Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae
TL;DR: In this article, a methodology was proposed to characterize most, if not all, phosphoproteins from a whole-cell lysate in a single experiment, and a total of 216 peptide sequences defining 383 sites of phosphorylation were determined.
Abstract: Protein kinases are coded by more than 2,000 genes and thus constitute the largest single enzyme family in the human genome. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine, and tyrosine residues. At least 30% of all proteins are thought to contain covalently bound phosphate. Despite the importance and widespread occurrence of this modification, identification of sites of protein phosphorylation is still a challenge, even when performed on highly purified protein. Reported here is methodology that should make it possible to characterize most, if not all, phosphoproteins from a whole-cell lysate in a single experiment. Proteins are digested with trypsin and the resulting peptides are then converted to methyl esters, enriched for phosphopeptides by immobilized metal-affinity chromatography (IMAC), and analyzed by nanoflow HPLC/electrospray ionization mass spectrometry. More than 1,000 phosphopeptides were detected when the methodology was applied to the analysis of a whole-cell lysate from Saccharomyces cerevisiae. A total of 216 peptide sequences defining 383 sites of phosphorylation were determined. Of these, 60 were singly phosphorylated, 145 doubly phosphorylated, and 11 triply phosphorylated. Comparison with the literature revealed that 18 of these sites were previously identified, including the doubly phosphorylated motif pTXpY derived from the activation loop of two mitogen-activated protein (MAP) kinases. We note that the methodology can easily be extended to display and quantify differential expression of phosphoproteins in two different cell systems, and therefore demonstrates an approach for "phosphoprofiling" as a measure of cellular states.
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TL;DR: The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
Abstract: Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
6,597 citations
TL;DR: A general mass spectrometric technology is developed and applied for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location to provide a missing link in a global, integrative view of cellular regulation.
3,404 citations
Cites background from "Phosphoproteome analysis by mass sp..."
..., hypothesis-free) analysis of in vivo phosphorylation (Aebersold and Mann, 2003; Chen and White, 2004; Ficarro et al., 2002; Mumby and Brekken, 2005; Rush et al., 2005; Salomon et al., 2003; Stover et al., 2004)....
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...…become a powerful technology for proteomics and a method of choice for unbiased (i.e., hypothesis-free) analysis of in vivo phosphorylation (Aebersold and Mann, 2003; Chen and White, 2004; Ficarro et al., 2002; Mumby and Brekken, 2005; Rush et al., 2005; Salomon et al., 2003; Stover et al., 2004)....
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TL;DR: A linear dynamic range over 2 orders of magnitude is demonstrated by using the number of spectra (spectral sampling) acquired for each protein by the data-dependent acquisition of peptides eluting into the mass spectrometer.
Abstract: Proteomic analysis of complex protein mixtures using proteolytic digestion and liquid chromatography in combination with tandem mass spectrometry is a standard approach in biological studies. Data-dependent acquisition is used to automatically acquire tandem mass spectra of peptides eluting into the mass spectrometer. In more complicated mixtures, for example, whole cell lysates, data-dependent acquisition incompletely samples among the peptide ions present rather than acquiring tandem mass spectra for all ions available. We analyzed the sampling process and developed a statistical model to accurately predict the level of sampling expected for mixtures of a specific complexity. The model also predicts how many analyses are required for saturated sampling of a complex protein mixture. For a yeast-soluble cell lysate 10 analyses are required to reach a 95% saturation level on protein identifications based on our model. The statistical model also suggests a relationship between the level of sampling observed for a protein and the relative abundance of the protein in the mixture. We demonstrate a linear dynamic range over 2 orders of magnitude by using the number of spectra (spectral sampling) acquired for each protein.
2,506 citations
TL;DR: Peptide sequence analysis using a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) is demonstrated and automated acquisition of high-quality, single-scan electron transfer dissociation MS/MS spectra of phosphopeptides separated by nanoflow HPLC is described.
Abstract: Peptide sequence analysis using a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion/ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS/MS spectra of phosphopeptides separated by nanoflow HPLC is described.
2,349 citations
TL;DR: A novel MS/MS-based analysis strategy using isotopomer labels, referred to as "tandem mass tags" (TMTs), for the accurate quantification of peptides and proteins is described, which allows peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods.
Abstract: A novel MS/MS-based analysis strategy using isotopomer labels, referred to as “tandem mass tags” (TMTs), for the accurate quantification of peptides and proteins is described. The new tags are designed to ensure that identical peptides labeled with different TMTs exactly comigrate in all separations. The tags require novel methods of quantification analysis using tandem mass spectrometry. The new tags and analysis methods allow peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods. The new TMTs permit simultaneous determination of both the identity and relative abundances of peptide pairs using a collision induced dissociation (CID)-based analysis method. Relative abundance measurements made in the MS/MS mode using the new tags are accurate and sensitive. Compared to MS-mode measurements, a very high signal-to-noise ratio is achieved with MS/MS based detection. The new tags should be applicable to a wide variety of peptide isolation ...
2,183 citations
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TL;DR: The approach described in this manuscript provides a convenient method to interpret tandem mass spectra with known sequences in a protein database.
6,317 citations
TL;DR: Extreme codon bias is seen for the Saccharomyces cerevisiae genes for the fermentative alcohol dehydrogenase isozyme I (ADH-I) and glyceraldehyde-3-phosphate dehydrogenased genes and a similar phenomenon is observed in the codon preferences of highly expressed genes in Escherichia coli.
1,490 citations
TL;DR: Evidence indicates that a novel class of proteins, known as targetting subunits, specifies the location, catalytic and regulatory properties of protein phosphatases and kinases, and thereby plays a key role in ensuring the fidelity ofprotein phosphorylation.
873 citations
TL;DR: A method for enriching phosphoserine/threonine-containing proteins from crude cell extracts and for subsequently identifying the phosphoproteins and sites of phosphorylation is described, which involves chemical replacement of the phosphate moieties by affinity tags and should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylated or dephosphorylation of serine/Threonine residues.
Abstract: The current progression from genomics to proteomics is fueled by the realization that many properties of proteins (e.g., interactions, post-translational modifications) cannot be predicted from DNA sequence1. Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry after two-dimensional electrophoretic separation, it can be difficult to elucidate low-abundance proteins of interest in the presence of a large excess of relatively abundant proteins2,3. Therefore, for effective proteome analysis it becomes critical to enrich the sample to be analyzed in subfractions of interest. For example, the analysis of protein kinase substrates can be greatly enhanced by enriching the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies4, the enrichment of phosphoserine/threonine-containing proteins has not been routinely possible. Here, we describe a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts, and for subsequently identifying the phosphoproteins and sites of phosphorylation. The method, which involves chemical replacement of the phosphate moieties by affinity tags, should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylation or dephosphorylation of serine/threonine residues. Our approach to phosphoprotein enrichment and mapping is based on site-specific modification of phosphoseryl/phosphothreonyl residues. Using the property that the phosphate moiety on such residues is labile at high pH 5‐14 , we chemically replace these phosphates by biotinylated moieties (Fig. 1A). These biotin groups can then be used as affinity handles for immobilized avidin enrichment of the formerly phosphorylated proteins from complex mixtures of proteins. Individual protein components in the enriched fraction can be separated by gel electrophoresis, digested with trypsin, and characterized by mass spectrometry. Alternatively, the entire protein mixture can be digested with trypsin, whereupon the biotinylated tryptic peptide fraction can be enriched with immobilized avidin and characterized by mass spectrometry. Under strongly alkaline conditions the phosphate moiety on phosphoseryl residues undergoes β-elimination to form the reactive dehydroalanyl residue 5‐10
775 citations
TL;DR: Hen egg albumin (ovalbumin) was fractionated into three components of varying phosphate contents and Porcine pepsin was purified in a similar manner to develop purification procedures for phosphoproteins.
765 citations