title: Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis.
abstract: Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis.
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M., Denmark.
authors.forename: E
authors.initials: E
authors.lastname: Sachon
authors.forename: S
authors.initials: S
authors.lastname: Mohammed
authors.forename: N
authors.initials: N
authors.lastname: Bache
authors.forename: O N
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D000588:Amines / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007202:Indicators and Reagents / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D008832:Microchemistry / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000588:Amines; D007202:Indicators and Reagents; D010748:Phosphopeptides; D011506:Proteins