Phosphotyrosine proteomic study of interferon alpha signaling pathway using a combination of immunoprecipitation and immobilized metal affinity chromatography

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.     

Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting

We describe fluorescence-based 2-D gel electrophoresis methods for visualization of low abundant, cancer relevant tyrosine phosphorylated (pTyr) proteins. The methods investigated were fluorescent Western blotting and two-dimensional difference gel electrophoresis (2-D DIGE) for detection of non-enriched and immunoaffinity enriched pTyr protein patterns. The same anti-phosphotyrosine specific antibody, 4G10, was used for both approaches. The results from fluorescent Western blotting of total proteins and from enriched CyDye DIGE pre-labeled pTyr proteins showed similar down regulation of phosphorylation upon treating of cells from a cancer model system (K562 chronic myeloid leukemia cells) with imatinib. This treatment introduced a known perturbation of phosphorylation that enabled testing of these new approaches to analyze variations in tyrosine phosphorylation levels. Enrichment of pTyr proteins was found highly advantageous for the outcome. Out of a simplified 2-D DIGE experiment of immunoaffinity enriched control and treated pTyr proteins, differential analysis as well as protein identification by mass spectrometry (MS) was possible.     

Tyrosine phosphoproteomics and identification of substrates of protein tyrosine phosphatase dPTP61F in Drosophila S2 cells by mass spectrometry-based substrate trapping strategy

Recent biochemical and genetic approaches have clearly defined the functional role of critical components in tyrosine phosphorylation-dependent signal transduction. These signaling modulators often exhibit evolutionarily conserved functions across various species. It has been proposed that if protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs), and thousands of their substrates could be identified and characterized, it would significantly advance our understanding of the underlying mechanisms that control animal development and physiological homeostasis. The fruit fly Drosophila melanogester has been used extensively as a model organism for investigating the developmental processes, but the state of its tyrosine phosphorylation is poorly characterized. In the current study, we used advanced mass spectrometry (MS)-based shotgun analyses to profile the tyrosine phosphoproteome of Drosophila S2 cells. Using immunoaffinity isolation of the phosphotyrosine (pTyr) subproteome from cells treated with pervanadate followed by enrichment of phosphopeptides, we identified 562 nonredundant pTyr sites in 245 proteins. Both this predefined pTyr proteome subset and the total cell lysates were then used as sample sources to identify potential substrates of dPTP61F, the smallest member in terms of amino acid number and molecular weight in the Drosophila PTP family and the ortholog of human PTP1B and T Cell-PTP, by substrate trapping. In total, 20 unique proteins were found to be specifically associated with the trapping mutant form of dPTP61F, eluted by vanadate (VO4(3-)), and identified by MS analyses. Among them, 16 potential substrates were confirmed as tyrosine phosphorylated proteins, including a receptor PTK PDGF/VEGF receptor, a cytosolic PTK Abl, and several components of SCAR/WAVE complex, which may work in coordination to control actin dynamics. Thus, our data suggest that dPTP61F plays a central role in counteracting PTK-mediated signaling pathways as well as in regulating actin reorganization and remodeling through tyrosine dephosphorylation of critical substrates in Drosophila cells.     

High-throughput kinase assay based on surface plasmon resonance suitable for native protein substrates

We report a novel in vitro high-throughput (HTP) kinase assay using surface plasmon resonance (SPR). In vitro tyrosine phosphorylation was performed in a microtiter plate, after which the substrate was captured with an antibody on a sensor chip and phosphotyrosine (pTyr) was detected with an anti-pTyr antibody. The capture and pTyr detection steps were performed using a Biacore A100, which is a sensitive and high-performance flow-cell-based SPR biosensor. This system allowed multiple sample processing (1000 samples/day) and high-quality data sampling. We compared the abilities of the HTP-SPR method and a standard radioisotope assay by measuring the phosphorylation of several substrate proteins by the Fyn tyrosine kinase. Similar results were obtained with both methods, suggesting that the HTP-SPR method is reliable. Therefore, the HTP-SPR method described in this study can be a powerful tool for a variety of screening analyses, such as kinase activity screening, kinase substrate profiling, and kinase HTP screening of kinase inhibitors.     

Monoclonal antibodies to an artificial immunogen, specifically demonstrating phosphotyrosine-containing proteins

The importance of protein phosphorylation at tyrosyl hydroxy groups in the control of cell proliferation has recently been established. For identification of tyrosine-phosphorylated proteins, monoclonal antibodies (Mabs) against artificial immunogens containing O-phosphotyrosine (pTyr) or tripeptide pTyr-Gly-Gly as haptens were generated; the haptens were coupled to carrier proteins (bovine serum albumin, human immunoglobulin, keyhole limpet hemocyanin). After immunization of mice with pTyr coupled to keyhole limpet hemocyanin, Mabs were generated which were highly specific for pTyr and did not cross-react with O-phosphoserine, O-phosphothreonine, tyrosine or nucleoside-5'-monophosphates. The Mabs specifically react with tyrosinephosphorylated proteins in the Rous sarcoma virus-transformed rat XC-cell.     

Tyrosine phosphorylation of the Rab24 GTPase in cultured mammalian cells

Several members of the large family of Rab GTPases have been shown to function in vesicular trafficking in mammalian cells. However, the exact role of Rab24 remains poorly defined. Rab24 differs from other Rab proteins in that it has a low intrinsic GTPase activity and is not efficiently prenylated. Here we report an additional unique property of Rab24; i.e., the protein can undergo tyrosine phosphorylation when overexpressed in cultured cells. Immunoblot analyses with specific anti-phosphotyrosine monoclonal antibodies revealed the presence of phosphotyrosine (pTyr) on myc-Rab24 in whole cell lysates and immunoprecipitated samples. No pTyr was detected on other overexpressed myc-tagged GTPases (H-Ras, Rab1b, Rab6, Rab11 or Rab13). Comparisons of myc-Rab24 in the soluble and particulate fractions from HEK293 and HEp-2 cells indicated that the cytosolic pool of Rab24 was more heavily phosphorylated than the membrane pool. Treatment of transfected cells with the broad-spectrum tyrosine kinase inhibitor, genistein, as well as the specific Src-family kinase inhibitor, PP2, eliminated the pTyr signal from Rab24. In contrast the receptor tyrosine kinase inhibitor, tyrphostin A25, had no effect. Tyrosine phosphorylation of Rab24 was reduced by alanine substitution of two unique tyrosines, one found in a strong consensus phosphorylation motif (Y [Formula: see text] ) in the hypervariable domain (Y172) and the other falling within the GXXXGK(S/T) motif known as the P-loop (Y17). The latter region is known to influence GTP hydrolysis in Rab proteins, so the phosphorylation of Y17 could contribute to the low intrinsic GTPase activity of Rab24. This is the first report of tyrosine phosphorylation in any member of the Ras superfamily and it raises the possibility that this type of modification could influence Rab24 targeting and interactions with effector protein complexes.     

A one-step method to identify MAP kinase residues involved in inactivation by tyrosine- and dual-specificity protein phosphatases

MAP kinases (MAPKs) are enzymes directly involved in the control of cellular homeostasis in response to external cues, from differentiation and developmental processes to cell transformation. The activation status of MAPKs, both in magnitude and in duration, reflects the balance of phosphorylation at their Thr and Tyr regulatory residues by specific MAPK kinases and their dephosphorylation by inactivating protein serine/threonine phosphatases (PPs) and protein tyrosine phosphatases (PTPs). The dephosphorylation of MAPKs by PTPs relies on molecular docking between the two enzymes at specific interaction sites. Here we outline a one-step method to identify ERK1/2 and p38α mutations that prevent binding and inactivation by PTPs (tyrosine- or dual-specificity phosphatases) based on the use of anti-pTyr antibodies and cell lysis buffers lacking or containing the broad PTP inhibitor sodium orthovanadate (Na₃VO₄).     

The role of mass spectrometry in proteome studies

Mass spectrometry (MS) is an important tool in modern protein chemistry. In proteome analyses the expression of hundreds or thousands of proteins can be monitored at the same time. First, complex protein mixtures are separated by two-dimensional gel electrophoresis (2-DE) and then individual proteins are identified by using MS followed by database searches. Recent developments in this field have made it possible to do automated, high-throughput protein identification that is needed in proteome analyses. MS can also be used to characterize post-translational modifications in proteins and to study protein complexes. This review will introduce the current MS methods used in proteome studies, and discuss their advantages and disadvantages. New instrumental MS developments are also presented that are useful in these analyses.     

基于SH2超亲体的微量样品酪氨酸磷酸化蛋白质组学技术研究进展及应用

酪氨酸磷酸化是生物体内一种重要的蛋白质磷酸化修饰类型,参与细胞信号转导、细胞迁移、凋亡等众多的生命活动过程。在磷酸化蛋白质组学研究中,由于酪氨酸磷酸化蛋白丰度低且有时起始样品量有限,传统的磷酸化蛋白质组富集方法用于酪氨酸磷酸化肽富集效率较低。微量样品的制备技术以及SH2超亲体的引入正在改变酪氨酸磷酸化蛋白质组研究的现状,现对此进行综述。     

Comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry

There is significant interest in characterization of the human plasma proteome due to its potential for providing biomarkers applicable to clinical diagnosis and treatment and for gaining a better understanding of human diseases. We describe here a strategy for comparative proteome analyses of human plasma, which is applicable to biomarker identifications for various disease states. Multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) has been applied to make comparative proteome analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Peptide peak areas and the number of peptide identifications for each protein were used to evaluate the reproducibility of LC-MS/MS and to compare relative changes in protein concentration between the samples following LPS treatment. A total of 804 distinct plasma proteins (not including immunoglobulins) were confidently identified with 32 proteins observed to be significantly increased in concentration following LPS administration, including several known inflammatory response or acute-phase mediators such as C-reactive protein, serum amyloid A and A2, LPS-binding protein, LPS-responsive and beige-like anchor protein, hepatocyte growth factor activator, and von Willebrand factor, and thus, constituting potential biomarkers for inflammatory response.     

Quantitative Proteome-Property Relationships (QPPRs). Part 1: finding biomarkers of organic drugs with mean Markov connectivity indices of spiral networks of blood mass spectra

Numerical parameters of the molecular networks, also referred as Topological Indices or Connectivity Indices (CIs), have been used in Bioorganic and Medicinal Chemistry to find Quantitative Structure-Activity, Property or Toxicity Relationship (QSAR, QSPR and QSTR) models. QSPR models generally use CIs as inputs to predict the biological activity of compounds. However, the literature does not evidence a great effort to find QSAR-like models for other biologically and chemically relevant systems. For instance, blood proteome constitutes a protein-rich information reservoir, since the serum proteome Mass Spectra (MS) represents a potential information source for the early detection of Biomarkers for diseases and/or drug-induced toxicities. The concept of mass spectrum network (MS network) for a single protein is already well-known. However, there are no reported results on the use of CIs for a MS network of a whole proteome to explore MS patterns. In this work, we introduced for the first time a novel network representation and the CIs for the MS of blood proteome samples. The new network bases on Randic's Spiral network have been previously introduced for protein sequences. The new MS CIs, called here Spiral Markov Connectivity (SMC(k)) of the MS Spiral graph can be calculated with the software MARCH-INSIDE, combining network and Markov model theory. The SMC(k) values could be used to seek QSAR-like models, called in this work Quantitative Proteome-Property Relationships (QPPRs). We calculate the SMC(k) values for 62 blood samples and fit a QPPR model by discriminating proteome MS, typical of individuals susceptible to suffer drug-induced cardiotoxicity from control samples. The accuracy, sensitivity, and specificity values of the QPPR model were between 73.08% and 87.5% in training and validation series. This work points to QPPR models as a powerful tool for MS detection of biomarkers in proteomics.     

Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.

The binding of tyrosine phosphorylated targets by SH2 domains is required for propagation of many cellular signals in higher eukaryotes; however, the determinants of phosphotyrosine (pTyr) recognition by SH2 domains are not well understood. In order to identify the attributes of pTyr required for high affinity interaction with SH2 domains, the binding of the SH2 domain of the Src kinase (Src SH2 domain) to a dephosphorylated peptide, a phosphoserine-containing peptide, and the amino acid pTyr was studied using titration calorimetry and compared with the binding of a high affinity tyrosyl phosphopeptide. The dephosphorylated peptide and the phosphoserine containing peptide both bind extremely weakly to the Src SH2 domain (DeltaGo (dephosphorylated)=-3.6 kcal/mol, DeltaGo (phosphoserine) >-3.7 kcal/mol); however, the DeltaGo value of pTyr binding is more favorable (-4.7 kcal/mol, or 50 % of the entire binding free energy of a high affinity tyrosyl phosphopeptide). These results indicate that both the phosphate and the tyrosine ring of the pTyr are critical determinants of high affinity binding. Alanine mutagenesis was also used to evaluate the energetic contribution to binding of ten residues located in the pTyr-binding site. Mutation of the strictly conserved Arg betaB5 resulted in a large increase in DeltaGo (DeltaDeltaGo=3.2 kcal/mol) while elimination of the other examined residues each resulted in a significantly smaller (DeltaDeltaGo<1.4 kcal/mol) reduction in affinity, indicating that Arg betaB5 is the single most important determinant of pTyr recognition. However, mutation of Cys betaC3, a residue unique to the Src SH2 domain, surprisingly increased affinity by eightfold (DeltaDeltaGo=-1.1 kcal/mol). Using a double mutant cycle analysis, it was revealed that residues of the pTyr-binding pocket are not coupled to the peptide residues C-terminal to the pTyr. In addition, comparison of each residue's DeltaDeltaGo value upon mutation with that residue's sequence conservation among SH2 domains revealed only a modest correlation between a residue's energetic contribution to pTyr recognition and its conservation throughout evolution. The results of this investigation highlight the importance of a single critical interaction, the buried ionic bond between the phosphate of the pTyr and Arg betaB5 of the SH2 domain, driving the binding of SH2 domains to tyrosine phosphorylated targets.     

Strategy for surveying the proteome using affinity proteomics and mass spectrometry

Antibody‐based microarrays is a rapidly evolving technology that has gone from the first proof‐of‐concept studies to more demanding proteome profiling applications, during the last years. Miniaturized microarrays can be printed with large number of antibodies harbouring predetermined specificities, capable of targeting high‐ as well as low‐abundant analytes in complex, nonfractionated proteomes. Consequently, the resolution of such proteome profiling efforts correlate directly to the number of antibodies included, which today is a key limiting factor. To overcome this bottleneck and to be able to perform in‐depth global proteome surveys, we propose to interface affinity proteomics with MS‐based read‐out, as outlined in this technical perspective. Briefly, we have defined a range of peptide motifs, each motif being present in 5–100 different proteins. In this manner, 100 antibodies, binding 100 different motifs commonly distributed among different proteins, would potentially target a protein cluster of 10⁴ individual molecules, i.e. around 50% of the nonredundant human proteome. Notably, these motif‐specific antibodies would be directly applicable to any proteome in a specie independent manner and not biased towards abundant proteins or certain protein classes. The biological sample is digested, exposed to these immobilized antibodies, whereby motif‐containing peptides are specifically captured, enriched and subsequently detected and identified using MS.     

Plasma proteome dynamics: analysis of lipoproteins and acute phase response proteins with 2H2O metabolic labeling

Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a (2)H(2)O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis. (2)H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with (2)H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.     

A sensitive magnetic bead method for the detection and identification of tyrosine phosphorylation in proteins by MALDI‐TOF/TOF MS

Phosphorylation is one of the most important PTMs and is estimated to occur on 30% of the mammalian proteome. Its perturbed regulation has been implicated in many pathologies. The rarity of phosphotyrosine compared with phosphoserine or phosphothreonine is prompting the development of more sensitive approaches because proteomic technologies that are currently used to assess tyrosine phosphorylation in proteins are inadequate, identifying only a fraction of the predicted tyrosine phosphoproteome. Here we describe the development of a reproducible, high‐sensitivity methodology for the detection and mapping of phosphotyrosine residues by MS. The anti‐phosphotyrosine antibody 4G10 was coupled covalently to super para‐magnetic beads or by affinity to super para‐magnetic beads with protein G covalently attached. Using this approach, we successfully enriched phosphotyrosine peptides mixed with non‐phosphorylated peptides at a ratio of up to 1:200, enabling detection at a level representing the highest sensitivity reported for tyrosine phosphorylation. The beads were subsequently used to enrich tyrosine phosphopeptides from a digest of the in vitro‐phosphorylated recombinant β‐intracellular region of the granulocyte‐macrophage colony‐stimulating factor receptor, which was subsequently analysed by MALDI‐TOF/TOF MS. Our results define this methodology as a sensitive approach for tyrosine phosphoproteome analysis.     

Targeted analysis of tyrosine phosphorylation by immuno-affinity enrichment of tyrosine phosphorylated peptides prior to mass spectrometric analysis

Tyrosine phosphorylation is a key process that regulates seminal biological functions, hence, deregulation of this mechanism is an underlying cause of several diseases including cancer and immunological disorders. Due to its low abundance, tyrosine phosphorylation is typically under-represented in most of the global MS-based phosphoproteomic studies. Here, we describe a selective approach based on immuno-affinity purification using specific antibodies to enrich tyrosine phosphorylated peptides from a complex proteolytic digest. LC-MS/MS analysis is subsequently used for peptide identification allowing the exact localization of the phosphorylated residue within the sequence. Using this approach more than 1000 non-redundant phosphotyrosine peptides can be identified in less than 6h of MS analysis, reflecting the high sensitivity and specificity of the technique. The identified tyrosine phosphorylated peptides can be used to study different biological aspects of tyrosine signaling and disease.     

High Throughput Identification of Monoclonal Antibodies to Membrane Bound and Secreted Proteins Using Yeast and Phage Display

Antibodies are ubiquitous and essential reagents for biomedical research. Uses of antibodies include quantifying proteins, identifying the temporal and spatial pattern of expression in cells and tissue, and determining how proteins function under normal or pathological conditions. Specific antibodies are only available for a small portion of the proteome, limiting study of those proteins for which antibodies do not exist. The technologies to generate target-specific antibodies need to be improved to obtain high quality antibodies to the proteome at reasonable cost. Here we show that renewable, validated, and standardized monoclonal antibodies can be generated at high throughput, without the need for antigen production or animal immunizations. In this study, 60 protein domains from 24 selected secreted proteins were expressed on the surface of yeast and used for selection of phage antibodies, over 400 monoclonal antibodies were identified within 3 weeks. A subset of these antibodies was validated for binding to cancer cells that overexpress the target protein by flow cytometry or immunohistochemistry. This approach will be applicable to many of the membrane-bound and the secreted proteins, 20–40% of the proteome, accelerating the timeline for Ab generation while reducing the cost.     

Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling

Background

Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/Principal Findings

We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/Significance

This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.     

Systematic Evaluation of Immobilized Trypsin‐Based Fast Protein Digestion for Deep and High‐Throughput Bottom‐Up Proteomics

Immobilized trypsin (IM) has been recognized as an alternative to free trypsin (FT) for accelerating protein digestion 30 years ago. However, some questions of IM still need to be answered. How does the solid matrix of IM influence its preference for protein cleavage and how well can IM perform for deep bottom‐up proteomics compared to FT? By analyzing Escherichia coli proteome samples digested with amine or carboxyl functionalized magnetic bead–based IM (IM‐N or IM‐C) or FT, it is observed that IM‐N with the nearly neutral solid matrix, IM‐C with the negatively charged solid matrix, and FT have similar cleavage preference considering the microenvironment surrounding the cleavage sites. IM‐N (15 min) and FT (12 h) both approach 9000 protein identifications (IDs) from a mouse brain proteome. Compared to FT, IM‐N has no bias in the digestion of proteins that are involved in various biological processes, are located in different components of cells, have diverse functions, and are expressed in varying abundance. A high‐throughput bottom‐up proteomics workflow comprising IM‐N‐based rapid protein cleavage and fast CZE‐MS/MS enables the completion of protein sample preparation, CZE‐MS/MS analysis, and data analysis in only 3 h, resulting in 1000 protein IDs from the mouse brain proteome.