Enhanced ionization of phosphorylated peptides during MALDI TOF mass spectrometry.
Although alpha-cyano-4-hydroxycinnamic acid functions as an excellent matrix for the analysis of most peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry, the ionization of phosphorylated peptides is usually suppressed by nonphosphorylated peptides. As an alternative matrix, 2',4',6'-trihydroxyacetophenone (THAP) with diammonium citrate was found to overcome this problem for the MALDI TOF mass spectrometric analysis of proteolytic digests of phosphorylated proteins. Specifically, the abundances of phosphorylated peptides in tryptic digests of bovine beta-casein and protein kinase C (PKC)-treated mouse cardiac troponin I were enhanced more than 10-fold using THAP during positive ion MALDI TOF mass spectrometry. The protonated molecules of phosphorylated peptides were sufficiently abundant that postsource decay TOF mass spectrometry was used to confirm the number of phosphate groups in each peptide. Finally, tryptic digestion followed by analysis using MALDI TOF mass spectrometry with THAP as the matrix facilitated the identification of a unique phosphorylation site in PKC-treated troponin I.
Yang
Xiaofeng
X
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Wu
Huaping
H
Kobayashi
Tomoyoshi
T
Solaro
R John
RJ
van Breemen
Richard B
RB
D000098:Acetophenones; D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D007477:Ions / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019210:Troponin I / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000098:Acetophenones; D002364:Caseins; D007477:Ions; D010748:Phosphopeptides; D019210:Troponin I; C107067:2,4,6-trihydroxyacetophenone; D011493:Protein Kinase C; D014357:Trypsin