined using UniProt database according to their accession numbers. Analyzing the available information about the identified membrane proteins, we found that, these proteins have a certain distribution or are shuttled between organelles: 119 proteins are from plasma membrane and a range of proteins are from other cell components, such as nucleus (11 proteins), endoplasmic reticulum (16 proteins), Golgi apparatus (23 proteins), and mitochondrion (7 proteins).

Post-translational modifications of membrane proteins

In this study, 359 post-translational modifications (PTMs) of membrane proteins were found in 187 proteins based on UniProt database; the other 30 membrane proteins do not have post translational modifications. Among the total of membrane proteins having PTMs, proteins with one PTM (77 proteins – 41.2%) and two PTMs (66 proteins – 35.3%) are the majority, while proteins with 5 PTMs make up the smallest group (4 proteins – 2.1%).

Among 359 modifications found in the identified membrane proteins, phosphorylation was the most common modification, with 136 phosphoproteins. Follow-up was glycosylation, with 96 glycoproteins. The third and fourth common groups were proteins with dilsufide bonds (48 proteins) and acetylation sites (34 proteins). Lipoproteins, palmitoylated proteins, nitrated proteins, ubiquitinated proteins… contributed to a small fraction of proteins with modifications (Fig. 3).

Figure 3

Components of post translational modifications (PTMs) of membrane proteins in human serum.

Notes: A total of 359 PTMs were found in 217 identified membrane proteins. The most common modifications are phosphorylation and glycosylation.

Discussions

In membrane proteomics, former studies are often inclined to use tissue/cell samples. Using membrane protein extraction by organic acid and NanoLC-MS/MS, Da Cruz et al found 182 membrane proteins from mouse liver mitochondrial inner membrane.21 Another study used membrane proteins extraction from pancreatic cancer cells, SDS-PAGE, in-gel digestion and NanoLC-ESI-MS/MS and detected more than 200 proteins.18 In addition, a large number (862) of membrane proteins were found in the brain cortex.19 The difference in the number of detected membrane proteins from various tissue types can be due to the specificity of each tissue, as well as fractionation and enrichment methods of each study. Combining data from four different sources, an early survey of the plasma by Anderson et al found 212 proteins with at least one TMD with multiple origins,1 which is similar to our results. Probably, there might be more membrane proteins in the serum that have not been detected yet because of their low abundance compared to other serum proteins.

Phosphorylation and glycosylation are the two most common modifications of protein in animal cells. Phosphorylation modifies the structure and function of many proteins. An upset balance between phosphorylation and dephosphorylation is the cause of many diseases.22 Glycosylation was estimated to be found in over 50% of human proteins,23 and also of interest because some diseases were found to be related to glycan structural alterations.24,25 Our results with the high proportion of phosphoproteins and glycoproteins showed the potential of membrane proteins in serum for diagnosis and treatment of disease.

Conclusion

By using NanoLC-ESI-MS/MS technologies and bioinformatics tools, a data set of 217 membrane proteins from normal human serum was identified. Some characterization such as subcellular distribution, molecular weigh, post translational modification, transmembrane domain (TMD) and average of hydrophobicity of the identified proteins were also given. 129 proteins (59%) have at least one transmembrane domain, and 187 proteins (86%) have post-translational modifications.

Table 1

List of identified membrane proteins in human serum.

No.	Accession number	Protein name	Score	Peptide match	Molecular weight	Subcellular location	PTM	Hydrophobicity value	TMDs
1	Q8TC27	A disintegrin and metalloprotease domain 32	43	5	90328	Unclear	Disulfide bond, Glycoprotein, Phosphoprotein	−0.312452	2
2	Q9H2U9	A disintegrin and metalloproteinase 7	35	9	88036	Unclear	Disulfide bond, Glycoprotein	−0.414589	2
3	Q9BZC7	ABC transporter ABCA2	61	16	272140	Membrane	Glycoprotein, Phosphoprotein	−0.065216	14
4	Q8IZY2	ABC transporter ABCA7	46	6	236328	Plasma membrane, Endosome membrane, Golgi apparatus membrane	Disulfide bond, Glycoprotein	0.055080	12
5	Q99758	ABC3	41	23	192862	Plasma membrane	Phosphoprotein	0.089026	13
6	P00519	Abl protein	46	3	123852	Nucleus membrane	Lipoprotein, Myristate, Phosphoprotein	−0.597169	0
7	O00763	Acetyl-CoA carboxylase	60	6	281569	Endomembrane	Phosphoprotein	−0.227218	1
8	O00763	Acetyl-CoA carboxylase 2	54	6	278361	Endomembrane	Phosphoprotein	−0.227218	1
9	O14617	Adapter-related protein complex 3 delta 1 subunit variant	62	9	145094	Golgi apparatus membrane	Phosphoprotein	−0.491761	1
10	O95996	Adenomatosis polyposis coli 2	49	24	245966	Golgi apparatus membrane	Unknown	−0.529135	0
11	Q13813	Alpha II spectrin	70	17	285689	Extracellular space	Acetylation, Phosphoprotein	−0.790330	0
12	P02763	Alpha-1-acid Glycoprotein 1 precursor	90	37	23579	Unclear	Disulfide bond, Glycoprotein, Lipoprotein, Palmitate, Phosphoprotein	−0.535821	1
13	Q9UHC3	Amiloride-sensitive cation channel 3 isoform c	48	8	61376	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.265348	2
14	Q07837	Amino acid transport protein	56	11	79240	Plasma membrane	Disulfide bond, Glycoprotein	−0.441082	1
15	Q07075	Aminopeptidase A	50	15	109689	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.390491	1
16	Q5Y190	Anchor protein	53	7	458487	Plasma membrane	Unknown	−0.188033	20
17	Q96Q91	Anion exchanger AE4	47	4	105149	Plasma membrane	Glycoprotein, Phosphoprotein	0.143947	11
18	Q9P2R3	ANKHZN	54	7	129534	Endosome membrane	Acetylation	−0.154490	0
19	P02647	Apolipoprotein A-I preproprotein	245	45	30759	Plasma membrane	Glycation, Glycoprotein, Lipoprotein, Palmitate, Phosphoprotein	−0.717228	0
20	Q12797	Aspartyl(asparaginyl)beta-hydroxylase; HAAH	42	3	86294	Endoplasmic reticulum membrane	Glycoprotein, Phosphoprotein	−0.788128	1
21	Q86UQ4	ATP binding cassette, sub-family A (ABC1), member 13	50	12	580524	Plasma membrane	Unknown	−0.010508	14
22	Q96J66	ATP-binding cassette protein C11 isoform A	40	18	151566	Plasma membrane	Glycoprotein	0.191607	12
23	Q96J66	ATP-binding cassette transporter MRP8	47	5	155872	Plasma membrane	Glycoprotein	0.191607	12
24	Q9HC28	ATP-binding cassette, sub-family A, member 2 isoform b	77	39	275244	Lysosome membrane	Unknown	−0.064204	14
25	Q92887	ATP-binding cassette, sub-family C (CFTR/MRP), member 2	36	68	175237	Plasma membrane	Glycoprotein, Phosphoprotein	0.091068	13
26	Q9UMD9	Autoantigen	55	8	154929	Plasma membrane	Disulfide bond, Glycoprotein, Hydroxylation, Phosphoprotein	−0.573080	1
27	Q13563	Autosomal dominant polycystic kidney disease type II protein	47	4	110462	Endoplasmic reticulum membrane	Glycoprotein	−0.328512	8
28	P50851	Beige-like protein; CDC4L protein	57	18	320076	Plasma membrane	Acetylation, Phosphoprotein	−0.201817	0
29	Q76KP1	Beta-1,4-N-acetyl-galactosaminyl transferase 4	39	10	116954	Golgi apparatus membrane	Glycoprotein	−0.597691	0
30	Q01082	Beta-spectrin	43	10	275259	Plasma membrane	Acetylation, Glycoprotein, Phosphoprotein	−0.766116	0
31	Q01082	Beta-spectrin 2 isoform 2	39	8	251948	Plasma membrane	Acetylation, Glycoprotein, Phosphoprotein	−0.766116	0
32	Q15413	Brain ryanodine receptor	73	24	556937	Endoplasmic reticulum	Glycoprotein	−0.278727	5
33	O14514	Brain-specific angiogenesis inhibitor 1	51	14	176900	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.311806	8
34	O60241	Brain-specific angiogenesis inhibitor 2	70	11	174738	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.211924	9
35	O60242	Brain-specific angiogenesis inhibitor 3	59	7	176123	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.211863	9
36	Q9HCU4	Cadherin EGF LAG seven-pass G-type receptor 2	44	7	322214	Plasma membrane	Disulfide bond, Glycoprotein, Hydroxylation	−0.270751	8
37	Q9NYQ7	Cadherin EGF LAG seven-pass G-type receptor 3	51	10	362937	Plasma membrane	Disulfide bond, Glycoprotein, Hydroxylation	−0.271258	8
38	Q9H251	Cadherin-23	74	6	370095	Plasma membrane	Glycoprotein, Phosphoprotein	−0.142278	2
39	Q59FJ3	Calcium channel, voltage-dependent, N type, alpha 1B subunit	49	8	264553	Unclear	Unknown	−0.264964	12
40	Q9HCF6	Calcium-permeable store-operated channel TRPM3b	72	11	180008	Unclear	Unknown	−0.302195	6
41	Q08499	cAMP-specific phoshodiesterase HPDE4D3 variant	42	6	76872	Unclear	Phosphoprotein	−0.674043	0
42	Q96P48	Centaurin delta 2 isoform a variant	41	9	166159	Plasma membrane, Golgi apparatus, Membrane	Phosphoprotein	−0.422553	0
43	Q15700	Channel associated protein of synapse	35	8	97896	Plasma membrane	Lipoprotein, Palmitate, Phosphoprotein	−0.519310	0
44	Q96QT4	Channel-kinase 1	65	11	214583	Plasma membrane	Phosphoprotein	−0.270456	6
45	P51788	Chloride channel protein 2	41	8	99402	Plasma membrane	Unknown	0.132517	11
46	P00751	Complement factor B preproprotein	175	13	86847	Plasma membrane	Acetylation	−0.501047	0
47	P17927	Complement receptor 1	38	8	230417	Plasma membrane	Disulfide bond, Glycoprotein, Pyrrolidone carboxylic acid	−0.320010	3
48	P10606	COX5B	64	178	13914	Mitochondrion membrane	Acetylation	−0.297674	0
49	Q7Z407	CUB and Sushi multiple domains 3 isoform 1	36	13	414007	Plasma membrane	Disulfide bond, Glycoprotein	−0.260239	1
50	Q8IY37	DEAH (Asp-Glu-Ala-His) box polypeptide 37	40	4	130547	Unclear	Unknown	−0.415385	0
51	Q6IC98	Death-inducing-protein	49	14	66766	Mitochondrion membrane	Phosphoprotein	−0.431833	3
52	Q14185	Dedicator of cytokinesis 1	52	6	216208	Unclear	Phosphoprotein	−0.430617	0
53	Q5VWQ8	Disabled homolog 2 interacting protein isoform 2	37	8	118547	Plasma membrane	Phosphoprotein	−0.578302	0
54	O75923	Dysferlin isoform 8	43	4	239254	Vesicle	Phosphoprotein	−0.400145	1
55	P22413	Ectonucleotide pyrophosphatase/phosphodiesterase 1	36	6	107024	Plasma membrane	Disulfide bond, Glycoprotein	−0.456000	1
56	Q8WYP5	ELYS transcription factor-like protein TMBS62	43	4	258191	Nuclear membrane	Acetylation, Phosphoprotein	−0.448765	0
57	Q541P7	EPH receptor B4 precursor	35	4	109741	Plasma membrane	Unknown	−0.235562	2
58	Q15375	Ephrin receptor EphA7	55	10	113735	Plasma membrane	Glycoprotein, Phosphoprotein	−0.284268	2
59	Q15375	Ephrin receptor EphA7 variant	61	10	113994	Plasma membrane	Glycoprotein, Phosphoprotein	−0.284268	2
60	P54762	Ephrin receptor EphB1 precursor	45	20	111297	Plasma membrane	Glycoprotein, Phosphoprotein	−0.214431	2
61	P29323	Ephrin type-B receptor 2	36	11	119128	Plasma membrane	Lipoprotein, Myristate,Palmitate, Phosphoprotein	−0.293460	2
62	Q9NZJ5	Eukaryotic translation initiation factor 2-alpha kinase 3	48	11	126095	Endoplasmic reticulum membrane	Glycoprotein, Phosphoprotein	−0.450717	2
63	Q16099	Excitatory amino acid receptor 1; kainate receptor subunit EAA1	41	9	108529	Plasma membrane	Glycoprotein	−0.046653	4
64	Q86XX4	Extracellular matrix protein FRAS1	45	10	453936	Plasma membrane	Glycoprotein	−0.295608	2
65	Q9NYQ8	FAT tumor suppressor 2 precursor	44	7	482097	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.249645	2
66	Q6V0I7	Fat-like cadherin FATJ protein	43	10	354108	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.262940	2
67	Q9NZM1	Fer-1 like protein 3	66	11	234737	Nuclear membrane, plasma membrane	Acetylation, Phosphoprotein	−0.456091	1
68	Q59F30	Fibroblast growth factor receptor 4 variant	38	5	114772	Unclear	Unknown	0.017312	6
69	P42345	FK506 binding protein 12-rapamycin associated protein 1	40	10	290759	Golgi membrane, endoplasmic reticulum membrane, mitochondrial outer membrane	Acetylation, Phosphoprotein	−0.192586	0
70	Q9Y2I7	FYVE finger-containing phosphoinositide kinase	66	18	239581	Endosome membrane	Acetylation, Phosphoprotein	−0.534366	0
71	Q86SQ6	G protein-coupled receptor 123	35	6	139868	Plasma membrane	Glycoprotein	−0.317749	6
72	Q5T848	G protein-coupled receptor 158	53	8	136886	Plasma membrane	Glycoprotein, Isopeptide bond, Phosphoprotein, Ubl conjugation	−0.480988	7
73	O75899	GABBR2 protein	42	5	100861	Plasma membrane	Glycoprotein, Phosphoprotein	−0.149734	8
74	Q14789	Giantin	44	13	377273	Golgi apparatus membrane	Disulfide bond, Phosphoprotein	−0.936847	1
75	P42261	Glutamate receptor type 1	42	7	102270	Plasma membrane, Endoplasmic reticulum membrane	Glycoprotein, Lipoprotein, Palmitate, Phosphoprotein	−0.199227	5
76	Q9Y3R0	Glutamate receptor-interacting protein 1	46	5	123203	Plasma membrane, Endoplasmic reticulum membrane	Phosphoprotein	−0.362500	0
77	Q14789	Golgi antigen gcp372	44	8	373440	Golgi apparatus membrane	Disulfide bond, Phosphoprotein	−0.936847	1
78	Q08378	Golgi autoantigen, golgin subfamily a, 3	69	17	167765	Golgi apparatus membrane	Acetylation, Phosphoprotein	−0.834781	0
79	Q08378	Golgin-160	57	16	167810	Golgi apparatus membrane	Acetylation, Phosphoprotein	−0.834781	0
80	Q6PRD1	GPR158-like 1 receptor	54	11	260635	Plasma membrane	Glycoprotein	−0.583101	7
81	Q8WXG9	G-protein coupled receptor 98	69	21	694181	Plasma membrane	Unknown	Too long	0
82	Q8IWJ2	GRIP and coiled-coil domain-containing 2 isoform a	53	15	196873	Golgi apparatus membrane	Phosphoprotein	−0.918825	0
83	O15068	Guanine nucleotide exchange factor DBS	37	4	129340	Plasma membrane	Phosphoprotein	−0.552067	0
84	P52272	Heterogeneous nuclear ribonucleoprotein M isoform a	44	13	77749	Plasma membrane	Acetylation, Phosphoprotein, Ubl conjugation	−0.341643	0
85	O43166	High-risk human papilloma viruses E6 oncoproteins targeted protein E6TP1 alpha; putative GAP protei	59	12	198570	Plasma membrane	Phosphoprotein	−0.671840	0
86	P35523	Human ClC-1 muscle chloride channel	37	7	109696	Plasma membrane	Unknown	0.034919	11
87	Q14643	Human type 1 inositol 1,4,5-trisphosphate receptor	43	8	309942	Endoplasmic reticulum membrane	Glycoprotein, Phosphoprotein	−0.315448	6
88	Q9HCF6	Hypothetical protein	72	10	175211	Unclear	Unknown	−0.300924	6
89	Q9P1Z9	Hypothetical protein	43	14	199652	Unclear	Phosphoprotein	−0.647996	2
90	A6NI73	Immunoglobulin-like transcript 11 protein	39	4	27186	Unclear	Disulfide bond, Glycoprotein	−0.267224	2
91	Q14571	Inositol 1,4,5-trisphosphate receptor type 2	50	6	311074	Plasma membrane	Acetylation, Phosphoprotein	−0.266755	7
92	Q6P9B9	Integrator complex subunit 5	38	5	109239	Nucleus membrane	Phosphoprotein	0.109029	2
93	Q15811	Intersectin long form	40	7	196293	Plasma membrane	Acetylation, Phosphoprotein	−0.668043	0
94	Q9H4E7	IRF4-binding protein	48	7	74422	Plasma membrane	Acetylation, Phosphoprotein	−0.946435	0
95	Q92508	KIAA0233	50	17	234888	Endoplasmic reticulum membrane	Glycoprotein	−0.002357	23
96	Q13023	KIAA0311	40	7	259702	Nucleus membrane	Unknown	−0.675982	0
97	Q96P48	KIAA0782 protein	45	4	145325	Plasma membrane, Golgi apparatus membrane	Phosphoprotein	−0.422553	0
98	Q9Y2H9	KIAA0973 protein	61	34	173192	Plasma membrane	Phosphoprotein	−0.569363	0
99	Q96RV3	KIAA0995 protein	42	7	109401	Plasma membrane	Glycoprotein	−0.284538	13
100	Q9BZ29	KIAA1058 protein	42	7	241774	Plasma membrane	Phosphoprotein	−0.349204	0
101	Q5T848	KIAA1136 protein	49	11	66642	Plasma membrane	Glycoprotein, Isopeptide bond, Phosphoprotein, Ubl conjugation	−0.480988	7
102	Q5T4S7	KIAA1307 protein	53	7	188524	Unclear	Acetylation, Phosphoprotein	Too long	0
103	Q9BZ72	KIAA1457 protein	43	11	151395	Plasma membrane	Phosphoprotein	−0.405561	0
104	Q9P1Z9	KIAA1529 protein	46	6	196058	Unclear	Phosphoprotein	−0.647996	2
105	Q6KCM7	KIAA1896 protein	45	7	63937	Mitochondrial membrane	Unknown	−0.099147	0
106	Q16787	Laminin alpha 3 subunit isoform 1	45	7	375652	Unclear	Disulfide bond, Glycoprotein	−0.381368	0
107	P11047	Laminin B2 chain	54	7	183195	Unclear	Disulfide bond, Glycoprotein	−0.615042	0
108	Q9Y6N6	Laminin gamma 3 chain precursor	40	6	177756	Unclear	Disulfide bond, Glycoprotein	−0.465018	0
109	Q13449	LAMP	41	13	37798	Plasma membrane	Disulfide bond, GPI-anchor, Glycoprotein, Lipoprotein, Phosphoprotein	−0.278106	2
110	P42704	Leucine-rich PPR-motif containing protein	52	7	146306	Nuclear inner membrane	Acetylation	−0.206097	0
111	Q86UK5	Limbin	41	5	148825	Plasma membrane	Glycoprotein	−0.445185	2
112	P50851	Lipopolysaccharide-responsive and beige-like anchor protein	63	10	321639	Plasma membrane	Acetylation, Phosphoprotein	−0.201817	0
113	Q9HCF6	Long transient receptor potential channel 3	75	10	196157	Plasma membrane	Unknown	−0.300924	6
114	Q9NZR2	Low density lipoprotein receptor related protein-deleted in tumor	51	9	534844	Plasma membrane	Disulfide bond, Glycoprotein	−0.489888	2
115	Q5JRA6	Melanoma inhibitory activity family, member 3	52	7	214255	Endoplasmic reticulum membrane	Glycoprotein, Phosphoprotein	−0.887466	3
116	Q86UL8	Membrane associated guanylate kinase, WW and PDZ domain containing 2	39	5	159454	Plasma membrane	Phosphoprotein	−0.668455	0
117	Q13421	Mesothelin	45	4	68652	Plasma membrane	Cleavage on pair of basic residues, GPI-anchor, Glycoprotein, Lipoprotein	−0.105714	2
118	A1L467	Met proto-oncogene isoform b precursor	48	6	157779	Plasma membrane	Unknown	−0.144388	2
119	Q8IWA4	Mitofusin 1 precursor	38	10	84892	Mitochondrial membrane	Unknown	−0.267476	2
120	Q6UVY6	Monooxygenase X	36	9	63474	Endoplasmic reticulum membrane	Disulfide bond, Glycoprotein	−0.260848	2
121	Q8WXI7	Mucin 16	38	7	747071	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	Too long	1
122	O75970	Multiple PDZ domain protein	60	7	222792	Plasma membrane	Phosphoprotein	−0.233203	0
123	Q9NZM1	Myoferlin	42	6	231092	Vesicle membrane, nuclear membrane, plasma membrane	Acetylation, Phosphoprotein	−0.456091	1
124	B2RTY4	Myosin-IXa	54	9	294918	Unclear	Phosphoprotein	−0.623980	0
125	Q8NFP9	Neurobeachin	53	6	330066	Plasma membrane	Phosphoprotein	−0.188358	2
126	P21359	Neurofibromin isoform 1	43	10	322760	Plasma membrane	Disulfide bond,GPI-anchor, Glycoprotein, Lipoprotein	−0.136950	4
127	Q9H2E3	Neuropilin-2b(5)	53	6	102975	Plasma membrane	Unknown	−0.440066	2
128	Q13423	Nicotinamide nucleotide transhydrogenase	85	9	114564	Mitochondrial membrane	Acetylation	0.299079	13
129	Q59GR1	Niemann-Pick disease, type C1 variant	36	5	145931	Plasma membrane	Unknown	0.216447	13
130	Q13224	N-methyl-D-aspartate receptor subunit NR3	53	16	168067	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.388342	6
131	Q13813	Nonerythroid alpha-spectrin	59	7	284905	Unclear	Acetylation, Phosphoprotein	−0.790330	0
132	Q9UM47	Notch homolog 3	43	8	256640	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.304826	3
133	Q5STG5	Notch homolog 4 (Drosophila)	51	7	221594	Unclear	Unknown	−0.287183	3
134	P46531	Notch1 preproprotein	38	11	286350	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.420119	3
135	Q92823	NrCAM protein	37	11	144138	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.379601	2
136	Q8WXH0	NUANCE	104	29	801683	Nuclear membrane	Acetylation, Phosphoprotein	Too long	0
137	Q5VU65	Nucleoporin 210 kDa-like isoform 1	43	10	211668	Unclear	Glycoprotein	0.050265	3
138	Q13017	p190-B	45	6	173000	Unclear	Nitration, Phosphoprotein	−0.592345	0
139	Q5T4S7	p600	121	21	580607	Unclear	Acetylation, Phosphoprotein	Too long	0
140	O60245	PCDH7 (BH-Pcdh)c	40	4	130999	Plasma membrane	Glycoprotein, Phosphoprotein	−0.390365	2
141	Q9BXM0	Periaxin	59	8	155248	Plasma membrane	Phosphoprotein	−0.225736	0
142	O75747	Phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing gamma polypeptide	38	9	167814	Unclear	Phosphoprotein	−0.322423	1
143	O00443	Phosphoinositide 3-kinase	43	9	192271	Plasma membrane	Acetylation, Phosphoprotein	−0.302136	0
144	O00750	PI-3 kinase	46	5	184101	Plasma membrane	Phosphoprotein	−0.363037	0
145	Q6T4P5	Plasticity-related protein 2	43	12	79523	Membrane	Glycoprotein	−0.178273	6
146	O75051	Plexin A2	37	13	214220	Cell membrane	Glycoprotein, Phosphoprotein	−0.181468	3
147	Q9Y4D7	Plexin-D1	48	9	215381	Cell membrane, Membrane	Glycoprotein	−0.159013	2
148	Q15142	Polycystic kidney disease-associated protein	57	5	396273	Integral to membrane	Unknown	0.022760	14
149	Q8TDX9	Polycystin-1 L1	35	4	319453	Integral to membrane	Glycoprotein	−0.136856	7
150	P13942	Pro-a2(XI)	51	10	160545	Cell membrane	Disulfide bond, Glycoprotein, Sulfation	−0.805186	0
151	P08123	Pro-alpha 2(I) collagen	51	9	129858	Plasma membrane	Glycoprotein, Hydroxylation, Pyrrolidone carboxylic acid	−0.648098	0
152	P02647	Proapolipoprotein	664	75	28944	Plasma membrane	Glycation, Glycoprotein, Lipoprotein, Palmitate, Phosphoprotein	−0.717228	0
153	Q07954	Prolow-density lipoprotein receptor-related protein 1	66	13	523119	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.510386	2
154	Q05655	Protein kinase C-delta 13	37	4	78624	Plasma membrane	Phosphoprotein	−0.377958	0
155	Q8TF72	Protein Shroom 3	41	8	218125	Plasma membrane	Phosphoprotein	−0.801652	0
156	Q96QU1	Protocadherin 15	59	13	217261	Plasma membrane	Phosphoprotein	−0.245473	2
157	Q96QU1	Protocadherin 15 isoform CD1-4 precursor	48	12	217303	Plasma membrane	Phosphoprotein	−0.245473	2
158	Q9UN70	Protocadherin 43	45	5	98230	Plasma membrane	Glycoprotein	−0.261670	2
159	Q14517	Protocadherin Fat 1	52	9	509384	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.294946	2
160	Q9NYQ8	Protocadherin Fat 2	59	7	482172	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein	−0.249645	2
161	Q86V60	PTPRM protein	39	8	158434	Unclear	Unknown	−0.385540	1
162	Q8WZA2	RAPGEF4 protein	53	13	100339	Unclear	Phosphoprotein	−0.333927	0
163	P23467	Receptor-type tyrosine-protein phosphatase beta	50	26	225497	Plasma membrane	Glycoprotein, Phosphoprotein	−0.372159	2
164	Q13332	Receptor-type tyrosine-protein phosphatase S	43	9	218155	Plasma membrane	Disulfide bond, Glycoprotein	−0.401798	2
165	Q86UR5	Regulating synaptic membrane exocytosis 1	40	6	190154	Plasma membrane	Phosphoprotein	−0.922459	0
166	Q5T4S7	Retinoblastoma-associated factor 600	124	20	580547	Unclear	Acetylation, Phosphoprotein	Too long	0
167	Q5T5U3	Rho GTPase-activating protein 21	41	8	218567	Golgi apparatus membrane, vesicle membrane	Phosphoprotein	−0.827338	0
168	Q13464	Rho-associated, coiled-coil containing protein kinase 1	48	6	159102	Golgi apparatus membrane	Unknown	−0.909454	0
169	Q5T5U3	Rho-GTPase activating protein 10	41	5	218563	Golgi apparatus membrane, vesicle membrane	Phosphoprotein	−0.827338	0
170	Q5JTH9	Ribosomal RNA processing 12 homolog (S. cerevisiae)	56	9	145067	Nuclear membrane	Phosphoprotein	−0.225366	1
171	Q86UR5	RIM long form	48	7	164927	Plasma membrane	Phosphoprotein	−0.922459	0
172	P21817	Ryanodine receptor 1	61	11	55204	Endoplasmic reticulum membrane	Glycoprotein, Phosphoprotein	−0.314300	7
173	Q92736	Ryanodine receptor 2	70	10	569626	Plasma membrane	Glycoprotein, Phosphoprotein	−0.324300	7
174	Q15413	Ryanodine receptor 3	71	24	557794	Endoplasmic reticulum membrane	Glycoprotein	−0.278727	5
175	O15027	SEC16 homolog A	36	5	115891	Endoplasmic reticulum membrane, Golgi apparatus membrane	Acetylation, Phosphoprotein	−0.606607	0
176	A4QN19	SEC16A protein	40	2	116196	Endoplasmic reticulum membrane, Golgi apparatus membrane	Acetylation, Phosphoprotein	−0.606607	0
177	Q9H2E6	Sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A	36	4	115608	Plasma membrane	Disulfide bond, Glycoprotein	−0.448543	2
178	O94921	Serine/threonine protein kinase PFTAIRE-1	41	8	53609	Plasma membrane	Phosphoprotein	−0.470362	0
179	Q8NGB0	Seven transmembrane helix receptor	49	10	157779	Unclear	Unknown	−0.275069	6
180	Q9Y566	SH3 and multiple ankyrin repeat domains protein 1	49	7	225738	Plasma membrane	Phosphoprotein	−0.539239	0
181	Q9UPX8	SH3 and multiple ankyrin repeat domains protein 2	54	6	135115	Plasma membrane	Glycoprotein, Phosphoprotein	−0.608572	0
182	Q7Z5N4	Sidekick-like protein 1	39	4	242926	Plasma membrane	Disulfide bond, Glycoprotein	−0.314144	1
183	P21817	Skeletal muscle ryanodine receptor	78	9	570517	Plasma membrane	Glycoprotein, Phosphoprotein, S-nitrosylation	Too long	0
184	O94813	Slit homolog 2	47	13	175803	Plasma membrane	Disulfide bond, Glycoprotein	−0.323414	0
185	O94813	SLIT2	40	7	175329	Plasma membrane	Disulfide bond, Glycoprotein	−0.323414	0
186	Q9C0H9	SNAP-25-interacting protein	42	6	127182	Plasma membrane	Phosphoprotein	−0.644456	0
187	Q9Y6M7	Sodium bicarbonate cotransporter2	44	8	115215	Plasma membrane	Glycoprotein, Phosphoprotein	−0.130725	10
188	Q53ZR1	Solute carrier family 12 (sodium/potassium/chloride transporters), member 2	64	13	132048	Plasma membrane	Unknown	0.069720	11
189	Q9HBR0	Solute carrier family 38, member 10 isoform b	40	5	84069	Unclear	Phosphoprotein	−0.363718	11
190	Q9NRC6	Spectrin beta chain, brain 4	72	14	419259	Unclear	Unknown	−0.576588	0
191	Q01082	Spectrin, beta, non-erythrocytic 1 isoform 1 variant	48	8	276797	Plasma membrane	Acetylation, Glycoprotein, Phosphoprotein	−0.766116	0
192	Q8WWQ8	Stabilin 2 precursor	57	9	288323	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein, Proteoglycan	−0.268563	2
193	Q8WWQ8	Stabilin-2	46	9	288329	Plasma membrane	Disulfide bond, Glycoprotein, Phosphoprotein, Proteoglycan	−0.268563	2
194	Q8IVL0	Steerin3 protein	37	5	256885	Nuclear membrane	Phosphoprotein	−0.638784	0
195	Q4LDE5	Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1	52	10	401915	Unclear	Disulfide bond, Glycoprotein	−0.320528	1
196	Q9Y4G6	Talin-2	50	11	273723	Plasma membrane	Acetylation, Phosphoprotein	−0.218412	0
197	Q9H2K2	Tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 2	41	4	128492	Golgi apparatus membrane	ADP-ribosylation	−0.317066	0
198	O60343	TBC1 domain family, member 4	36	5	148068	Plasma membrane	Phosphoprotein	−0.553083	1
199	Q15643	Thyroid receptor-interacting protein 11	38	7	228184	Golgi apparatus membrane	Phosphoprotein	−0.843506	0
200	P12270	Tpr	54	10	265840	Nuclear membrane	Acetylation, Phosphoprotein	−0.968726	0
201	Q9BX84	Transient receptor potential cation channel subfamily M member 6 variant c	79	20	232975	Unclear	Phosphoprotein	−0.369882	5
202	Q8N6Q1	Transmembrane and coiled-coil domains 5A	47	5	34495	Unclear	Unknown	−0.563888	1
203	O95271	TRF1-interacting ankyrin-related ADP-ribose polymerase	47	12	143690	Golgi apparatus membrane	ADP-ribosylation, Phosphoprotein	−0.327506	0
204	Q15643	Trip230	92	12	228116	Unclear	Phosphoprotein	−0.843506	0
205	Q9BX84	Truncated transient receptor potential cation channel subfamily M member 6 variant a	57	13	224977	Plasma membrane	Phosphoprotein	−0.369882	5
206	Q8NFA0	Ubiquitin specific protease 32	47	8	183821	Unclear	Lipoprotein, Phosphoprotein, Prenylation	−0.474689	0
207	P55916	Uncoupling protein 3 isoform UCP3S	36	15	30219	Mitochondrion membrane	Unknown	0.074679	0
208	P17927	Unnamed protein product	41	10	230401	Plasma membrane	Disulfide bond, Glycoprotein, Pyrrolidone carboxylic acid	−0.320010	3
209	P53420	Unnamed protein product	65	10	126774	Vesicle membrane	Acetylation, Phosphoprotein	−0.659231	1
210	P11717	Unnamed protein product	48	14	281089	Plasma membrane	Acetylation, Disulfide bond, Glycoprotein, Phosphoprotein	−0.368528	3
211	Q6ZUB1	Unnamed protein product	36	10	158870	Unclear	Glycoprotein	−0.709205	1
212	O75110	Unnamed protein product	39	10	108198	Unclear	Phosphoprotein	0.062178	9
213	Q9NRW7	Vacuolar protein sorting	41	6	65388	Endosome membrane, Golgi apparatus membrane	Unknown	−0.310526	0
214	Q86Y38	Xylosyltransferase I	41	12	108357	Endoplasmic reticulum membrane, Golgi apparatus membrane	Disulfide bond, Glycoprotein	−0.540564	0
215	Q13433	Zinc transporter ZIP6	41	5	84685	Plasma membrane	Glycoprotein, Phosphoprotein	−0.421987	6
216	Q13439	256 kD golgin	59	9	256666	Golgi apparatus membrane	Phosphoprotein	−1.052981	0
217	Q8N1I0	Dedicator of cytokinesis protein 4	49	11	225206	Endomembrane	Phosphoprotein	−0.371464	0