A Strategy for Identification and Quantitation of Phosphopeptides by Liquid Chromatography/Tandem Mass Spectrometry

Tsay, Yeou Guang; Wang, Yan‐Hsiung; Chiu, Chi-Ming; Shen, Bo; Lee, Sheng-Chung

doi:10.1006/abio.2000.4837

Cited by 70 publications

(47 citation statements)

References 42 publications

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“…In-gel Tryptic Digestion and Mass Spectrometry-In-gel tryptic digestion was performed with the previously described procedure (13). The peptides resuspended in 0.1% formic acid were analyzed using a quadrupole ion trap mass spectrometer (Esquire 3000 plus, Bruker-Daltonics, Leipzig, Germany) interfaced with a high-pressure liquid chromatograph system (UltiMate, LC Packings, San Francisco, CA).…”

Section: Methodsmentioning

confidence: 99%

Reversible Acetylation Regulates Salt-inducible Kinase (SIK2) and Its Function in Autophagy*

Yang

Tan

Chen

et al. 2013

Journal of Biological Chemistry

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Background: Salt-inducible kinase (SIK) 2 is an AMP-activated protein kinase family kinase that mediates hormonal and nutrient signaling but has no known link to cellular stress response. Results: p300/CBP and HDAC6 reciprocally regulates Lys-53 acetylation of SIK2, consequently impacting its activity and function in autophagosome maturation. Conclusion: SIK2 kinase activity, via a acetylation-based regulatory switch, contributes to autophagy progression. Significance: SIK2 may be linked to neurodegenerative or protein aggregate disorders.

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Section: Methodsmentioning

confidence: 99%

Reversible Acetylation Regulates Salt-inducible Kinase (SIK2) and Its Function in Autophagy*

Yang

Tan

Chen

et al. 2013

Journal of Biological Chemistry

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“…Label-free approaches to quantitative proteomics have gained prominence in recent years since no additional chemistry or sample preparation steps are required. These include biostatistical profiling [8,9] in clinical proteomics whereby peptide ions that show intensity changes over many samples are recorded and represent potential protein biomarkers, methods for comparing the ratios of integrated peak areas/intensities of phosphorylated to nonphosphorylated peptide ions between two samples [10,11], a strategy to assess the stoichiometry of phosphorylation sites [12], and the popular method of quantifying protein levels from different samples based on the number of MS/MS spectra that identify the protein of interest (spectral counting) [13][14][15][16][17].We demonstrate a method to survey relative protein quantity between samples that is an extension to the spectral counting technique whereby the average of the TIC for all of the MS/MS spectra that identify a protein is used as a quantitative measure. Each spectral count gets a unique abundance value rather than equivalent values of "1" as with spectral counting.…”

mentioning

confidence: 99%

“…These include biostatistical profiling [8,9] in clinical proteomics whereby peptide ions that show intensity changes over many samples are recorded and represent potential protein biomarkers, methods for comparing the ratios of integrated peak areas/intensities of phosphorylated to nonphosphorylated peptide ions between two samples [10,11], a strategy to assess the stoichiometry of phosphorylation sites [12], and the popular method of quantifying protein levels from different samples based on the number of MS/MS spectra that identify the protein of interest (spectral counting) [13][14][15][16][17].…”

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confidence: 99%

A label‐free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen

et al. 2008

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In order to assess the biological function of proteins and their modifications for understanding signaling mechanisms within cells as well as specific biomarkers to disease, it is important that quantitative information be obtained under different experimental conditions. Stable isotope labeling is a powerful method for accurately determining changes in the levels of proteins and PTMs; however, isotope labeling experiments suffer from limited dynamic range resulting in signal change ratios of less than ,20:1 using most commercial mass spectrometers. Label-free approaches to relative quantification in proteomics such as spectral counting have gained popularity since no additional chemistries are needed. Here, we show a label-free method for relative quantification based on the TIC from peptide MS/MS spectra collected from data-dependent runs can be used effectively as a quantitative measure and expands the dynamic range over isotope labeling experiments allowing for abundance differences up to ,60:1 in a screen for proteins that bind to phosphotyrosine residues. Keywords:Differential expression / Quantitative analysis / Shotgun proteomics / Signal transduction profiling 994 Proteomics 2008, 8, 994-999 Methods for acquiring quantitative proteomics data are continually developing with very accurate stable isotope labeling (SIL) and label-free approaches. SIL provides chemically equivalent but isotopically different internal standards for each peptide/protein for direct comparison of mass spectral signal intensities that represent relative abundance. Common SIL strategies include protein level labeling strategies such as stable isotope labeling of amino acids in cell culture (SILAC) [1], a global method whereby all translated proteins have isotope labels metabolically incorporated at selected amino acid residues, and isotope-coded affinity tags (ICAT) [2], a technique that labels cysteine residues at the protein level. Peptide level labeling strategies include multiplexed isobaric tags for relative and absolute quantification (iTRAQ) [3], global internal standard technology (GIST) [4], a global post-digestion labeling method that labels primary amine groups (peptide N-terminus and lysine residues) and an extension of GIST called in-gel stable isotope labeling (ISIL) [5,6], a method that labels primary amine groups of proteins (protein N-terminus and lysine residues) directly from gel separated samples. Each isotope labeling strategy has its advantages and disadvantages depending upon the experimental questions but most suffer from an experimental Proteomics 2008, 8, 994-999 Cell Biology 995 dynamic range limitation of ,20:1. Protein level differences greater than this limit usually suffer from very large errors in ratio determination [7]. Label-free approaches to quantitative proteomics have gained prominence in recent years since no additional chemistry or sample preparation steps are required. These include biostatistical profiling [8,9] in clinical proteomics whereby peptide ions that show intensity chang...

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“…A few researchers have suggested that a comparison of mass spectrometric signals of phosphorylated peptides with internal controls (i.e., an unmodified peptide derived from the same protein) can provide adequate quantitation. The difference between these reported methods seems to be in the choice of internal standard, and whether it should be the corresponding unphosphorylated peptide [25,26] or a peptide derived from the same protein that does not contain any modifications [27]. Ideally, one would want the internal standard to have physical and chemical properties that are most similar to that of the phosphorylated peptide to be quantitated.…”

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Studies of ligand-induced site-specific phosphorylation of epidermal growth factor receptor

Guo

Kozlosky

Ericsson

et al. 2003

J. Am. Soc. Mass Spectrom.

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The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in the regulation of growth in many animal cells, including cancer cells. Phosphorylation of specific tyrosine residues within the cytoplasmic domain of EGFR is part of the initial activation process that occurs upon ligand binding, and these phosphotyrosine residues subsequently serve as docking sites for intracellular signaling molecules. To study the phosphorylation on each individual site, EGFR generated from a human epidermoid carcinoma cell line (A431) was analyzed by mass spectrometry. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) was used to identify the tryptic phosphopeptides and their sites of phosphorylation (Y992, Y1045, Y1068, Y1086, S1142, Y1148, and Y1173). Ion intensities for the phosphorylated and unphosphorylated tryptic peptides containing the sites of phosphorylation were measured, and the intensity ratios were used to assess the degree of phosphorylation at each site. Ligand concentrations were varied for epidermal growth factor (EGF) and transforming growth factor alpha (TGF␣) as stimuli, and all of the EGFR tyrosine sites were consequently found to exhibit increased levels of phosphorylation, although at different rates and to different extents. Phosphorylation of Y992 appeared to plateau at lower concentrations of ligand, whereas the other sites continued to have increased phosphorylation throughout a wide range of concentrations. Only small differences could be detected between the EGF and the TGF␣-induced EGFR phosphorylation. Pretreatment of A431 cells with a small molecule EGFR inhibitor nearly eliminated the ligand-induced phosphorylation on all of the sites except

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A Strategy for Identification and Quantitation of Phosphopeptides by Liquid Chromatography/Tandem Mass Spectrometry

Cited by 70 publications

References 42 publications

Reversible Acetylation Regulates Salt-inducible Kinase (SIK2) and Its Function in Autophagy*

Reversible Acetylation Regulates Salt-inducible Kinase (SIK2) and Its Function in Autophagy*

A label‐free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen

Studies of ligand-induced site-specific phosphorylation of epidermal growth factor receptor

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