Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

The amplified peptidome: the new treasure chest of candidate biomarkers

Mass spectrometric analysis of the low-molecular weight (LMW) range of the serum/plasma proteome is revealing the existence of large numbers of previously unknown peptides and protein fragments predicted to be derived from low-abundance proteins. This raises the question of why such low abundance molecules would be retained at detectable levels in the circulation, instead of being rapidly cleared and excreted. Theoretical models of biomarker production and association with serum carrier proteins have been developed to elucidate the mechanisms governing biomarker half-life in the bloodstream. These models predict that the vast majority of LMW biomarkers exist in association with circulating high molecular mass carrier proteins. Moreover, the total serum/plasma concentration of the biomarker is largely determined by the clearance rate of the carrier protein, not the free-phase biomarker clearance itself. These predictions have been verified experimentally using molecular mass fractionation of human serum before mass spectrometry sequence analysis. These principles have profound implications for biomarker discovery and measurement.     

Human plasma proteome analysis by reversed sequence database search and molecular weight correlation based on a bacterial proteome analysis

In shotgun proteomics, proteins can be fractionated by 1-D gel electrophoresis and digested into peptides, followed by liquid chromatography to separate the peptide mixture. Mass spectrometry generates hundreds of thousands of tandem mass spectra from these fractions, and proteins are identified by database searching. However, the search scores are usually not sufficient to distinguish the correct peptides. In this study, we propose a confident protein identification method for high-throughput analysis of human proteome. To build a filtering protocol in database search, we chose Pseudomonas putida KT2440 as a reference because this bacterial proteome contains fewer modifications and is simpler than the human proteome. First, the P. putida KT2440 proteome was filtered by reversed sequence database search and correlated by the molecular weight in 1-D-gel band positions. The characterization protocol was then applied to determine the criteria for clustering of the human plasma proteome into three different groups. This protein filtering method, based on bacterial proteome data analysis, represents a rapid way to generate higher confidence protein list of the human proteome, which includes some of heavily modified and cleaved proteins.     

Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery

The low molecular weight plasma proteome and its biological relevance are not well defined; therefore, experiments were conducted to directly sequence and identify peptides observed in plasma and serum protein profiles. Protein fractionation, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) profiling, and liquid-chromatography coupled to MALDI tandem mass spectrometry (MS/MS) sequencing were used to analyze the low molecular weight proteome of heparinized plasma. Four fractionation techniques using functionally derivatized 96-well plates were used to extract peptides from plasma. Tandem TOF was successful for identifying peptides up to m/z 5500 with no prior knowledge of the sequence and was also used to verify the sequence assignments for larger ion signals. The peptides (n>250) sequenced in these profiles came from a surprisingly small number of proteins (n approximately 20), which were all common to plasma, including fibrinogen, complement components, antiproteases, and carrier proteins. The cleavage patterns were consistent with those of known plasma proteases, including initial cleavages by thrombin, plasmin and complement proteins, followed by aminopeptidase and carboxypeptidase activity. On the basis of these data, we discuss limitations in biomarker discovery in the low molecular weight plasma or serum proteome using crude fractionation coupled to MALDI-MS profiling.     

液质联用多反应监测法定量目标多肽或蛋白质

为建立优化的血浆内源性多肽提取方法,并且构建目标多肽和蛋白质的质谱定量方 法,本研究考察了超滤法、有机溶剂沉淀法和固相萃取法对血浆内源性多肽的提取效果 ,并通过Tricine-SDS-PAGE对提取效果进行比较.通过液相色谱串联质谱多反应监测 （MRM）分析,建立了多肽标准品ESAT-6定量方法,并将ESAT-6定量建立的液相色谱和质谱条件应用于蛋白质的定量,对多肽和蛋白质MRM定量的标准曲线进行了考 察.Tricine-SDS-PAGE结果表明,乙腈沉淀法是最佳的血浆内源性多肽提取方法,低分子量的多肽可以得到很好的富集,且能有效地去除高分子蛋白质的污染.液相色谱串联 质谱MRM法检测血浆内提取的多肽,标准曲线的线性较好,相关系数为0.999.另外,采 用MRM法对胶内分离的蛋白质进行定量,标准曲线的线性相关系数为0.995.综上所述, 本研究构建了一种简单有效的血浆多肽提取方法,通过液质联用MRM法成功地实现了目标多肽和蛋白质定量测定.该定量方法可以推广应用于复杂样品中的多肽和蛋白质的定 量分析.     

Analysis of the human serum proteome

Changes in serum proteins that signal histopathological states, such as cancer, are useful diagnostic and prognostic biomarkers. Unfortunately, the large dynamic concentration range of proteins in serum makes it a challenging proteome to effectively characterize. Typically, methods to deplete highly abundant proteins to decrease this dynamic protein concentration range are employed, yet such depletion results in removal of important low abundant proteins. A multi-dimensional peptide separation strategy utilizing conventional separation techniques combined with tandem mass spectrometry (MS/MS) was employed for a proteome analysis of human serum. Serum proteins were digested with trypsin and resolved into 20 fractions by ampholyte-free liquid phase isoelectric focusing. These 20 peptide fractions were further fractionated by strong cation-exchange chromatography, each of which was analyzed by microcapillary reversed-phase liquid chromatography coupled online with MS/MS analysis. This investigation resulted in the identification of 1444 unique proteins in serum. Proteins from all functional classes, cellular localization, and abundance levels were identified. This study illustrates that a majority of lower abundance proteins identified in serum are present as secreted or shed species by cells as a result of signalling, necrosis, apoptosis, and hemolysis. These findings show that the protein content of serum is quite reflective of the overall profile of the human organism and a conventional multidimensional fractionation strategy combined with MS/MS is entirely capable of characterizing a significant fraction of the serum proteome. We have constructed a publicly available human serum proteomic database (http://bpp.nci.nih.gov) to provide a reference resource to facilitate future investigations of the vast archive of pathophysiological content in serum. These authors contributed equally to this work.     

Effect of immunoaffinity depletion of human serum during proteomic investigations

Controversy exists regarding the proper mining of the human serum proteome. Because of the analytical challenges of accurately measuring samples containing a very large dynamic range of protein concentrations, current practices have employed depletion of the highly abundant housekeeping serum proteins, such as albumin and immunoglobins. There is question as to the selectivity of depletion, namely, is there loss of other non abundant serum proteins along with albumin, haptoglobin and other commonly depleted proteins. In this study, human serum was analyzed with and without immunoaffinity depletion of the six most abundant proteins by multidimensional liquid chromatography tandem mass spectrometry. Two replicates of each experiment were conducted and compared against one another. In both depleted and nondepleted replicates there was a 73% and 72% overlap of identified peptides and a 64% and 78% overlap of identified proteins, respectively. Of 262 unique proteins identified in the four experiments, 82 were found in common to all four experiments, 142 unique to the depleted serum, and 38 unique to the nondepleted serum. Although serum depletion of highly abundant proteins significantly increased the number of proteins identified, both the degree of sample complexity and this depletion method resulted in a nonselective loss of other proteins.     

Analysis of low-abundance, low-molecular-weight serum proteins using mass spectrometry.

To detect diseases early in the general population, new diagnostic approaches are needed that have adequate sensitivity and specificity. Recent studies have used mass spectrometry to identify a serum proteomic pattern for breast and ovarian cancer. Serum contains 60-80 mg/mL protein, but 57-71% of this is serum albumin, and 8-26% are gamma-globulins. These large proteins must be depleted before smaller, less-abundant proteins can be detected using mass spectrometry, but because serum albumin is known to act as a carrier for smaller proteins, removal of these molecules using columns or filtration may result in the loss of molecules of interest. The objective of this study was to develop a reproducible method to deplete serum samples of high-abundance proteins in order to analyze the less-abundant proteins present in serum. We used organic solvents to precipitate the large proteins out of solution. We also predicted that this would cause many smaller proteins to dissociate from their carrier molecules, allowing for detection of a larger number of peptides and small proteins. These treated samples were analyzed using capillary liquid chromatography coupled with electrospray ionization mass spectrometry. Analysis demonstrated reproducible results. Acetonitrile treatment clearly released many carrier-bound molecular species and was superior to ultrafiltration alone for serum proteomic analysis.     

Comparison of blood serum peptide enrichment methods by Tricine SDS-PAGE and mass spectrometry

Characterisation of blood serum peptides can provide valuable information on physiological and pathological processes. However, the analysis of raw serum samples by MS results in the identification of a limited number of peptides. In order to improve sensitivity, many peptide enrichment methods have been proposed during the last ten years. Here, we present a comparison of fractionation methods aimed to simplify analysis of small proteins and peptides in blood serum, one of the most promising sources of putative biomarkers. Specifically, three methods based on ultrafiltration, differential precipitation, and peptide ligand libraries (ProteoMiner) were evaluated for the enrichment of peptides and low molecular weight proteins, as demonstrated by Tricine SDS-PAGE and subsequent LC-MS/MS (GeLC-MS/MS). As a result, differential solubilisation (DS) allowed the identification of the highest number of peptides. Moreover, the DS method enabled also the quantitative comparison of samples, producing fundamental information in biomarker discovery approaches.     

Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry

Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.     

The low molecular weight proteome of Halobacterium salinarum

Systematic investigation of low molecular weight proteins (LMW, below 20 kDa) in the archaeon Halobacterium salinarum resulted in a 6-fold enhancement of the identification rate, reaching 35% of the theoretical proteome in that size range. This was achieved by optimization of common protocols for protein analysis with general applicability. LMW proteins were rapidly and effectively enriched by filter membrane centrifugation followed by tricine SDS-PAGE. Without staining and with significantly shortened digestion protocols, LMW proteins were identified using an FT-ICR mass spectrometer which allows reliable protein identification by MS3 of a single peptide. In addition to a series of technical challenges, small proteins may show low gene expression levels as suggested by their low average codon adaptation index. Twenty functionally uncharacterized proteins contain a characteristic DNA/RNA binding zinc finger motif which underlines the biological relevance of the small proteome and the necessity of their analysis for systems biology.     

Development of Gel-Filter Method for High Enrichment of Low-Molecular Weight Proteins from Serum

The human serum proteome has been extensively screened for biomarkers. However, the large dynamic range of protein concentrations in serum and the presence of highly abundant and large molecular weight proteins, make identification and detection changes in the amount of low-molecular weight proteins (LMW, molecular weight ≤ 30kDa) difficult. Here, we developed a gel-filter method including four layers of different concentration of tricine SDS-PAGE-based gels to block high-molecular weight proteins and enrich LMW proteins. By utilizing this method, we identified 1,576 proteins (n = 2) from 10 μL serum. Among them, 559 (n = 2) proteins belonged to LMW proteins. Furthermore, this gel-filter method could identify 67.4% and 39.8% more LMW proteins than that in representative methods of glycine SDS-PAGE and optimized-DS, respectively. By utilizing SILAC-AQUA approach with labeled recombinant protein as internal standard, the recovery rate for GST spiked in serum during the treatment of gel-filter, optimized-DS, and ProteoMiner was 33.1 ± 0.01%, 18.7 ± 0.01% and 9.6 ± 0.03%, respectively. These results demonstrate that the gel-filter method offers a rapid, highly reproducible and efficient approach for screening biomarkers from serum through proteomic analyses.     

Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.     

Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p < 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.     

Mass spectrometric identification of serum peptides employing derivatized poly(glycidyl methacrylate/divinyl benzene) particles and mu-HPLC

Biomarkers play a key role in preclinical screening and diagnosis of a disease. Various support materials are utilized for this task, in combination with MALDI-TOF-MS. The way to effectively bind serum contents and their profiling is well-elaborated by the material-enhanced laser desorption ionization (MELDI) approach. In this particular work, focus is placed on the development of a strategy to identify low molecular weight serum peptides. Poly(GMA/DVB) is derivatized in a way to achieve an affinity termed as immobilized metal ion affinity chromatography (IMAC). Iminodiacetic acid (IDA) is used as a chelating ligand, whereas copper (Cu2+) acts as a metal ion for complexing peptides and proteins out of blood serum. Polymer binds the serum compounds over a broad mass range, which includes low mass peptides and high mass albumin (66 kDa). Bound contents are eluted from material by an acetonitrile/trifluoroacetic acid mixture, which proves the reversible nature of metal and amino acid linkage. Polystyrene/divinyl benzene (PS/DVB) monolithic capillary column is used for fractionation through RP-HPLC, prior to the target spotting. The tandem TOF fragment ion mass spectra of each fraction is acquired and used to search against the Swiss-Prot database, using the Mascot search engine for the identification of peptides.     

尿液蛋白富集方法的比较

人尿液中蛋白含量低,在进行质谱分析时易被高丰度蛋白掩盖。因此,发展高效和高选择性的富集方法,是实现尿蛋白标记物深度覆盖的必要前提。探究不同实验方法对尿液蛋白富集和尿蛋白质组的影响尤为重要。本研究采用超滤法、硝酸纤维素膜富集法和饱和硫酸铵沉淀法,等体积各处理5例健康志愿者和膀胱癌患者10 mL尿液样本,富集尿液蛋白,SDS-PAGE分离尿蛋白,比较不同方法纯化的效率;通过质谱分析,比较不同纯化方法的肽段鉴定效果,确定针对尿液蛋白质组蛋白的最佳富集方法。相对于超滤和硝酸纤维素膜富集法,饱和硫酸铵沉淀法成功地应用于健康人尿蛋白的富集和质谱检测,在保证回收蛋白质量的前提下,可减少高丰度白蛋白的干扰,富集更多低丰度蛋白,提高了质谱鉴定的灵敏度。综上所述,饱和硫酸铵提取尿蛋白的效果较好,该方法具有大规模处理尿液、提高蛋白质组学筛选临床诊断标记物研究的应用潜力。     

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.     

Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.     

Comprehensive peptidome analysis of mouse livers by size exclusion chromatography prefractionation and nanoLC-MS/MS identification

Peptidome analysis has received increasing attention in recent years. Cancer diagnosis by serum peptidome has also been reported by peptides' profiling for discovery of peptide biomarkers. Tissue, which may have a higher biomarker concentration than blood, has not been investigated extensively by means of peptidome analysis. Here, a method for the peptidome analysis of mouse liver was developed by the combination of size exclusion chromatography (SEC) prefractionation with nano-liquid chromatography-tamdem mass spectrometry (nanoLC-MS/MS) analysis. The extracted peptides from mouse liver were separated according to their molecular weight using a size exclusion column. MALDI-TOF MS was used to characterize the molecular weight distribution of the peptides in fractions eluted from the SEC column. The low molecular weight (LMW) (MW < 3000 Da) peptides in the collected fractions were directly analyzed by LC-MS/MS which resulted in the identification of 1181 unique peptides (from 371 proteins). The high molecular weight (HMW) (MW > 3000 Da) peptides in the early two fractions from the SEC column were first digested with trypsin, and the resulted digests were then analyzed by LC-MS/MS, which led to the identification of 123 and 127 progenitor proteins of the HMW peptides in fractions 1 and 2, respectively. Analysis of the peptides' cleavage sites showed that the peptides are cleaved in regulation, which may reflect the protease activity and distribution in body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery.     

Effect of 2MEGA labeling on membrane proteome analysis using LC-ESI QTOF MS

One of the challenges associated with large-scale proteome analysis using tandem mass spectrometry (MS/MS) and automated database searching is to reduce the number of false positive identifications without sacrificing the number of true positives found. In this work, a systematic investigation of the effect of 2MEGA labeling (N-terminal dimethylation after lysine guanidination) on the proteome analysis of a membrane fraction of an Escherichia coli cell extract by 2-dimensional liquid chromatography MS/MS is presented. By a large-scale comparison of MS/MS spectra of native peptides with those from the 2MEGA-labeled peptides, the labeled peptides were found to undergo facile fragmentation with enhanced a1 or a1-related (a(1)-17 and a(1)-45) ions derived from all N-terminal amino acids in the MS/MS spectra; these ions are usually difficult to detect in the MS/MS spectra of nonderivatized peptides. The 2MEGA labeling alleviated the biased detection of arginine-terminated peptides that is often observed in MALDI and ESI MS experiments. 2MEGA labeling was found not only to increase the number of peptides and proteins identified but also to generate enhanced a1 or a1-related ions as a constraint to reduce the number of false positive identifications. In total, 640 proteins were identified from the E. coli membrane fraction, with each protein identified based on peptide mass and sequence match of one or more peptides using MASCOT database search algorithm from the MS/MS spectra generated by a quadrupole time-of-flight mass spectrometer. Among them, the subcellular locations of 336 proteins are presently known, including 258 membrane and membrane-associated proteins (76.8%). Among the classified proteins, there was a dramatic increase in the total number of integral membrane proteins identified in the 2MEGA-labeled sample (153 proteins) versus the unlabeled sample (77 proteins).