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.
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.
Kang
Jeong-Hun
JH
CREST, Japan Science and Technology Corporation, Kawaguchi, Japan.
Toita
Riki
R
Oishi
Jun
J
Niidome
Takuro
T
Katayama
Yoshiki
Y
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
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