Peptide ladder sequencing by mass spectrometry using a novel, volatile degradation reagent.
A conceptually novel approach to protein sequencing involves the generation of ragged-end polypeptide chains followed by mass spectroscopic analysis of the resulting nested set of fragments. We report here on the synthesis and development of a volatile isothiocyanate (trifluoroethylisothiocyanate) that allows the identification of several consecutive residues starting with a few picomoles of peptide. The nested set of peptides is generated simply by adding equal aliquots of starting peptide each cycle and driving both the coupling and cleavage reactions to completion. No additional reagents are required to act as chain terminators and retention of the peptide terminal amine allows for subsequent modification with quaternary ammonium alkyl NHS esters to improve sensitivity. Complex washing procedures are not required each cycle, as reagents and by-products are efficiently removed under vacuum, eliminating extractive loss. Multiple peptide samples can be processed simultaneously, with each degradation cycle completed in 35-40 min. The inherent simplicity of the process should allow for easy automation and permit rapid processing of samples in parallel.
Bartlet-Jones
M
M
Protein Sequencing Laboratory, Imperial Cancer Research Fund, London, UK.
Jeffery
W A
WA
Hansen
H F
HF
Pappin
D J
DJ
D000595:Amino Acid Sequence; D007202:Indicators and Reagents; D017879:Isothiocyanates; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013861:Thiocyanates / Q000138:chemical synthesis / Q000737:chemistry
D007202:Indicators and Reagents; D017879:Isothiocyanates; D010455:Peptides; D000644:Quaternary Ammonium Compounds; D013861:Thiocyanates; C005441:phenylisothiocyanate