Site-specific Phosphorylation Events Research Articles

A basic phosphoproteomic-DIA workflow integrating precise quantification of phosphosites in systems biology.

Phosphorylation is one of the most important post-translational modifications (PTMs) of proteins, governing critical protein functions. Most human proteins have been shown to undergo phosphorylation, and phosphoproteomic studies have been widely applied due to recent advancements in high-resolution mass spectrometry technology. Although the experimental workflow for phosphoproteomics has been well-established, it would be useful to optimize and summarize a detailed, feasible protocol that combines phosphoproteomics and data-independent acquisition (DIA), along with follow-up data analysis procedures due to the recent instrumental and bioinformatic advances in measuring and understanding tens of thousands of site-specific phosphorylation events in a single experiment. Here, we describe an optimized Phos-DIA protocol, from sample preparation to bioinformatic analysis, along with practical considerations and experimental configurations for each step. The protocol is designed to be robust and applicable for both small-scale phosphoproteomic analysis and large-scale quantification of hundreds of samples for studies in systems biology and systems medicine.

Phosphoproteomic Analysis of Primary Myeloma Patient Samples Identifies Distinct Phosphorylation Signatures Correlating with Chemo-Sensitivity Profiles in an Ex Vivo Drug Sensitivity Testing Platform

Introduction: Multiple myeloma (MM) is characterized by the clonal expansion of plasma cells in the bone marrow resulting in end-organ damage. Despite an extensive increase in the five-year survival rate in recent years, MM is still considered an incurable disease as patients will repeatedly relapse and develop resistance to current chemotherapies. A key focus for the personalization of myeloma therapy is understanding the biological mechanisms of drug resistance and identifying clinically useful biomarkers of therapeutic response. Highly efficient techniques for the enrichment of phosphorylated peptides followed by high resolution mass spectrometry facilitates the quantitation of thousands of site-specific phosphorylation events. Here, we have performed a phosphoproteomic analysis on MM cell lysates stratified based on their ex vivo drug response profiles to advance our understanding of drug resistance mechanisms.Materials and Methods: CD138 + plasma cells were isolated from 20 adult MM patient bone marrow aspirates at diagnosis (n=7) or relapse (n=13). Samples were grouped based on ex vivo drug sensitivity and resistance testing (DSRT) as follows: highly sensitive (Group I), sensitive (Group II), resistant (Group III), highly resistant (Group IV) [1]. For the phosphoproteomic analysis, peptides were generated and purified using the filter aided sample preparation (FASP) protocol. Peptide tandem mass tag (TMT) labelling, Fe 3+ immobilized metal ion affinity chromatography (IMAC), synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) was performed. Nonenriched peptides were used for proteomic analysis. Resulting data was analysed using MaxQuant, followed by normalization of phosphosite intensities using the internal reference scale (IRS) method, and statistical analysis using Perseus. Functional enrichment and kinase enrichment analyses were performed on significant phosphoproteins using g:profiler and KEA2, respectively.Results: Our quantitative MS-based phosphoproteomic analysis identified 2,945 phosphorylation sites on 2,232 phosphopeptides from 690 phosphoproteins. Of these phosphorylation sites, 176 were significantly changed between all four DSRT groups and 267 were significantly changed between Group I and Group IV (False Discovery Rate (FDR) < 0.05). Hierarchical clustering was performed to highlight the distinct phosphoproteomic profiles associated with each DSRT group, of which the very sensitive (Group I) and very resistant (Group IV) subgroups demonstrated a well-defined separation (Fig. 1A, 1B). KEGG pathway analysis and gene ontology (GO) analysis of significantly increased phosphorylated proteins in Group IV compared to Group I MM patients demonstrated an increased phosphorylation of proteins associated with tight junctions, the Rap1 signalling pathway and the phosphatidylinositol signalling system; indicating an upregulation of cell adhesion associated processes in drug resistant MM. Phosphoproteins increased in abundance in Group I compared to Group IV MM patients revealed an increased phosphorylation of proteins involved in translation and RNA processing including the spliceosome, RNA transport and RNA binding pathways (Fig. 1C). We identified filamin A serine 2152, RAS guanyl-releasing protein 2 serine 576 and proto-oncogene tyrosine-protein kinase Src serine 17 as increased in Group IV MM, and nuclease-sensitive element-binding protein 1 (YBX1) serine 165, CD44 serine 697 and Bcl2-associated agonist of cell death (BAD) serine 99 as increased in Group I MM. KEA of the upregulated phosphoproteome in Group IV revealed an enrichment of cyclin dependent kinase 1 (CDK1) and ribosomal s6 kinases (RPS6K) whereas casein kinase 2 (CK2) and the glycolysis-associated kinases were enriched in Group I (Fig. 1D).Conclusion: Our study has generated a phosphoproteomic dataset demonstrating distinct phosphorylation signatures associated with drug sensitivity in clinical MM plasma cells. The identification of phosphorylation events associated with drug resistance provides a basis for further exploration of these events and associated signalling pathways to further understand drug resistance mechanisms in MM and identify potential biomarkers of therapeutic response and targets for drug re-sensitization in MM.

Alterations in the CO2 availability induce alterations in the phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria are the only prokaryotes that perform plant-like oxygenic photosynthesis. They evolved an inorganic carbon-concentrating mechanism to adapt to low CO2 conditions. Quantitative phosphoproteomics was applied to analyze regulatory features during the acclimation to low CO2 conditions in the model cyanobacterium Synechocystis sp. PCC 6803. Overall, more than 2500 proteins were quantified, equivalent to c.70% of the Synechocystis theoretical proteome. Proteins with changing abundances correlated largely with mRNA expression levels. Functional annotation of the noncorrelating proteins revealed an enrichment of key metabolic processes fundamental for maintaining cellular homeostasis. Furthermore, 105 phosphoproteins harboring over 200 site-specific phosphorylation events were identified. Subunits of the bicarbonate transporter BCT1 and the redox switch protein CP12 were among phosphoproteins with reduced phosphorylation levels at lower CO2 , whereas the serine/threonine protein kinase SpkC revealed increased phosphorylation levels. The corresponding ΔspkC mutant was characterized and showed decreased ability to acclimate to low CO2 conditions. Possible phosphorylation targets of SpkC including a BCT1 subunit were identified by phosphoproteomics. Collectively, our study highlights the importance of posttranscriptional regulation of protein abundances as well as posttranslational regulation by protein phosphorylation for the successful acclimation towards low CO2 conditions in Synechocystis and possibly among cyanobacteria.

A data-independent acquisition-based global phosphoproteomics system enables deep profiling

Phosphoproteomics can provide insights into cellular signaling dynamics. To achieve deep and robust quantitative phosphoproteomics profiling for minute amounts of sample, we here develop a global phosphoproteomics strategy based on data-independent acquisition (DIA) mass spectrometry and hybrid spectral libraries derived from data-dependent acquisition (DDA) and DIA data. Benchmarking the method using 166 synthetic phosphopeptides shows high sensitivity (<0.1 ng), accurate site localization and reproducible quantification (~5% median coefficient of variation). As a proof-of-concept, we use lung cancer cell lines and patient-derived tissue to construct a hybrid phosphoproteome spectral library covering 159,524 phosphopeptides (88,107 phosphosites). Based on this library, our single-shot streamlined DIA workflow quantifies 36,350 phosphosites (19,755 class 1) in cell line samples within two hours. Application to drug-resistant cells and patient-derived lung cancer tissues delineates site-specific phosphorylation events associated with resistance and tumor progression, showing that our workflow enables the characterization of phosphorylation signaling with deep coverage, high sensitivity and low between-run missing values.

Cell cycle checkpoint control: The cyclin G1/Mdm2/p53 axis emerges as a strategic target for broad-spectrum cancer gene therapy - A review of molecular mechanisms for oncologists.

Basic research in genetics, biochemistry and cell biology has identified the executive enzymes and protein kinase activities that regulate the cell division cycle of all eukaryotic organisms, thereby elucidating the importance of site-specific protein phosphorylation events that govern cell cycle progression. Research in cancer genomics and virology has provided meaningful links to mammalian checkpoint control elements with the characterization of growth-promoting proto-oncogenes encoding c-Myc, Mdm2, cyclins A, D1 and G1, and opposing tumor suppressor proteins, such as p53, pRb, p16INK4A and p21WAF1, which are commonly dysregulated in cancer. While progress has been made in identifying numerous enzymes and molecular interactions associated with cell cycle checkpoint control, the marked complexity, particularly the functional redundancy, of these cell cycle control enzymes in mammalian systems, presents a major challenge in discerning an optimal locus for therapeutic intervention in the clinical management of cancer. Recent advances in genetic engineering, functional genomics and clinical oncology converged in identifying cyclin G1 (CCNG1 gene) as a pivotal component of a commanding cyclin G1/Mdm2/p53 axis and a strategic locus for re-establishing cell cycle control by means of therapeutic gene transfer. The purpose of the present study is to provide a focused review of cycle checkpoint control as a practicum for clinical oncologists with an interest in applied molecular medicine. The aim is to present a unifying model that: i) clarifies the function of cyclin G1 in establishing proliferative competence, overriding p53 checkpoints and advancing cell cycle progression; ii) is supported by studies of inhibitory microRNAs linking CCNG1 expression to the mechanisms of carcinogenesis and viral subversion; and iii) provides a mechanistic basis for understanding the broad-spectrum anticancer activity and single-agent efficacy observed with dominant-negative cyclin G1, whose cytocidal mechanism of action triggers programmed cell death. Clinically, the utility of companion diagnostics for cyclin G1 pathways is anticipated in the staging, prognosis and treatment of cancers, including the potential for rational combinatorial therapies.

SimPhospho: a software tool enabling confident phosphosite assignment.

MotivationMass spectrometry combined with enrichment strategies for phosphorylated peptides has been successfully employed for two decades to identify sites of phosphorylation. However, unambiguous phosphosite assignment is considered challenging. Given that site-specific phosphorylation events function as different molecular switches, validation of phosphorylation sites is of utmost importance. In our earlier study we developed a method based on simulated phosphopeptide spectral libraries, which enables highly sensitive and accurate phosphosite assignments. To promote more widespread use of this method, we here introduce a software implementation with improved usability and performance.ResultsWe present SimPhospho, a fast and user-friendly tool for accurate simulation of phosphopeptide tandem mass spectra. Simulated phosphopeptide spectral libraries are used to validate and supplement database search results, with a goal to improve reliable phosphoproteome identification and reporting. The presented program can be easily used together with the Trans-Proteomic Pipeline and integrated in a phosphoproteomics data analysis workflow.Availability and implementationSimPhospho is open source and it is available for Windows, Linux and Mac operating systems. The software and its user’s manual with detailed description of data analysis as well as test data can be found at https://sourceforge.net/projects/simphospho/.Supplementary information Supplementary data are available at Bioinformatics online.

Targeted mass spectrometry: An emerging powerful approach to unblock the bottleneck in phosphoproteomics.

Following the rapid expansion of the proteomics field, the investigation of post translational modifications (PTM) has become extremely popular changing our perspective of how proteins constantly fine tune cellular functions. Reversible protein phosphorylation plays a pivotal role in virtually all biological processes in the cell and it is one the most characterized PTM up to date. During the last decade, the development of phosphoprotein/phosphopeptide enrichment strategies and mass spectrometry (MS) technology has revolutionized the field of phosphoproteomics discovering thousands of new site-specific phosphorylations and unveiling unprecedented evidence about their modulation under distinct cellular conditions. The field has expanded so rapidly that the use of traditional methods to validate and characterize the biological role of the phosphosites is not feasible any longer. Targeted MS holds great promise for becoming the method of choice to study with high precision and sensitivity already known site-specific phosphorylation events. This review summarizes the contribution of large-scale unbiased MS analyses and highlights the need of targeted MS-based approaches for follow-up investigation. Additionally, the article illustrates the biological relevance of protein phosphorylation by providing examples of disease-related phosphorylation events and emphasizes the benefits of applying targeted MS in clinics for disease diagnosis, prognosis and drug-response evaluation.

Development of SH2 probes and pull-down assays to detect pathogen-induced, site-specific tyrosine phosphorylation of the TLR adaptor SCIMP.

Protein tyrosine phosphorylation guides many molecular interactions for cellular functions. SCIMP is a transmembrane adaptor protein (TRAP) family member that mediates selective proinflammatory cytokine responses generated by pathogen-activated Toll-like receptor (TLR) pathways in macrophages. TLR activation triggers SCIMP phosphorylation and selective phosphorylation of distinct tyrosine residues on this adaptor offers the potential for regulating or biasing inflammatory responses. To analyze site-specific phosphorylation events, we developed three probes based on the SH2 domains of known SCIMP effectors, and used them for pull-downs from macrophage extracts. CRISPR-mediated SCIMP-deficient RAW264.7 macrophage-like cells were reconstituted with various phosphorylation-deficient (Y58F, Y96F, Y120F) SCIMPs, and used to demonstrate the specificity of LPS/TLR4-induced, site-specific phosphorylation of SCIMP for the temporal recruitment of the effectors Grb2, Csk and SLP65. Our findings reveal potential for differential SCIMP phosphorylation and specific effectors to influence TLR signaling and inflammatory programs. Furthermore, the use of Csk-SH2 pull-downs to identify additional known and new Csk targets in LPS-activated macrophages reveals the wider utility of our SH2 probes.

Characterization of Receptor-Associated Protein Complex Assembly in Interleukin (IL)-2- and IL-15-Activated T-Cell Lines.

It remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. We have previously dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells, unveiling subtle differences that may contribute to their functional dichotomy. In this study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is highly orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and -independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2 receptor β subunit (IL-2Rβ). We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Overall, our study sheds new light into the initial molecular events triggered by IL-2 and IL-15 and constitutes a further step toward a better understanding of the early signaling aspects of the two closely related cytokines in T-lymphocytes.

The Peripheral Blood Eosinophil Proteome

A system-wide understanding of biological processes requires a comprehensive knowledge of the proteins in the biological system. The eosinophil is a type of granulocytic leukocyte specified early in hematopoietic differentiation that participates in barrier defense, innate immunity, and allergic disease. The proteome of the eosinophil is largely unannotated with under 500 proteins identified. We now report a map of the nonstimulated peripheral blood eosinophil proteome assembled using two-dimensional liquid chromatography coupled with high-resolution mass spectrometry. Our analysis yielded 100,892 unique peptides mapping to 7,086 protein groups representing 6,813 genes as well as 4,802 site-specific phosphorylation events. We account for the contribution of platelets that routinely contaminate purified eosinophils and report the variability in the eosinophil proteome among five individuals and proteomic changes accompanying acute activation of eosinophils by interleukin-5. Our deep coverage and quantitative analyses fill an important gap in the existing maps of the human proteome and will enable the strategic use of proteomics to study eosinophils in human diseases.

Integrating titania enrichment, iTRAQ labeling, and Orbitrap CID‐HCD for global identification and quantitative analysis of phosphopeptides

Recent advances in MS instrumentation and progresses in phosphopeptide enrichment, in conjunction with more powerful data analysis tools, have facilitated unbiased characterization of thousands of site-specific phosphorylation events. Combined with stable isotope labeling by amino acids in cell culture metabolic labeling, these techniques have made it possible to quantitatively evaluate phosphorylation changes in various physiological states in stable cell lines. However, quantitative phosphoproteomics in primary cells and tissues remains a major technical challenge due to the lack of adequate techniques for accurate quantification. Here, we describe an integrated strategy allowing for large scale quantitative profiling of phosphopeptides in complex biological mixtures. In this technique, the mixture of proteolytic peptides was subjected to phosphopeptide enrichment using a titania affinity column, and the purified phosphopeptides were subsequently labeled with iTRAQ reagents. After further fractionation by strong-cation exchange, the peptides were analyzed by LC-MS/MS on an Orbitrap mass spectrometer, which collects CID and high-energy collisional dissociation (HCD) spectra sequentially for peptide identification and quantitation. We demonstrate that direct phosphopeptide enrichment of protein digests by titania affinity chromatography substantially improves the efficiency and reproducibility of phosphopeptide proteomic analysis and is compatible with downstream iTRAQ labeling. Conditions were optimized for HCD normalized collision energy to balance the overall peptide identification and quantitation using the relative abundances of iTRAQ reporter ions. Using this approach, we were able to identify 3557 distinct phosphopeptides from HeLa cell lysates, of which 2709 were also quantified from HCD scans.

Maximal entropy inference of oncogenicity from phosphorylation signaling

Point mutations in the phosphorylation domain of the Bcr-Abl fusion oncogene give rise to drug resistance in chronic myelogenous leukemia patients. These mutations alter kinase-mediated signaling function and phenotypic outcome. An information theoretic analysis of the correlation of phosphoproteomic profiling and transformation potency of the oncogene in different mutants is presented. The theory seeks to predict the leukemic transformation potency from the observed signaling by constructing a distribution of maximal entropy of site-specific phosphorylation events. The theory is developed with special reference to systems biology where high throughput measurements are typical. We seek sets of phosphorylation events most contributory to predicting the phenotype by determining the constraints on the signaling system. The relevance of a constraint is measured by how much it reduces the value of the entropy from its global maximum, where all events are equally likely. Application to experimental phospho-proteomics data for kinase inhibitor-resistant mutants shows that there is one dominant constraint and that other constraints are not relevant to a similar extent. This single constraint accounts for much of the correlation of phosphorylation events with the oncogenic potency and thereby usefully predicts the trends in the phenotypic output. An additional constraint possibly accounts for biological fine structure.

Stat1 is an inhibitor of Ras-MAPK signaling and Rho small GTPase expression with implications in the transcriptional signature of Ras transformed cells

The signal transducer and activator of transcription Stat1 plays an indispensable role in the regulation of innate immunity and tumor immuno-surveillance. The anti-tumor activity of Stat1 is largely dependent on its ability to function downstream of interferon (IFN) signaling. However, anti-tumor functions of Stat1 that are independent of IFN signaling have started to emerge. For example, we recently reported that the anti-tumor activity of Stat1 in Ras transformation is affected by Stat1 phosphorylation at tyrosine (Y) 701 and serine (S) 727, both of which determine p27Kip1 expression and function [PLoS ONE 2008;3:e3476]. Herein, we provide further evidence that these site-specific phosphorylation events of Stat1 regulate the inhibition of the Ras-MAPK pathway and expression of RhoA, Cdc42 and Rac1 GTPases in Ras transformed cells. Site-specific Stat1 phosphorylation also controls the transcriptional activities of Stat3 and Stat5 and is capable of orchestrating a complex regulatory network that influences the expression of genes involved in cell cycle control, cell-cell adhesion and signal transduction. These findings further substantiate the notion that Stat1 as a master regulator of Ras-mediated transformation, a property that is intimately affected by its phosphorylation at Y701 and S727.

Quantitative phosphoproteomics – an emerging key technology in signal‐transduction research

Protein phosphorylation is the most important type of reversible post-translational modification involved in the regulation of cellular signal-transduction processes. In addition to controlling normal cellular physiology on the molecular level, perturbations of phosphorylation-based signaling networks and cascades have been implicated in the onset and progression of various human diseases. Recent advances in mass spectrometry-based proteomics helped to overcome many of the previous limitations in protein phosphorylation analysis. Improved isotope labeling and phosphopeptide enrichment strategies in conjunction with more powerful mass spectrometers and advances in data analysis have been integrated in highly efficient phosphoproteomics workflows, which are capable of monitoring up to several thousands of site-specific phosphorylation events within one large-scale analysis. Combined with ongoing efforts to define kinase-substrate relationships in intact cells, these major achievements have considerable potential to assess phosphorylation-based signaling networks on a system-wide scale. Here, we provide an overview of these exciting developments and their potential to transform signal-transduction research into a technology-driven, high-throughput science.

Kinase-Selective Enrichment Enables Quantitative Phosphoproteomics of the Kinome across the Cell Cycle

Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.

Quantitative measurement of epidermal growth factor receptor–mitogen-activated protein kinase signal transduction using a nine-plex, peptide-based immunoassay

Aberrant epidermal growth factor receptor (EGFR, ErbB1) signaling is implicated in cell transformation, motility, and invasion in a variety of cell types, and EGFR is the target of several anticancer drugs. However, the kinetics of EGFR signaling and the individual contributions of site-specific phosphorylation events remain largely unknown. A peptide-based, multiplex immunoassay approach was developed to simultaneously measure both total and phosphorylated protein in a single sample. The approach involves the proteolytic digestion of proteins prior to the isolation and quantitation of site-specific phosphorylation events within an individual protein. Quantitation of phosphorylated and total proteins, in picomolar to nanomolar concentrations, were interpolated from standard curves generated with synthetic peptides that correspond to the peptide targets used in the immunoassays. In this study, a bead-based, nine-plex immunoassay measuring total and phosphorylated protein was constructed to measure temporal, site-specific phosphorylation of key members of the EGFR pathway (ErbB1 receptor, MEK1, MEK2, ERK1, and ERK2) in A431 cells stimulated with epidermal growth factor. The effect of MEK inhibition on this pathway was determined using a known MEK kinase inhibitor, SL327. The results reported herein are the first quantitative measurements of site-specific phosphorylation events and total proteins in a single sample, at the same time representing a new paradigm for standardized protein and phosphorylation analysis using multiplexed, peptide-based, sandwich immunoassays.

Reference-facilitated Phosphoproteomics: FAST AND RELIABLE PHOSPHOPEPTIDE VALIDATION BY μLC-ESI-Q-TOF MS/MS

Recent advances in instrument control and enrichment procedures have enabled us to quantify large numbers of phosphoproteins and record site-specific phosphorylation events. An intriguing problem that has arisen with these advances is to accurately validate where phosphorylation events occur, if possible, in an automated manner. The problem is difficult because MS/MS spectra of phosphopeptides are generally more complicated than those of unmodified peptides. For large scale studies, the problem is even more evident because phosphorylation sites are based on single peptide identifications in contrast to protein identifications where at least two peptides from the same protein are required for identification. To address this problem we have developed an integrated strategy that increases the reliability and ease for phosphopeptide validation. We have developed an off-line titanium dioxide (TiO(2)) selective phosphopeptide enrichment procedure for crude cell lysates. Following enrichment, half of the phosphopeptide fractionated sample is enzymatically dephosphorylated, after which both samples are subjected to LC-MS/MS. From the resulting MS/MS analyses, the dephosphorylated peptide is used as a reference spectrum against the original phosphopeptide spectrum, in effect generating two peptide spectra for the same amino acid sequence, thereby enhancing the probability of a correct identification. The integrated procedure is summarized as follows: 1) enrichment for phosphopeptides by TiO(2) chromatography, 2) dephosphorylation of half the sample, 3) LC-MS/MS-based analysis of phosphopeptides and corresponding dephosphorylated peptides, 4) comparison of peptide elution profiles before and after dephosphorylation to confirm phosphorylation, and 5) comparison of MS/MS spectra before and after dephosphorylation to validate the phosphopeptide and its phosphorylation site. This phosphopeptide identification represents a major improvement as compared with identifications based only on single MS/MS spectra and probability-based database searches. We investigated an applicability of this method to crude cell lysates and demonstrate its application on the large scale analysis of phosphorylation sites in differentiating mouse myoblast cells.

The Role of Tau Phosphorylation in the Pathogenesis of Alzheimers Disease

The microtubule-associated protein tau, which is abundantly expressed in neurons, is deposited in cells in an abnormally phosphorylated state as fibrillar lesions in numerous neurodegenerative diseases, with the most notable being Alzheimer's disease. Tau plays a crucial role in the neuron as it binds and stabilizes microtubules, and can regulate axonal transport; functions that are regulated by site-specific phosphorylation events. In pathological conditions such as Alzheimer's disease and other tauopathies, tau is abnormally phosphorylated, and that this contributes to its dysfunction. Given the increasing evidence that a disruption in the normal phosphorylation state of tau followed by conformational changes plays a key role in the pathogenic events that occur in Alzheimer's disease and other tauopathies; it is critical to elucidate the regulation of tau phosphorylation. This review focuses on recent literature pertaining to the regulation of tau phosphorylation and function, and the role that a dysregulation of tau phosphorylation may play in the neuronal dysfunction in Alzheimer's disease.

An integrated strategy for identification and relative quantification of site‐specific protein phosphorylation using liquid chromatography coupled to MS2/MS3

Reversible and differential multisite protein phosphorylation is an important mechanism controlling the activity of cellular proteins. Here we describe a robust and highly selective approach for the identification and relative quantification of site-specific phosphorylation events. This integrated strategy has three major parts: visualisation of phosphorylated proteins using fluorescently stained polyacrylamide gels, determination of the phosphorylation site(s) using automatic MS3 triggered by the loss of phosphoric acid, and relative quantification of phosphorylation by integrating MS2- and MS3-extracted ion traces using a fast-scanning, linear ion trap mass spectrometer. As a test case, recombinant sucrose-phosphate synthase (SPS) from Arabidopsis thaliana (At5g1110) was used for identification and quantification of site-specific phosphorylation. The identified phosphorylation site of the actively expressed protein coincides with the major regulatory in vivo phosphorylation site in spinach SPS. Site-specific differential in vitro phosphorylation of native protein was demonstrated after incubation of the recombinant protein with cold-adapted plant leaf extracts from A. thaliana, suggesting regulatory phosphorylation events of this key enzyme under stress response.

Tau phosphorylation: physiological and pathological consequences

The microtubule-associated protein tau, abundant in neurons, has gained notoriety due to the fact that it is deposited in cells as fibrillar lesions in numerous neurodegenerative diseases, and most notably Alzheimer's disease. Regulation of microtubule dynamics is the most well-recognized function of tau, but it is becoming increasingly evident that tau plays additional roles in the cell. The functions of tau are regulated by site-specific phosphorylation events, which if dysregulated, as they are in the disease state, result in tau dysfunction and mislocalization, which is potentially followed by tau polymerization, neuronal dysfunction and death. Given the increasing evidence that a disruption in the normal phosphorylation state of tau plays a key role in the pathogenic events that occur in Alzheimer's disease and other neurodegenerative conditions, it is of crucial importance that the protein kinases and phosphatases that regulate tau phosphorylation in vivo as well as the signaling cascades that regulate them be identified. This review focuses on recent literature pertaining to the regulation of tau phosphorylation and function in cell culture and animal model systems, and the role that a dysregulation of tau phosphorylation may play in the neuronal dysfunction and death that occur in neurodegenerative diseases that have tau pathology.