title: Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS.
abstract: Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-alpha. Substantial increases of phosphorylation were observed for Thr(446) upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser(1078). This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.
authors.affiliation: Department of Kinesiology, Arizona State University, P.O. Box 874501, 85287-4501, Tempe, AZ, USA.
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Yi
authors.affiliation: Department of Kinesiology, Arizona State University, P.O. Box 874501, 85287-4501, Tempe, AZ, USA.
authors.forename: Moulun
authors.initials: M
authors.lastname: Luo
authors.affiliation: Department of Kinesiology, Arizona State University, P.O. Box 874501, 85287-4501, Tempe, AZ, USA. Lawrence.Mandarino@asu.edu.|the Department of Kinesiology, Arizona State University, Tempe, Arizona. Lawrence.Mandarino@asu.edu.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Mandarino
authors.affiliation: Department of Medicine, University of Texas Health Center at San Antonio, San Antonio, Texas, USA.
authors.forename: Sara M
authors.initials: SM
authors.lastname: Reyna
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Carroll
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.
authors.forename: Susan T
authors.initials: ST
authors.lastname: Weintraub
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013912:Threonine / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha; D013912:Threonine; D012694:Serine