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Quantification of phosphoprotein variants in stem cells by mass spectrometry and a novel, digital high‐resolution isoelectric focusing approach.
Author(s) -
Biringer Roger Gregory,
Rosenblatt Michael,
Rampalli Krystal,
Hughes Douglas,
Rogers John,
Hühmer Andreas
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.862.1
Subject(s) - phosphoprotein , chemistry , isobaric labeling , mass spectrometry , isoelectric focusing , phosphorylation , chromatography , orbitrap , tandem mass spectrometry , tandem mass tag , protein phosphorylation , proteomics , computational biology , quantitative proteomics , biochemistry , protein kinase a , biology , protein mass spectrometry , enzyme , gene
Isoelectric focusing (IEF) is often used as a high resolution fractionation technique to separate proteins of interest from complex cellular background. We have combined a digital IEF approach with a multiplexed quantitation strategy based on isobaric mass labels to resolve and quantify protein expression and phosphoprotein changes in mesenchymal stem cells during differentiation into neurons. Proteins were labeled with Tandem Mass Tags (TMT), separated by digital IEF and quantified by complimentary approaches. Accurate relative quantities of phosphoprotein variants were determined using TMT labeling and LTQ‐Orbitrap‐based LCMS methodologies confirming phosphorylation events previously observed. Alternatively, Western blots and high content analysis with phospho‐specific and pan‐specific antibodies verified these phosphorylation changes in Erk1/2, Stat1, and Pak2 kinase, and several other proteins relevant to neural differentiation. The flexibility of the digital IEF approach enabled comparison of both methods. However, isobaric labeling and high resolution mass spectrometry provided resolution, throughput, and accuracy for phosphoprotein variant quantification superior to traditional methods.