title: Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts.
abstract: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used successfully to detect phosphorylation sites in proteins. Applications may be limited by the low response of phosphopeptides compared to nonphosphorylated peptides in MALDI MS. The addition of ammonium salts to the matrix/analyte solution substantially enhances the signal for phosphopeptides. In examples shown for equimolar mixtures, the phosphorylated peptide peaks become the largest peaks in the spectrum upon ammonium ion addition. This can allow for the identification of phosphopeptides in an unfractionated proteolytic digestion mixture. Sufficient numbers of protonated phosphopeptides can be generated such that they can be subjected to postsource decay analysis, in order to confirm the number of phosphate groups present. The approach works well with the common MALDI matrices such as alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, and with ammonium salts such as diammonium citrate and ammonium acetate.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing 48824, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Asara
authors.forename: J
authors.initials: J
authors.lastname: Allison
mesh_terms: D000595:Amino Acid Sequence; D006868:Hydrolysis; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D000644:Quaternary Ammonium Compounds; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
chemical_list: D007202:Indicators and Reagents; D010748:Phosphopeptides; D000644:Quaternary Ammonium Compounds; D014357:Trypsin