Label-free Relative Quantification of Co-eluting Isobaric Phosphopeptides of Insulin Receptor Substrate-1 by HPLC-ESI-MS/MS

Paul Langlais, Lawrence J. Mandarino, Zhengping Yi

    Research output: Contribution to journalArticle

    24 Citations (Scopus)

    Abstract

    Intracellular signal transduction is often regulated by transient protein phosphorylation in response to external stimuli. Insulin signaling is dependent on specific protein phosphorylation events, and analysis of insulin receptor substrate-1 (IRS-1) phosphorylation reveals a complex interplay between tyrosine, serine, and threonine phosphorylation. The phospho-specific antibody-based quantification approach for analyzing changes in site-specific phosphorylation of IRS-1 is difficult due to the dearth of phospho-antibodies compared with the large number of known IRS-1 phosphorylation sites. We previously published a method detailing a peak area-based mass spectrometry approach, using precursor ions for peptides, to quantify the relative abundance of site-specific phosphorylation in the absence or presence of insulin. We now present an improvement wherein site-specific phosphorylation is quantified by determining the peak area of fragment ions respective to the phospho-site of interest. This provides the advantage of being able to quantify co-eluting isobaric phosphopeptides (differentially phosphorylated versions of the same peptide), allowing for a more comprehensive analysis of protein phosphorylation. Quantifying human IRS-1 phosphorylation sites at Ser303, Ser323, Ser330, Ser348, Ser527, and Ser531 shows that this method is linear (n = 3; r 2 = 0.85 ± 0.05, 0.96 ± 0.01, 0.96 ± 0.02, 0.86 ± 0.07, 0.90 ± 0.03, 0.91 ± 0.04, respectively) over an approximate 10-fold range of concentrations and reproducible (n = 4; coefficient of variation = 0.12, 0.14, 0.29, 0.30, 0.12, 0.06, respectively). This application of label-free, fragment ion-based quantification to assess relative phosphorylation changes of specific proteins will prove useful for understanding how various cell stimuli regulate protein function by phosphorylation.

    Original languageEnglish (US)
    Pages (from-to)1490-1499
    Number of pages10
    JournalJournal of the American Society for Mass Spectrometry
    Volume21
    Issue number9
    DOIs
    StatePublished - Sep 2010

    Fingerprint

    Insulin Receptor Substrate Proteins
    Phosphopeptides
    Phosphorylation
    Labels
    High Pressure Liquid Chromatography
    Ions
    Proteins
    Phospho-Specific Antibodies
    Insulin
    Signal transduction
    Peptides
    Threonine
    Serine
    Mass spectrometry
    Tyrosine
    Signal Transduction
    Mass Spectrometry

    ASJC Scopus subject areas

    • Structural Biology
    • Spectroscopy

    Cite this

    Label-free Relative Quantification of Co-eluting Isobaric Phosphopeptides of Insulin Receptor Substrate-1 by HPLC-ESI-MS/MS. / Langlais, Paul; Mandarino, Lawrence J.; Yi, Zhengping.

    In: Journal of the American Society for Mass Spectrometry, Vol. 21, No. 9, 09.2010, p. 1490-1499.

    Research output: Contribution to journalArticle

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    abstract = "Intracellular signal transduction is often regulated by transient protein phosphorylation in response to external stimuli. Insulin signaling is dependent on specific protein phosphorylation events, and analysis of insulin receptor substrate-1 (IRS-1) phosphorylation reveals a complex interplay between tyrosine, serine, and threonine phosphorylation. The phospho-specific antibody-based quantification approach for analyzing changes in site-specific phosphorylation of IRS-1 is difficult due to the dearth of phospho-antibodies compared with the large number of known IRS-1 phosphorylation sites. We previously published a method detailing a peak area-based mass spectrometry approach, using precursor ions for peptides, to quantify the relative abundance of site-specific phosphorylation in the absence or presence of insulin. We now present an improvement wherein site-specific phosphorylation is quantified by determining the peak area of fragment ions respective to the phospho-site of interest. This provides the advantage of being able to quantify co-eluting isobaric phosphopeptides (differentially phosphorylated versions of the same peptide), allowing for a more comprehensive analysis of protein phosphorylation. Quantifying human IRS-1 phosphorylation sites at Ser303, Ser323, Ser330, Ser348, Ser527, and Ser531 shows that this method is linear (n = 3; r 2 = 0.85 ± 0.05, 0.96 ± 0.01, 0.96 ± 0.02, 0.86 ± 0.07, 0.90 ± 0.03, 0.91 ± 0.04, respectively) over an approximate 10-fold range of concentrations and reproducible (n = 4; coefficient of variation = 0.12, 0.14, 0.29, 0.30, 0.12, 0.06, respectively). This application of label-free, fragment ion-based quantification to assess relative phosphorylation changes of specific proteins will prove useful for understanding how various cell stimuli regulate protein function by phosphorylation.",
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