Multiphosphorylation-Dependent Recognition of Anti-pS2 Antibodies against RNA Polymerase II C-Terminal Domain Revealed by Chemical Synthesis.

Phosphorylation is a major constituent of the CTD code, which describes the set of post-translational modifications on 52 repeats of a YSPTSPS consensus heptad that orchestrates the binding of regulatory proteins to the C-terminal domain (CTD) of RNA polymerase II. Phospho-specific antibodies are used to detect CTD phosphorylation patterns. However, their recognition repertoire is underexplored due to limitations in the synthesis of long multiphosphorylated peptides. Herein, we describe the development of a synthesis strategy that provides access to multiphosphorylated CTD peptides in high purity without HPLC purification for immobilization onto microtiter plates. Native chemical ligation was used to assemble 12 heptad repeats in various phosphoforms. The synthesis of >60 CTD peptides, 48-90 amino acids in length and containing up to 6 phosphosites, enabled a detailed and rapid analysis of the binding characteristics of different anti-pSer2 antibodies. The three antibodies tested showed positional selectivity with marked differences in the affinity of the antibodies for pSer2-containing peptides. Furthermore, the length of the phosphopeptides allowed a systematic analysis of the multivalent chelate-type interactions. The absence of multivalency-induced binding enhancements is probably due to the high flexibility of the CTD scaffold. The effect of clustered phosphorylation proved to be more complex. Recognition of pSer2 by anti-pSer2-antibodies can be prevented and, perhaps surprisingly, enhanced by the phosphorylation of "bystander" amino acids in the vicinity. The results have relevance for functional analysis of the CTD in cell biological experiments.

Immunopeptidomic Analysis of the Phosphopeptidome Displayed by HLA Class I Molecules.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a powerful tool that allows the characterization of MHC-associated peptidomes. In addition to conventional nonmodified peptides, these complex mixtures also contain phosphorylated species, which may be of great interest for personalized cancer immunotherapy. Here, we provide a detailed protocol to identify phosphopeptides displayed by human HLA class I molecules consisting of four main steps: (1) immunopurification of MHC-I molecules, (2) phosphopeptide enrichment, (3) LC-MS/MS analysis, and (4) MS/MS ion search.

Tumor Infiltrating Lymphocytes Target HLA-I Phosphopeptides Derived From Cancer Signaling in Colorectal Cancer.

There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients' tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients' tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies.

Generation of Phosphopeptide-Specific T Cell Lines as Tools for Melanoma Immunotherapy.

The importance of tumor-associated antigen-specific T cells in the effective control of cancer has been highlighted by recent advances in cancer immunotherapies that target the programmed cell death-1 (PD-1) pathway or that utilize modified T cell receptors. Phosphopeptide-specific T cells are of interest because they recognize a new class of tumor antigens that are derived from proteins relevant for cancer development and growth. These T cell lines or their antigen receptors can be used in combination with other forms of therapy to improve the immune response and survival of cancer patients. We describe here a protocol for the generation of human and transgenic murine phosphopeptide-specific T cells lines as tools for investigating T cell reactivity against melanoma phosphoantigens displayed by HLA-A*0201.

MHC-restricted phosphopeptide antigens: preclinical validation and first-in-humans clinical trial in participants with high-risk melanoma.

BACKGROUND: Phosphorylated peptides presented by MHC molecules represent a new class of neoantigens expressed on cancer cells and recognized by CD8 T-cells. These peptides are promising targets for cancer immunotherapy. Previous work identified an HLA-A*0201-restricted phosphopeptide from insulin receptor substrate 2 (pIRS2) as one such target. The purpose of this study was to characterize a second phosphopeptide, from breast cancer antiestrogen resistance 3 (BCAR3), and to evaluate safety and immunogenicity of a novel immunotherapic vaccine comprising either or both of these phosphorylated peptides. METHODS: Phosphorylated BCAR3 protein was evaluated in melanoma and breast cancer cell lines by Western blot, and recognition by T-cells specific for HLA-A*0201-restricted phosphorylated BCAR3 peptide (pBCAR3126-134) was determined by 51Cr release assay and intracellular cytokine staining. Human tumor explants were also evaluated by mass spectrometry for presentation of pIRS2 and pBCAR3 peptides. For the clinical trial, participants with resected stage IIA-IV melanoma were vaccinated 6 times over 12 weeks with one or both peptides in incomplete Freund's adjuvant and Hiltonol (poly-ICLC). Adverse events (AEs) were coded based on National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) V.4.03, with provision for early study termination if dose-limiting toxicity (DLT) rates exceeded 33%. The enrollment target was 12 participants evaluable for immune response to each peptide. T-cell responses were assessed by interferon-γ ELISpot assay. RESULTS: pBCAR3 peptides were immunogenic in vivo in mice, and in vitro in normal human donors, and T-cells specific for pBCAR3126-134 controlled outgrowth of a tumor xenograft. The pIRS21097-1105 peptide was identified by mass spectrometry from human hepatocellular carcinoma tumors. In the clinical trial, 15 participants were enrolled. All had grade 1 or 2 treatment-related AEs, but there were no grade 3-4 AEs, DLTs or deaths on study. T-cell responses were induced to the pIRS21097-1105 peptide in 5/12 patients (42%, 90% CI 18% to 68%) and to the pBCAR3126-134 peptide in 2/12 patients (17%, 90% CI 3% to 44%). CONCLUSION: This study supports the safety and immunogenicity of vaccines containing the cancer-associated phosphopeptides pBCAR3126-134 and pIRS21097-1105, and the data support continued development of immune therapy targeting phosphopeptides. Future studies will define ways to further enhance the magnitude and durability of phosphopeptide-specific immune responses. TRIAL REGISTRATION NUMBER: NCT01846143.

Murine xenograft bioreactors for human immunopeptidome discovery.

The study of peptides presented by MHC class I and class II molecules is limited by the need for relatively large cell numbers, especially when studying post-translationally modified or otherwise rare peptide species. To overcome this problem, we pose the hypothesis that human cells grown as xenografts in immunodeficient mice should produce equivalent immunopeptidomes as cultured cells. Comparing human cell lines grown either in vitro or as murine xenografts, we show that the immunopeptidome is substantially preserved. Numerous features are shared across both sample types, including peptides and proteins featured, length distributions, and HLA-binding motifs. Peptides well-represented in both groups were from more abundant proteins, or those with stronger predicted HLA binding affinities. Samples grown in vivo also recapitulated a similar phospho-immunopeptidome, with common sequences being those found at high copy number on the cell surface. These data indicate that xenografts are indeed a viable methodology for the production of cells for immunopeptidomic discovery.

Prevalence and significance of anti-saccharomyces cerevisiae antibodies in primary Sjögren's syndrome.

OBJECTIVES: Saccharomyces cerevisiae is a common yeast used in the food industry. IgG and IgA antibodies against the phosphopeptidomannan of the S. cerevisiae cell wall (ASCA) are a well known marker of disease severity in Crohn's disease. Moreover, a number of studies assessed ASCA in several systemic and organ-specific autoimmune diseases postulating molecular mimicry as a possible link between ASCA and autoimmunity. However, since they have never been tested in primary Sjögren's syndrome (pSS), the purpose of this study was to investigate these antibodies in a large cohort of pSS patients, compared to healthy donors (HD), and their significance as potentially helpful biomarker in a clinical setting. METHODS: ASCA IgG+IgA were assessed with ASCA screen dot for Blue Diver instrument (Alphadia sa/nv, Belgium). The comparison between the aminoacid sequence of mannan of S. cerevisiae and well characterised auto-antigens peculiar to pSS (52kD and 60kD Ro/SSA, La/SSB) was performed with the Basic Local Alignment Search Tool (BLAST). RESULTS: The prevalence of ASCA in our pSS cohort was 4.8%. We also reported that the ASCA target protein has a high similarity with Ro60/SSA protein further supporting the molecular mimicry hypothesis. Finally, we observed that ASCA positivity is associated with pSS specific clinical and serological features. ASCA+ pSS patients displayed a triple combination of circulating anti-Ro52/SSA, anti-Ro60/SSA and anti-La/SSB antibodies, associated with low complement and cutaneous involvement. CONCLUSIONS: Our data suggest a possible pathogenic/prognostic significance of ASCA in pSS.

Using Phospho-Peptides Immobilized on Magnetic Beads for Absorption Control in Immunohistochemistry.

Phospho-specific primary antibodies are used in immunohistochemistry (IHC) to detect phosphorylated sequences in proteins, in some cases they may also cross-react with non- or de-phosphorylated sequences. To rule out nonspecific staining, and to determine that the staining pattern is specific it is necessary to employ a so-called absorption control: phospho-specific primary antibodies are first incubated with phospho-peptide immunogen to block antibody binding sites, and this mixture is applied to tissue sections. If the antibody pre-blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific. However, if the staining does occur, it indicates that the antibody is nonspecific. The drawback of doing absorption by mixing the peptide with the antibody is that in solution such peptide-antibody complexes can dissociate unblocking the antibody which becomes capable of binding to cell and tissue targets, producing unwanted staining. To overcome this problem, we have developed a simple absorption control technique allowing for efficient blocking of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of peptide-antibody complex from the incubation mixture eliminating the risk of un-blocking primary antibodies via their dissociation from the blocking peptide.

IgG1 anti-cell wall and IgG2 anti-phosphopeptidomannan antibodies in the diagnosis of invasive candidiasis and heavy Candida colonization.

We conducted a retrospective study to evaluate the usefulness of immunoglobulin G (IgG) subclasses against Candida cell wall fragments (CW) and phosphopeptidomannan (PPM) for the diagnosis of invasive candidiasis (IC). We analyzed 54 patients with IC (n = 19), Candida heavy colonization (HC; n = 16), and controls (no IC or HC, n = 19).In nonneutropenic patients (n = 47), the sensitivity and specificity values of IgG1 anti-CW and IgG2 anti-PPM in IC were 88%, 59%, and 88%, 94%, respectively. The areas under the receiver operating characteristic curves were 0.69 (0.51-0.88) and 0.901 (0.78-1.02), respectively. IgG1 mean values (arbitrary units) and 95% confidence interval were 46 (20-71), 42 (-0.38 to 84) and 20 (8.3-32) in IC, HC, and in controls, respectively, and discriminated IC but not HC from controls (P = .032, and P = .77, respectively). IgG2 mean values were 26 (9.2-42), 19 (4.4-33), and 3.2 (0.28-6.6) in IC, HC, and in controls, respectively, and discriminated both IC and HC from controls (P < .0001 and P = .035, respectively) but did not separate IC from HC (P = .2). IgG2 showed positivity as early as one day after the IC diagnosis. Antibodies were detected in only two out of a total of seven neutropenic patients.For both IC and HC patients, the diagnostic performance of IgG2 anti-PPM was better than the one of IgG1 anti-CW. In nonneutropenic patients, IgG2 anti-PPM accurately identified not only IC patients but also HC patients at high risk for IC. This marker may help clinicians in the initiation of early preemptive therapy.

Quantitative phosphoproteomic analysis of IL-33-mediated signaling.

Interleukin-33 (IL-33) is a novel member of the IL-1 family of cytokines that plays diverse roles in the regulation of immune responses. IL-33 exerts its effects through a heterodimeric receptor complex resulting in the production and release of proinflammatory cytokines. A detailed understanding of the signaling pathways activated by IL-33 is still unclear. To gain insights into the IL-33-mediated signaling mechanisms, we carried out a SILAC-based global quantitative phosphoproteomic analysis that resulted in the identification of 7191 phosphorylation sites derived from 2746 proteins. We observed alterations in the level of phosphorylation in 1050 sites corresponding to 672 proteins upon IL-33 stimulation. We report, for the first time, phosphorylation of multiple protein kinases, including mitogen-activated protein kinase activated protein kinase 2 (Mapkapk2), receptor (TNFRSF) interacting serine-threonine kinase 1 (Ripk1), and NAD kinase (Nadk) that are induced by IL-33. In addition, we observed IL-33-induced phosphorylation of several protein phosphatases including protein tyrosine phosphatase, nonreceptor-type 12 (Ptpn12), and inositol polyphosphate-5-phosphatase D (Inpp5d), which have not been reported previously. Network analysis revealed an enrichment of actin binding and cytoskeleton reorganization that could be important in macrophage activation induced by IL-33. Our study is the first quantitative analysis of IL-33-regulated phosphoproteome. Our findings significantly expand the understanding of IL-33-mediated signaling events and have the potential to provide novel therapeutic targets pertaining to immune-related diseases such as asthma where dysregulation of IL-33 is observed. All MS data have been deposited in the ProteomeXchange with identifier PXD000984 (http://proteomecentral.proteomexchange.org/dataset/PXD000984).

MHC-restricted phosphopeptides from insulin receptor substrate-2 and CDC25b offer broad-based immunotherapeutic agents for cancer.

Cancer cells display novel phosphopeptides in association with MHC class I and II molecules. In this study, we evaluated two HLA-A2-restricted phosphopeptides derived from the insulin receptor substrate (IRS)-2 and the cell-cycle regulator CDC25b. These proteins are both broadly expressed in multiple malignancies and linked to cancer cell survival. Two phosphopeptides, termed pIRS-21097-1105 and pCDC25b38-46, served as targets of strong and specific CD8 T-cell memory responses in normal human donors. We cloned T-cell receptor (TCR) cDNAs from murine CD8 T-cell lines specific for either pIRS-21097-1105 or pCDC25b38-46. Expression of these TCRs in human CD8 T cells imparted high-avidity phosphopeptide-specific recognition and cytotoxic and cytokine-secreting effector activities. Using these cells, we found that endogenously processed pIRS-21097-1105 was presented on HLA-A2(+) melanomas and breast, ovarian, and colorectal carcinomas. Presentation was correlated with the level of the Ser(1100)-phosphorylated IRS-2 protein in metastatic melanoma tissues. The highest expression of this protein was evident on dividing malignant cells. Presentation of endogenously processed pCDC25b38-46 was narrower, but still evident on HLA-A2(+) melanoma, breast carcinoma, and lymphoblastoid cells. Notably, pIRS-21097-1105-specific and pCDC25b38-46-specific TCR-expressing human CD8 T cells markedly slowed tumor outgrowth in vivo. Our results define two new antigens that may be developed as immunotherapeutic agents for a broad range of HLA-A2(+) cancers.

In vivo phosphorylation of WRKY transcription factor by MAPK.

Plants activate signaling networks in response to diverse pathogen-derived signals, facilitating transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. Identification of phosphorylation targets of MAPK and in vivo detection of the phosphorylated substrates are important processes to elucidate the signaling pathway in plant immune responses. We have identified a WRKY transcription factor, which is phosphorylated by defense-related MAPKs, SIPK and WIPK. Recent evidence demonstrated that some group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are activated by MAPK-dependent phosphorylation. In this chapter, we describe protocols for preparation of anti-phosphopeptide antibodies, detection of activated MAPKs using anti-phospho-MAPK antibody, and activated WRKY using anti-phospho-WRKY antibody, respectively.

Method for the generation of antibodies specific for site and posttranslational modifications.

Protein phosphorylation plays critical roles in multiple aspects of cellular events. Site- and phosphorylation state-specific antibodies are indispensable to analyze spatially and temporally distribution of protein phosphorylation in cells. Such information provides some clues of its biological function. Here, we describe a strategy to design a phosphopeptide as an antigen for a site- and phosphorylation state-specific antibody. Importantly, this strategy is also applicable to the production of other types of antibodies, which specifically recognize the site-specific modification, such as acetylation, methylation, and proteolysis. This protocol also focuses on the screening for monoclonal version of a site- and phosphorylation state-specific antibody.

Quantitative phosphoproteomic analysis of RIP3-dependent protein phosphorylation in the course of TNF-induced necroptosis.

Tumor necrosis factor (TNF) induced cell death in murine fibrosarcoma L929 cells is a model system in studying programed necrosis (also known as necroptosis). Receptor interacting protein 3 (RIP3), a serine-threonine kinase, is known to play an essential role in TNF-induced necroptosis; however, the phosphorylation events initiated by RIP3 activation in necroptotic process is still largely unknown. Here, we performed a quantitative MS based analysis to compare TNF-induced changes in the global phosphoproteome of wild-type (RIP3(+/+) ) and RIP3-knockdown L929 cells at different time points after TNF treatment. A total of 8058 phosphopeptides spanning 6892 phosphorylation sites in 2762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 h. By comparing the phosphorylation sites in wild-type and RIP3-knockdown L929 cells, 174, 167, and 177 distinct phosphorylation sites were revealed to be dependent on RIP3 at the 0.5, 2, and 4 h time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3-dependent phosphorylation in lipopolysaccharide-stimulated peritoneal macrophages and TNF-treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report are highly relevant to the study of TNF-induced necroptosis of L929 cells.

MHC class I-associated phosphopeptides are the targets of memory-like immunity in leukemia.

Deregulation of signaling pathways is a hallmark of malignant transformation. Signaling-associated phosphoproteins can be degraded to generate cancer-specific phosphopeptides that are presented by major histocompatibility complex (MHC) class I and II molecules and recognized by T cells; however, the contribution of these phosphoprotein-specific T cells to immune surveillance is unclear. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8(+) T cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and human leukocyte antigen-matched primary leukemia cells ex vivo. Notably, healthy individuals showed robust CD8(+) T cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients. This reduction correlated with clinical outcome; however, immunity was restored after allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T cell adoptive transfer immunotherapies.

Nature-inspired design of motif-specific antibody scaffolds.

Aberrant changes in post-translational modifications (PTMs) such as phosphate groups underlie a majority of human diseases. However, detection and quantification of PTMs for diagnostic or biomarker applications often require PTM-specific monoclonal antibodies (mAbs), which are challenging to generate using traditional antibody-selection methods. Here we outline a general strategy for producing synthetic, PTM-specific mAbs by engineering a motif-specific 'hot spot' into an antibody scaffold. Inspired by a natural phosphate-binding motif, we designed and selected mAb scaffolds with hot spots specific for phosphoserine, phosphothreonine or phosphotyrosine. Crystal structures of the phospho-specific mAbs revealed two distinct modes of phosphoresidue recognition. Our data suggest that each hot spot functions independently of the surrounding scaffold, as phage display antibody libraries using these scaffolds yielded >50 phospho- and target-specific mAbs against 70% of target peptides. Our motif-specific scaffold strategy may provide a general solution for rapid, robust development of anti-PTM mAbs for signaling, diagnostic and therapeutic applications.

Identification of the lamin A/C phosphoepitope recognized by the antibody P-STM in mitotic HeLa S3 cells.

BACKGROUND: Lamins A and C, two major structural components of the nuclear lamina that determine nuclear shape and size, are phosphoproteins. Phosphorylation of lamin A/C is cell cycle-dependent and is involved in regulating the assembly-disassembly of lamin filaments during mitosis. We previously reported that P-STM, a phosphoepitope-specific antibody raised against the autophosphorylation site of p21-activated kinase 2, recognizes a number of phosphoproteins, including lamins A and C, in mitotic HeLa cells. RESULTS: Here, using recombinant proteins and synthetic phosphopeptides containing potential lamin A/C phosphorylation sites in conjunction with in vitro phosphorylation assays, we determined the lamin A/C phosphoepitope(s) recognized by P-STM. We found that phosphorylation of Thr-19 is required for generating the P-STM phosphoepitope in lamin A/C and showed that it could be created in vitro by p34cdc2/cyclin B kinase (CDK1)-catalyzed phosphorylation of lamin A/C immunoprecipitated from unsynchronized HeLa S3 cells. To further explore changes in lamin A/C phosphorylation in living cells, we precisely quantified the phosphorylation levels of Thr-19 and other sites in lamin A/C isolated from HeLa S3 cells at interphase and mitosis using the SILAC method and liquid chromatography-tandem mass spectrometry. The results showed that the levels of phosphorylated Thr-19, Ser-22 and Ser-392 in both lamins A and C, and Ser-636 in lamin A only, increased -2- to 6-fold in mitotic HeLa S3 cells. CONCLUSIONS: Collectively, our results demonstrate that P-STM is a useful tool for detecting Thr-19-phosphorylated lamin A/C in cells and reveal quantitative changes in the phosphorylation status of major lamin A/C phosphorylation sites during mitosis.

A simple competitive enzyme-linked immunosorbent assay for the specific detection of the multiphosphorylated 1-25 ß-casein fragment.

A specific and simple competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine bovine ß-casein phosphopeptides (ß-CPP) in casein phosphopeptides (CPP) or CPP complexes such as casein phosphopeptide amorphous calcium phosphate complexes added into dairy products. The method combines sample pretreatment designed for CPP enrichment and anti-ß-CPP(f(1-25)) monoclonal antibody 1A5 (mAb 1A5). The mAb 1A5 bound specifically to the tryptic phosphopeptides from ß-casein but not from αs1- or αs2-casein. Reactivity was also influenced by the extent of the phosphorylated form of serine residues. Based on the sequence-specific recognition and contribution of phosphorylated serine residues, the epitope of mAb 1A5 was found to reside within the cluster motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu and the surrounding residues in ß-CPP. The competitive ELISA developed here can be used as an alternative to specialised and expensive techniques such as mass spectrometry. In particular, it is suitable for the measurement of CPP or CPP complexes in dairy products, which contain closely related endogenous molecular species.

Using phospho-motif antibodies to determine kinase substrates.

Phosphorylation of substrates by protein kinases regulates a myriad of cellular processes, ranging from proliferation and migration to autophagy, senescence, and apoptosis. Kinase substrate selectivity is largely dependent on the amino acid sequence surrounding the phosphorylation site; therefore, substrate-directed, phosphorylation-state-sensitive, motif-specific ("phospho-motif") antibodies represent powerful tools to identify novel kinase substrates and to investigate mechanisms of substrate phosphorylation in many signaling pathways typically associated with human malignancies. Phospho-motif antibodies are engineered to recognize proteins that contain a phosphorylated residue in the context of a specific motif. They are raised against a library of phospho-peptides comprising both the phosphorylated residue and the surrounding residues that determine kinase specificity, with degenerate residues taking up the remaining positions. Currently, several categories of phospho-motif antibody are commercially available, which may be used to specifically detect Ser, Thr, Ser/Thr, or Tyr residues phosphorylated by different protein kinase families. These antibodies are commonly used in immunoprecipitation and/or immunoblotting protocols to determine kinase-induced substrate phosphorylation. This unit describes the use of phospho-motif antibodies to elucidate the kinase(s) responsible for phosphorylating substrate proteins.

The 4G10, pY20 and p-TYR-100 antibody specificity: profiling by peptide microarrays.

The reversible phosphorylation of tyrosine residues is one of the most frequent post-translational modifications regulating enzymatic activities and protein-protein interactions in eukaryotic cells. Cells responding to internal or external regulatory inputs modify their phosphorylation status and diseased cells can often be diagnosed by observing alterations in their qualitative or quantitative phosphorylation profile. As a consequence the ability to describe the phosphorylation profile of a cell is central to many approaches aiming at the characterisation of signalling pathways. Anti-phosphotyrosine (pY) antibodies are widely used as experimental tools to monitor the phosphorylation status of a cell. By using peptide microarray technology we have characterised the substrate specificity of three widely used pY antibodies. We report that they are more sensitive to sequence context than is generally assumed and that their sequence preferences differ.