Global proteome analysis of a human gastric carcinoma

An approach that combines analysis of global protein digests (GPDs) of various subcellular fractions with a novel chromatographic-based method to map protein expression profiles is described. The KATO III gastric carcinoma cell line was fractionated into membrane and cytosol fractions. Each subcellular fraction was digested with trypsin to yield complex mixtures of global protein tags (GPTs). These mixtures were fractionated by two dimensions of chromatography, and GPTs were sequenced by microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), using two further complementary dimensions of chromatography. Additionally, a novel method of protein expression profiling was used to map the KATO III human gastric carcinoma cell line. This method uses the cells' natural proteolytic processes to derive in vivo peptide tags that represent proteins of every functional class and from all subcellular compartments. In one example, expressed protein tags (EPTs) are naturally displayed on the surface of cells by multiligand receptors. Isolation and sequence identification of EPTs is an efficient approach for protein profiling that is complementary to GPT analysis. The EPT approach also provides a further unique subcellular fraction of the biological starting material. Isolation of the multiligand receptors was by immunoaffinity chromatography (IAC). In the current study, five individual peptide maps (two EPTs and three GPTs) of the KATO III cell line were fractionated by multimodal chromatography, and sequenced by on-line multimodal microcapillary LC-MS/MS. This analysis led to the identification of 4291 individual peptide sequences, which defined 1966 unique proteins expressed by this human carcinoma cell line.     

Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.     

Multivariate comparison between peptide mass fingerprints obtained by liquid chromatography-electrospray ionization-mass spectrometry with different trypsin digestion procedures

Peptide mass fingerprints were obtained for three different proteins using three different digestion procedures in triplicates with liquid chromatography coupled to electrospray ionization mass spectrometry. For each protein the results were compared with multivariate data analysis (cluster analysis, kernel principal component analysis) and pair-wise contrast evaluation. Clear systematic differences between the digestion procedures were established for all the proteins. The visual presentation of the pair-wise differences between procedures could to some extent be related to the protein fragments, although the main objective was to identify m/z and retention regions in the original peptide maps that should be subject to further exploration.     

Efficient peptide mapping and its application to identify embryo proteins in rice proteome analysis

Using direct N-terminal analysis, only 31 N-terminally unblocked proteins out of 100 rice embryo proteins could be identified. To obtain protein sequence information for the remaining 69 blocked proteins, we developed a simple, efficient and rapid method. Using this method, we determined the peptide maps of 20 proteins per day in 10 pmol amounts. Applying this method to rice proteome analysis, we determined the internal sequences of all 69 blocked proteins. A total of 28 proteins out of 100 analyzed showed sequence similarity to the proteins with known functions in the SWISS-PROT and NCBI databases. Alternatively, we also used peptide mass fingerprinting determined by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to identify the rice proteins separated by two-dimensional electrophoresis (2-DE). Although peptide-mass fingerprinting is a high-throughput method, we could not easily identify all the rice proteins or genes by this method, because the complete database information on rice, is not yet available and many proteins are post-translationally modified. Therefore, at present, the improved peptide mapping method as we report here is considered to be very useful in rice proteome analysis, especially for blocked proteins.     

Continuous sample deposition from reversed-phase liquid chromatography to tracks on a matrix-assisted laser desorption/ionization precoated target for the analysis of protein digests

Peptide mass fingerprinting by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) is one of the standard high-throughput methods for protein identification today. Traditionally this method has been based on spotting peptide mixtures onto MALDI targets. While this method works well for more abundant proteins, low-abundance proteins mixed with high-abundance proteins tend to go undetected due to ion suppression effects, instrumental dynamic range limitations and chemical noise interference. We present an alternative approach where liquid chromatography (LC) effluent is continuously collected as linear tracks on a MALDI target. In this manner the chromatographic separation is spatially preserved on the target, which enables generation of off-line LC-MS and LC-MS/MS data by MALDI. LC-MALDI sample collection provides improved sensitivity and dynamic range, spatial resolution of peptides along the sample track, and permits peptide mass mapping of low-abundance proteins in mixtures containing high-abundance proteins. In this work, standard and ribosomal protein digests are resolved and captured using LC-MALDI sample collection and analyzed by MALDI-TOF-MS.     

Large-scale muLC-MS/MS for silver- and Coomassie blue-stained polyacrylamide gels

2-DE combined with LC-MS/MS has become a routine, reliable protein separation and identification technology for proteome analysis. The demand for large-scale protein identifications after 2-DE separation requires a sensitive and high-throughput LC-MS/MS method. In this report, a simple, splitless, fully automated capillary LC-MS/MS system was described for the large-scale identification of proteins from gels stained with either silver or CBB. The gel samples were digested and peptides were extracted using an in-gel digestion workstation. The peptides were automatically introduced into a capillary column by an autosampler connected to an HPLC pump. A nanoLC pump was then used to deliver the gradient and elute the peptides from the capillary column directly into an LCQ IT mass spectrometer. Neither a peptide trapping setting nor a flow split is needed in this simple setup. The collected MS/MS spectra were then automatically searched by SEQUEST, and filtered and organized by DTASelect. Hundreds of silver-stained or CBB-stained Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens proteins separated by denaturing or nondenaturing 2-DE were digested and routinely analyzed using this fully automated muLC-MS/MS system. High peptide hits and sequence coverage were achieved for most CBB-stained gel spots. About 75% of the spots were found to contain multiple proteins. Although silver staining is not commonly thought to be optimal for MS analysis, protein identifications were successfully obtained from silver-stained 2-DE spots detected using methods with and without formaldehyde for protein fixation.     

The Utility of Accurate Mass and LC Elution Time Information in the Analysis of Complex Proteomes

The combination of mass and normalized elution time (NET) of a peptide identified by liquid chromatography-mass spectrometry (LC-MS) measurements can serve as a unique signature for that peptide. However, the specificity of an LC-MS measurement depends upon the complexity of the proteome (i.e., the number of possible peptides) and the accuracy of the LC-MS measurements. In this work, theoretical tryptic digests of all predicted proteins from the genomes of three organisms of varying complexity were evaluated for specificity. Accuracy of the LC-MS measurement of mass-NET pairs (on a 0 to 1.0 NET scale) was described by bivariate normal sampling distributions centered on the peptide signatures. Measurement accuracy (i.e., mass and NET standard deviations of +/-0.1, 1, 5, and 10 ppm, and +/-0.01 and 0.05, respectively) was varied to evaluate improvements in process quality. The spatially localized confidence score, a conditional probability of peptide uniqueness, formed the basis for the peptide identification. Application of this approach to organisms with comparatively small proteomes, such as Deinococcus radiodurans, shows that modest mass and elution time accuracies are generally adequate for confidently identifying most peptides. For more complex proteomes, more accurate measurements are required. However, the study suggests that the majority of proteins for even the human proteome should be identifiable with reasonable confidence by using LC-MS measurements with mass accuracies within +/-1 ppm and high efficiency separations having elution time measurements within +/-0.01 NET.     

Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides

A rapid means of identifying many components in an enriched mixture of proteins is enzymatic digestion of the entire protein fraction. This complex peptide mixture is then subjected to reversed-phase high performance liquid chromatography (HPLC) coupled on-line with a mass spectrometer capable of data-dependent ion selection for fragmentation (LC-tandem mass spectrometry; MS/MS). Thus, as many peptides as possible in the sample are fragmented to produce MS/MS spectra, which can then be searched against sequence databases. Ideally, one peptide from each protein in the mixture would be fragmented and identified. To this end, we employed an affinity selection method to capture cysteinyl peptides and thereby simplify the mixture. Both the captured cysteinyl and the noncysteinyl peptides are analyzed by LC-MS/MS, to increase the number of proteins identified. The method was tested on a limited set of standard proteins and applied to the analysis of a protein fraction obtained from isolated mitochondria treated with atractyloside. To further increase the number of different precursor ions selected for fragmentation, dynamic exclusion and ion selection from multiple narrow mass ranges of consecutive runs were employed.     

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

Disposable chromatography for a high-throughput nano-ESI/MS and nano-ESI/MS-MS platform

High-throughput proteomics has typically relied on protein identification based on MALDI-MS peptide maps of proteolytic digests of 2D-gel-separated proteins. This technique, however, requires significant sequence coverage in order to achieve a high level of confidence in the identification. Tandem MS data have the advantage of requiring fewer peptides (2) for high confidence identification, assuming adequate MS/MS sequence coverage. MALDI-MS/MS techniques are becoming available, but can still be problematic because of the difficulty of inducing fragment ions of a singly charged parent ion. Electrospray ionization, however, has the advantage of generating multiply charged species that are more readily fragmented during MS/MS analysis. Two electrospray/tandem mass spectrometry-based approaches, nanovial-ESI-MS/MS and LC-MS/MS, are used for high throughput proteomics, but much less often than MALDI-MS and peptide mass fingerprinting. Nanovial introduction entails extensive manual manipulation and often shows significant chemical background from the in-gel digest. LC-MS has the advantages that the chemical background can be removed prior to analysis and the analytes are concentrated during the separation, resulting in more abundant analyte signals. On the other hand, LC-MS can often be time intensive. Here, we report the incorporation of on-line sample clean-up and analyte concentration with a high-throughput, chip-based, robotic nano-ESI-MS platform for proteomics studies.     

A universal surrogate peptide to enable LC-MS/MS bioanalysis of a diversity of human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies

For the development of human antibody Fc (fraction crystallizable) region-containing therapeutic protein candidates, which can be either monoclonal antibodies (mAbs) or pharmacologically active proteins/peptides fused to the Fc region of human Immunoglobulin G (IgG), reliable quantification of these proteins in animal pharmacokinetic study plasma samples is critical. LC-MS/MS has emerged as a promising assay platform for this purpose. LC-MS/MS assays used for bioanalysis of human antibody Fc region-containing therapeutic protein candidates frequently rely upon quantification of a 'signature' surrogate peptide whose sequence is unique to the protein analyte of interest. One drawback of the signature peptide approach is that a new LC-MS/MS assay must be developed for each new human Fc region-containing therapeutic protein. To address this issue, we propose an alternative 'universal surrogate peptide' approach for the quantification of human antibody Fc region-containing therapeutic protein candidates in plasma samples from all nonclinical species. A single surrogate tryptic peptide was identified in the Fc region of most human antibody Fc-containing therapeutic protein candidates. An LC-MS-MS method based upon this peptide was shown to be capable of supporting bioanalysis of a diversity of human Fc region-containing therapeutic protein candidates in plasma samples of all commonly used animal species.     

Gel-free sample preparation for the nanoscale LC-MS/MS analysis and identification of low-nanogram protein samples

Protein identification at the low nanogram level could in principle be obtained by most nanoscale LC-MS/MS systems. Nevertheless, the complex sample preparation procedures generally required in biological applications, and the consequent high risk of sample losses, very often hamper practical achievement of such low levels. In fact, the minimal amount of protein required for the identification from a gel band or spot, in general, largely exceeds the theoretical limit of identification reachable by nanoscale LC-MS/MS systems. A method for the identification of low levels of purified proteins, allowing limits of identification down to 1 ng when using standard bore, 75 microm id nanoscale LC-MS/MS systems is here reported. The method comprises an offline two-step sample cleanup, subsequent to protein digestion, which is designed to minimize sample losses, allows high flexibility in the choice of digestion conditions and delivers a highly purified peptide mixture even from "real world" digestion conditions, thus allowing the subsequent nanoscale LC-MS/MS analysis to be performed in automated, unattended operation for long series. The method can be applied to the characterization of low levels of affinity purified proteins.     

超滤法结合纳升液相色谱-串联质谱技术分析漏出性胸腔积液中多肽组分

漏出性胸腔积液中的多肽和蛋白质等生物分子直接或间接地与机体特定的生理、病理状态相关,反映了肺部或者全身其它部位疾病的信息.本研究利用超滤法将漏出性胸腔积液中的多肽组分进行分离,经脱盐富集后,进行纳升液相色谱-串联质谱分析.结果表明,在漏出性胸腔积液中共鉴定到来源于52种蛋白的314条多肽,超过一半的肽段来源于纤维蛋白原,并且许多肽段具有阶梯序列的特征.此外,在来源于胶原蛋白和纤维蛋白原的多肽中还发现了大量的脯氨酸氧化修饰.基因本体论富集分析显示,漏出性胸腔积液多肽组分所属蛋白均具有胞外分泌的属性.本研究给出了漏出性胸腔积液中多肽组的序列、等电点、分子量、翻译后修饰等理化参数的分布特征,为进一步寻找肺部疾病相关的多肽标志物提供了可借鉴的参考数据和分析方法.     

A label-free differential proteomic analysis of mouse bronchoalveolar lavage fluid exposed to ultrafine carbon black

Ultrafine carbon black (ufCB) is a potential hazard to the lung. It causes changes in protein expression and it increases alveolar-capillary permeability in the lung. Label-free quantitative proteomic methods allow a sensitive and accurate analytical method for identifying and quantifying proteins in a protein mixture without chemically modifying the proteins. We used a label-free quantitative proteomic approach that combined and aligned LC-MS and LC-MS/MS spectra to analyze mouse bronchoalveolar lavage fluid (BALF) protein changes associated with exposure to ufCB. We developed a simple normalization method for quantification without spiking the internal standard. The intensities of unchanged peptides were used as normalization factors based on a statistical method to avoid the influence of peptides changed because of ufCB. LC-MS/MS spectra and then database searching were used to identify proteins. The relative abundances of the aligned peptides of identified proteins were determined using LC-MS spectra. We identified 132 proteins, of which 77 are reported for the first time. In addition, the expression of 15 inflammatory proteins and surfactant-associated proteins was regulated (i.e., 7 upregulated and 8 downregulated) compared with the controls. Several proteins not previously reported provide complementary information on the proteins present in mouse BALF, and they are potential biomarkers for the understanding of mechanisms involved in ufCB-induced lung disorders hypothesize that using the label-free quantitative proteomic approach introduced here is well suited for more rigorous, large-scale quantitative analysis of biological samples. We hypothesize that this label-free quantitative proteomic approach will be suited for a large-scale quantitative analysis of biological samples.     

Capillary electrophoresis/tandem mass spectrometry for analysis of proteins from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis

Capillary electrophoresis/time-of-flight mass spectrometry(CE/TOFMS) has been used for analysis of in-gel digests of protein spots excised from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). An off-line purification and preconcentration procedure with a Zip Tip is used before CE/TOFMS analysis which allows for detection of protein spots with <1 picomole of material from 2-D gels. The off-line procedure provides sufficient purification for analysis while maintaining the quality of the CE separation. Using this procedure, several proteins from Coomassie Blue and zinc negatively stained gels are identified by the peptide maps generated and database searching. CE/TOF tandem mass spectrometry is used for the confirmation of database searching results and structural analysis of peptides that do not match the expected peptide maps obtained from the database in order to identify structural modifications. Several modifications were pinpointed and identified by this method.     

Identification of microbial mixtures by LC-selective proteotypic-peptide analysis (SPA)

This paper describes a method--using a combination of LC-MS/MS of selected bacteria-specific peptides and database search--for determining the species of bacteria present in a mixture. We identified the proteotypic peptides that were associated with specific bacteria by searching protein databases for the LC-MS/MS data. The retention time windows for specific peptide markers were used as an extra constraint so that the peptide markers of many bacterial species could be analyzed in a single LC-selective proteotypic-peptide analysis (SPA). We performed LC-MS/MS analyses on the proteolytic digest of cell extracts and monitored only the selected marker peptide ions at given elution time windows. The corresponding bacterial species could be characterized when the selected peptides that eluted at expected elution windows were identified correctly from the database. We managed to identify up to eight bacterial species simultaneously during a single LC-MS/MS analysis, as well as bacteria mixed in various abundances. Two marker ions having similar values of m/z, but obtained from two different bacterial samples, which would otherwise be selected as precursors within mass tolerance and would complicate the MS/MS data, were time-resolved using LC and then used to correctly identify their bacterial sources. The coupling of selective MS/MS monitoring with separation methods, such as LC, provides a highly selective and accurate analytical method for characterizing complex mixtures of bacterial species.     

Peptidomic analysis of human acquired enamel pellicle

Human acquired enamel pellicle is the result of a selective interaction of salivary proteins and peptides with the tooth surface. In the present work, the characterization of the peptides as well as the type of interactions established with the enamel surface was performed. Peptides from in vivo bovine enamel implants in the human oral cavity were sequentially extracted using guanidine and trifluoroacetic acid solutions and the fractions obtained were analysed by LC-MS and LC-MS/MS. Based on the LC-MS data, six phosphorylated peptides were identified in an intact form, strongly adsorbed to the enamel surface. Data from the LC-MS/MS analyses allowed us to identified 30 fragment peptides non-covalently bonded to enamel [basic proline-rich proteins, histatins (1 and 3) and acidic proline-rich protein classes]. The tandem mass spectrometry experiments showed the existence of a pattern of amide bond cleavage for the different identified peptide classes suggesting a selective proteolytic activity. For histatins, a predominance of cleavage at Arg, Lys and His residues was observed, while for basic proline-rich proteins, cleavage at Arg and Pro residues prevailed. In the case of acidic proline-rich proteins, a clearly predominance of cleavage of the Gln-Gly amide bond was evident.     

Mass spectrometric study of the effects of hydrophobic surface chemistry and morphology on the digestion of surface-bound proteins

Our previous work has demonstrated that reversed-phase chromatographic micro-beads can be used to capture proteins from complex biological matrices and the surface-bound proteins can be enzymatically digested for protein identification by mass spectrometry (MS). Here we examine the peptides generated from digestion of proteins bound to various types of micro-bead surfaces in order to determine the effects of surface chemistry and surface morphology on the digestion process. Detailed examinations of site cleavages and sequence coverage are carried out for a tryptic digestion of cytochrome c adsorbed on reversed-phase polystyrene divinylbenzene (Poros R2 beads) versus C(18) bonded-phase silica beads. It is shown that although the surface does not completely hinder the digestion of cleavage sites of the protein, the digestion products are clearly different than those obtained from a solution digest. Specifically, a partial digestion results from surface digestion, resulting in a greater number of missed cleavages than a comparable solution digest. Subsequent comparisons of peptide mass maps generated from the digestion of various proteins on surfaces with altering chemistry (C(4), C(8), C(18), and R2 beads), or with different surface morphology, were performed. The results reveal that surface chemistry plays only a minor role in affecting the peptide mass maps, and surface morphology had no noticeable effects on the resulting peptide mass maps. It is also shown that the mass spectrometric detection method used to analyze the digested peptides can significantly influence the information content on cleavage sites and the extent of sequence coverage. The use of a combination of MALDI, LC/off-line MALDI, and LC/ESI MS is demonstrated to be crucial in revealing subtle changes in the peptide mass maps.     

Bottom-up proteomics of envelope proteins extracted from spinach chloroplast via high organic content CE-MS

A high organic content CE-MS/MS (HOCE-MS/MS) method was developed for the proteomic analysis of envelope proteins extracted from spinach leaves. Separation was performed in a 1-m long hydroxypropyl cellulose coated capillary, using 8% (v/v) formic acid in 70% (v/v) methanol and 22% water as the BGE. A flow-through microvial interface was used to couple the CE system with an Orbitrap Fusion Lumos mass spectrometer, and field-amplified sample stacking was used to improve the concentration sensitivity. Using this optimized method, 3579 peptides and 1141 proteins were identified using the Proteome Discoverer software with a 1% false discovery rate at the protein level. Relative to conventional aqueous CE, HOCE-MS did a better job of discovering hydrophobic peptides and provided more peptide and protein identifications. Relative to nano-LC-MS, it achieved comparable peptide and protein identification performance and detected peptides not identified by LC-MS: of the full set of peptides identified using the two techniques, 19% were identified only using HOCE-MS. It also outperformed nano-LC-MS with respect to the detection of low molecular weight peptides.     

Optimization of immunoaffinity enrichment and detection: toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry

Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment.