A picomole-scale method for charge derivatization of peptides for sequence analysis by mass spectrometry.
A highly activated ester containing a fixed positive charge, S-pentafluorophenyl [tris(2,4,6-trimethoxyphenyl)phosphonium]acetate bromide (TMPP-AcSC6F5 bromide), has been synthesized as a reagent for N-terminal modification of peptides. Stable in aqueous acetonitrile solution during extended storage, TMPP-AcSC6F5 bromide reacts with unprotected peptides through p-(dimethylamino)pyridine (DMAP)-promoted amidation in aqueous acetonitrile (15 min, ambient temperature) to form N-TMPP-Ac derivatives of peptides. These peptide derivatives are readily amenable to analysis by fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Greater than 90% conversion has been observed in transforming low-nanomole quantities of analyte using molar ratios of 1:5:10 (peptide/reagent/ DMAP). For reactions at the picomole level a slightly modified stoichiometry, with molar ratios of 1:10:500, is employed. Owing to the high reaction efficiency and the tolerance to moderate excess reagent and base during analysis by FAB- and MALDI-MS, the reaction mixture containing the modified peptides can be analyzed directly in most cases, without sample cleanup. Examples of the preparation and analysis of a variety of N-TMPP-acetyl-peptides (TMPP-Ac-peptides) ranging from hexamers to 15-mers are given. Collisionally activated dissociation tandem mass spectrometry of TMPP-Ac-derivatives showed dominant a-type ions, accompanied by d- and c-type ions in some cases, allowing sequence determination to be made in a straightforward manner.
Huang
Z H
ZH
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Wu
J
J
Roth
K D
KD
Yang
Y
Y
Gage
D A
DA
Watson
J T
JT
D000595:Amino Acid Sequence; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D017421:Sequence Analysis / Q000379:methods*
D007202:Indicators and Reagents; D010455:Peptides