Label-free relative quantification of co-eluting isobaric phosphopeptides of insulin receptor substrate-1 by HPLC-ESI-MS/MS |
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Authors: | Paul Langlais Lawrence J Mandarino Zhengping Yi |
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Institution: | 1.ASU/Mayo Center for Metabolic and Vascular Biology,Arizona State University,Tempe,USA;2.Department of Medicine,Mayo Clinic in Arizona,Scottsdale,USA;3.School of Life Sciences,Arizona State University,Tempe,USA |
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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 phosphospecific 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; r2 = 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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