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Martin R. Larsen - One of the best experts on this subject based on the ideXlab platform.
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tish a robust and sensitive global phosphoproteomics strategy employing a combination of tio2 simac and hilic
Journal of Proteomics, 2012Co-Authors: Kasper Engholmkeller, Pernille Birck, Joachim Størling, Flemming Pociot, Thomas Mandruppoulsen, Martin R. LarsenAbstract:Abstract Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, Phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO 2 Phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated “TiSH”). The advantages of the strategy include a high specificity and sample preparation workload reduction due to the TiO 2 pre-enrichment step, as well as low adsorptive losses. We demonstrate the capability of this strategy by quantitative investigation of early interferon-γ signaling in low quantities of insulinoma cells. We identified ~ 6600 unique Phosphopeptides from 300 μg of peptides/condition (22 unique Phosphopeptides/μg) in a duplex dimethyl labeling experiment, with an enrichment specificity > 94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-γ pathway, thereby supporting the utility of this global phosphoproteomics strategy. This strategy thus shows great potential for interrogating signaling networks from low amounts of sample with high sensitivity and specificity.
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TiSH - a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC
Journal of Proteomics, 2012Co-Authors: Pernille Birck, Joachim Størling, Thomas Mandrup-poulsen, Kasper Engholm-keller, Martin R. Larsen, Flemming PociotAbstract:Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, Phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO2Phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated "TiSH"). The advantages of the strategy include a high specificity and sample preparation workload reduction due to the TiO2pre-enrichment step, as well as low adsorptive losses. We demonstrate the capability of this strategy by quantitative investigation of early interferon-γ signaling in low quantities of insulinoma cells. We identified ~6600 unique Phosphopeptides from 300μg of peptides/condition (22 unique Phosphopeptides/μg) in a duplex dimethyl labeling experiment, with an enrichment specificity>94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-γ pathway, thereby supporting the utility of this global phosphoproteomics strategy. This strategy thus shows great potential for interrogating signaling networks from low amounts of sample with high sensitivity and specificity. © 2012 Elsevier B.V.
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evaluation of the impact of some experimental procedures on different Phosphopeptide enrichment techniques
Rapid Communications in Mass Spectrometry, 2007Co-Authors: Soren Skov Jensen, Martin R. LarsenAbstract:The complete characterization of phosphorylated proteins requires an efficient procedure for the enrichment of Phosphopeptides from amongst a complicated peptide mixture. The sensitivity of the traditional immobilized metal affinity chromatography (IMAC) approach is severely affected by various buffers, detergents and other reagents normally utilized in biochemical and cell biological procedures, and thus pre-purification steps such as reversed-phase chromatography is required prior to Phosphopeptide enrichment. Here we evaluate the use of different ‘non-Phosphopeptide-excluding compounds’ in the loading buffer for titanium dioxide (TiO2) chromatography and show that TiO2 is more robust and tolerant towards many reagents, including salts, detergents and other low molecular mass molecules, than conventional IMAC. In addition, we show that the inclusion of various detergents can enhance the efficiency of this enrichment method, as Phosphopeptides that otherwise adhere to plastic surfaces can be efficiently solubilized and subsequently purified. The TiO2 chromatography technique is also compared to zirconium dioxide chromatography for Phosphopeptide enrichment. Copyright © 2007 John Wiley & Sons, Ltd.
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highly selective enrichment of phosphorylated peptides using titanium dioxide
Nature Protocols, 2006Co-Authors: Tine E Thingholm, Ole Norregaard Jensen, Thomas J D Jorgensen, Martin R. LarsenAbstract:The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective Phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward Phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although Phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH ≥ 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of Phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).
Ruedi Aebersold - One of the best experts on this subject based on the ideXlab platform.
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confident site localization using a simulated Phosphopeptide spectral library
Journal of Proteome Research, 2015Co-Authors: Veronika Suni, Ruedi Aebersold, Susumu Y Imanishi, Alessio Maiolica, Garry L CorthalsAbstract:We have investigated if Phosphopeptide identification and simultaneous site localization can be achieved by spectral library searching. This allows taking advantage of comparison of specific spectral features, which would lead to improved discrimination of differential localizations. For building a library, we propose a spectral simulation strategy where all possible single phosphorylations can be simply and accurately (re)constructed on enzymatically dephosphorylated peptides, by predicting the diagnostic fragmentation events produced in beam-type CID. To demonstrate the performance of our approach, enriched HeLa Phosphopeptides were dephosphorylated with alkaline phosphatase and analyzed with higher energy collisional dissociation (HCD), which were then used for creating a spectral library of simulated Phosphopeptides. Spectral library searching using SpectraST was performed on data sets of synthetic Phosphopeptides and the HeLa Phosphopeptides, and subsequently compared to Mascot and Sequest database s...
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Reproducible isolation of distinct, overlapping segments of the phosphoproteome
Nature Methods, 2007Co-Authors: Bernd Bodenmiller, Lukas N Mueller, Markus Mueller, Bruno Domon, Ruedi AebersoldAbstract:The ability to routinely analyze and quantitatively measure changes in protein phosphorylation on a proteome-wide scale is essential for biological and clinical research. We assessed the ability of three common Phosphopeptide isolation methods (phosphoramidate chemistry (PAC), immobilized metal affinity chromatography (IMAC) and titanium dioxide) to reproducibly, specifically and comprehensively isolate Phosphopeptides from complex mixtures. Phosphopeptides were isolated from aliquots of a tryptic digest of the cytosolic fraction of Drosophila melanogaster Kc167 cells and analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry. Each method reproducibly isolated Phosphopeptides. The methods, however, differed in their specificity of isolation and, notably, in the set of Phosphopeptides isolated. The results suggest that the three methods detect different, partially overlapping segments of the phosphoproteome and that, at present, no single method is sufficient for a comprehensive phosphoproteome analysis.
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a systematic approach to the analysis of protein phosphorylation
Nature Biotechnology, 2001Co-Authors: Huilin Zhou, Julian D Watts, Ruedi AebersoldAbstract:Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities1,2,3. Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis4,5,6. Thus, there has been a substantial need for a more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures. Here we describe such an approach to protein phosphorylation analysis. It consists of three steps: (1) selective Phosphopeptide isolation from a peptide mixture via a sequence of chemical reactions, (2) Phosphopeptide analysis by automated liquid chromatography–tandem mass spectrometry (LC-MS/MS), and (3) identification of the phosphoprotein and the phosphorylated residue(s) by correlation of tandem mass spectrometric data with sequence databases. By utilizing various phosphoprotein standards and a whole yeast cell lysate, we demonstrate that the method is equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins, and is capable of selectively isolating and identifying Phosphopeptides present in a highly complex peptide mixture.
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identification of flow dependent endothelial nitric oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3 kinase inhibitor ly294002
Journal of Biological Chemistry, 1999Co-Authors: Byron Gallis, Ruedi Aebersold, Garry L Corthals, David R. Goodlett, Hiroto Ueba, Steven R Presnell, Daniel Figeys, David G Harrison, Bradford C Berk, Marshall A CorsonAbstract:Abstract Endothelial cells release nitric oxide (NO) acutely in response to increased laminar fluid shear stress, and the increase is correlated with enhanced phosphorylation of endothelial nitric-oxide synthase (eNOS). Phosphoamino acid analysis of eNOS from bovine aortic endothelial cells labeled with [32P]orthophosphate demonstrated that only phosphoserine was present in eNOS under both static and flow conditions. Fluid shear stress induced phosphate incorporation into two specific eNOS tryptic peptides as early as 30 s after initiation of flow. The flow-induced tryptic Phosphopeptides were enriched, separated by capillary electrophoresis with intermittent voltage drops, also known as “peak parking,” and analyzed by collision-induced dissociation in a tandem mass spectrometer. Two Phosphopeptide sequences determined by tandem mass spectrometry, TQpSFSLQER and KLQTRPpSPGPPPAEQLLSQAR, were confirmed as the two flow-dependent Phosphopeptides by co-migration with synthetic Phosphopeptides. Because the sequence (RIR)TQpSFSLQER contains a consensus substrate site for protein kinase B (PKB or Akt), we demonstrated that LY294002, an inhibitor of the upstream activator of PKB, phosphatidylinositol 3-kinase, inhibited flow-induced eNOS phosphorylation by 97% and NO production by 68%. Finally, PKB phosphorylated eNOS in vitro at the same site phosphorylated in the cell and increased eNOS enzymatic activity by 15–20-fold.
Roland S Annan - One of the best experts on this subject based on the ideXlab platform.
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Hydrophilic interaction chromatography for fractionation and enrichment of the phosphoproteome.
Methods in molecular biology (Clifton N.J.), 2009Co-Authors: Dean E Mcnulty, Roland S AnnanAbstract:Mass spectrometry-based protein phosphorylation analysis on a proteome-wide scale remains a formidable challenge, hampered by the complexity and dynamic range of protein expression on the global level and multi-site phosphorylation at substoichiometric ratios at the individual protein level. It is recognized that reduction of sample complexity or enrichment of the Phosphopeptide pool is a necessary prerequisite for global phospho-proteomics. Immobilized metal affinity chromatography (IMAC) and strong cation exchange chromatography, either alone or in tandem, have emerged as the most widely used chromatographic-based enrichment strategies. However, each is not without shortcomings. Both techniques provide little fractionation of phosphorylated species and are compromised by competition and co-elution of highly acidic peptides. Here, we describe a Phosphopeptide prefractionation scheme using hydrophilic interaction chromatography, which both enriches the Phosphopeptide pool and efficiently fractionates the remaining peptides. When used in front of IMAC, the selectivity of the metal affinity resin is improved to greater than 95%. The lack of significant numbers of nonphosphorylated peptides also allows for more efficient use of the mass spectrometer duty cycle in that the instrument spends nearly all of its time in sequencing the Phosphopeptides.
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a multidimensional electrospray ms based approach to Phosphopeptide mapping
Analytical Chemistry, 2001Co-Authors: Roland S Annan, Michael J Huddleston, Rati Verma, Raymond J Deshaies, Steven A CarrAbstract:A new, multidimensional electrospray MS-based strategy for Phosphopeptide mapping is described which eliminates the need to radiolabel protein with 32P or 33P. The approach utilizes two orthogonal MS scanning techniques, both of which are based on the production of Phosphopeptide-specific marker ions at m/z 63 and/or 79 in the negative ion mode. These scan methods are combined with liquid chromatography−electrospray mass spectrometry and nanoelectrospray MS/MS to selectively detect and identify Phosphopeptides in complex proteolytic digests. Low-abundance, low-stoichiometry phosphorylation sites can be selectively determined in the presence of an excess of nonphosphorylated peptides, even in cases where the signal from the Phosphopeptide is indistinguishable from background in the conventional MS scan. The strategy, which has been developed and refined in our laboratory over the past few years, is particularly well suited to phosphoproteins that are phosphorylated to varying degrees of stoichiometry on mu...
Gavin E Reid - One of the best experts on this subject based on the ideXlab platform.
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Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization
Journal of The American Society for Mass Spectrometry, 2012Co-Authors: Yali Lu, Xiao Zhou, Paul M. Stemmer, Gavin E ReidAbstract:An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded ‘fixed charge’ sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS^3, and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic Phosphopeptides with S , S ′-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized Phosphopeptides. Notably, 44% of the Phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of ‘light’ (S(CH_3)_2) and ‘heavy’ (S(CD_3)_2) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized Phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced Phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS^3 or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency.
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techniques for Phosphopeptide enrichment prior to analysis by mass spectrometry
Mass Spectrometry Reviews, 2009Co-Authors: Jamie D Dunn, Gavin E Reid, Merlin L BrueningAbstract:Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of Phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of Phosphopeptides prior to MS analysis. This review discusses the enrichment of Phosphopeptides with immobilized metal affinity chromatography, reversible covalent binding, and metal oxide affinity chromatography. Capture of Phosphopeptides on TiO2 seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of Phosphopeptides isolated from complex samples. Development of a standard series of Phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on-plate isolation prior to MALDI-MS are emerging as convenient methods for purification of small (µL) samples. On-plate enrichment can yield >70% recoveries of Phosphopeptides in mixtures of a few digested proteins and can avoid sample-handling steps, but this technique is likely limited to relatively simple samples such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:29–54, 2010
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Phosphopeptide enrichment using maldi plates modified with high capacity polymer brushes
Analytical Chemistry, 2008Co-Authors: Jamie D Dunn, Elizabeth A Igrisan, Amanda M Palumbo, Gavin E Reid, Merlin L BrueningAbstract:Matrix-assisted laser desorption/ionization plates coated with poly(2-hydroxyethyl methacrylate) (PHEMA) brushes that are derivatized with Fe(III)-nitrilotriacetate (NTA) complexes allow selective, efficient Phosphopeptide enrichment prior to analysis by mass spectrometry (MS). Fe(III)-NTA-PHEMA brushes (60 nm thick) have a Phosphopeptide binding capacity of 0.6 μg/cm2 and exhibit Phosphopeptide recoveries of over 70%, whereas much thinner polymer films containing Fe(III)-NTA afford a recovery of only 20%, and a monolayer of Fe(III)-NTA shows a recovery of just 10%. Recoveries are determined by comparing signals from enriched unlabeled Phosphopeptides with those of their deuterium-labeled analogues that were added to the plate just prior to addition of matrix. Mass spectra of Phosphopeptide-containing samples enriched using Fe(III)-NTA-PHEMA-modified plates also demonstrate higher recoveries or fewer interfering peaks than corresponding spectra obtained with enrichment using several commercially available...
John R Yates - One of the best experts on this subject based on the ideXlab platform.
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optimizing tio2 based Phosphopeptide enrichment for automated multidimensional liquid chromatography coupled to tandem mass spectrometry
Analytical Chemistry, 2007Co-Authors: Greg T Cantin, Teresa R Shock, Sung Kyu Park, Hiten D Madhani, John R YatesAbstract:An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO2 in the enrichment of Phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of Phosphopeptides, an optimized set of loading, wash, and elution conditions were realized for TiO2. A comparison of TiO2 with other resins used for Phosphopeptide enrichment, Fe(III)-IMAC and ZrO2, was also carried out using tryptic digests of both simple and moderately complex protein mixtures; where TiO2 was shown to be superior in performance.
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automatic validation of Phosphopeptide identifications from tandem mass spectra
Analytical Chemistry, 2007Co-Authors: Bingwen Lu, Sung Kyu Park, Cristian I Ruse, Tao Xu, John R YatesAbstract:We developed and compared two approaches for automated validation of Phosphopeptide tandem mass spectra identified using database searching algorithms. Phosphopeptide identifications were obtained through SEQUEST searches of a protein database appended with its decoy (reversed sequences). Statistical evaluation and iterative searches were employed to create a high-quality data set of Phosphopeptides. Automation of postsearch validation was approached by two different strategies. By using statistical multiple testing, we calculate a p value for each tentative peptide phosphorylation. In a second method, we use a support vector machine (SVM; a machine learning algorithm) binary classifier to predict whether a tentative peptide phosphorylation is true. We show good agreement (85%) between postsearch validation of Phosphopeptide/spectrum matches by multiple testing and that from support vector machines. Automatic methods conform very well with manual expert validation in a blinded test. Additionally, the algo...
