title: Effect of the addition of diammonium citrate to alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix for the detection of phosphorylated peptide in phosphorylation reactions using cell and tissue lysates.
abstract: The ionization of phosphorylated peptides is usually suppressed by non-phosphorylated peptides when alpha-cyano-4-hydroxycinnamic acid (CHCA) is used as a matrix for matrix-assisted laser desorption/ionization-time-of-Flight (MALDI-TOF) mass spectrometry analysis. In the present study, we examined the effect of diammonium citrate addition to the CHCA matrix on the detection of phosphorylated peptides. Substrates for protein kinase C (PKC) and c-Src were synthesized and phosphorylated by reaction with cell and tissue lysate samples. The addition of diammonium citrate to the CHCA matrix increased the sensitivity for distinguishing phosphorylated peptides from background noise. However, the effect depended on substrate concentration.
authors.affiliation: CREST, Japan Science and Technology Corporation, Kawaguchi, Japan.
authors.forename: Jeong-Hun
authors.initials: JH
authors.lastname: Kang
authors.forename: Riki
authors.initials: R
authors.lastname: Toita
authors.forename: Jun
authors.initials: J
authors.lastname: Oishi
authors.forename: Takuro
authors.initials: T
authors.lastname: Niidome
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Katayama
mesh_terms: D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D045744:Cell Line, Tumor; D019343:Citric Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D003373:Coumaric Acids / Q000737:chemistry*; D008297:Male; D008545:Melanoma / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014020:Tissue Extracts / Q000737:chemistry / Q000378:metabolism*; D019061:src-Family Kinases
chemical_list: D003373:Coumaric Acids; D010455:Peptides; D000644:Quaternary Ammonium Compounds; D014020:Tissue Extracts; C007175:alpha-cyano-4-hydroxycinnamate; D019343:Citric Acid; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C426729:diammonium hydrogen citrate