On-line derivatization of peptides for improved sequence analysis by micro-column liquid chromatography coupled with electrospray ionization-tandem mass spectrometry.
An on-line method has been developed for the derivatization and coupled liquid chromatography (LC)/electrospray ionization (ESI) MS analysis of peptides at the femtomol level. Peptides are reacted with N-succinimidyl-2(3-pyridyl)acetate (SPA) in buffered aqueous medium at pH7 following loading on a precolumn (PC) in a microcolumn switching system. The fast-hydrolysing reagent is dissolved in dry methanol and mixed, in a 3 vol% ratio, with a buffer just before reaching the sample on the PC. Following the reaction and wash, the N-pyridylacetyl (PA) derivatives formed are transferred to the analytical micro-high-performance (HP) LC column for separation and subsequent ESI tandem MS analysis. The reaction is nearly quantitative and takes place selectively at the N-terminal and lysine amino functions, the latter providing a chemical means to distinguish between isobaric residues lysine and glutamine. In some cases, a minor reaction was observed with the tyrosine hydroxyl group. The N-terminal PA group was able to alter the collision-induced fragmentation pathway of peptides in favour of the formation of abundant b-type ions, a helpful feature for sequence elucidation of unknown peptides, particularly with quadrupole ion trap instruments.
Cárdenas
M S
MS
Department of Analytical Chemistry, Faculty of Sciences, University of Córdoba, Spain.
van der Heeft
E
E
de Jong
A P
AP
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D017421:Sequence Analysis / Q000379:methods
D007202:Indicators and Reagents; D010455:Peptides