Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry.
We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.
Aebersold
R
R
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Bures
E J
EJ
Namchuk
M
M
Goghari
M H
MH
Shushan
B
B
Covey
T C
TC
D001331:Automation; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D017879:Isothiocyanates; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D012680:Sensitivity and Specificity; D017421:Sequence Analysis / Q000379:methods*; D013861:Thiocyanates / Q000737:chemistry*; D013997:Time Factors
D007202:Indicators and Reagents; D017879:Isothiocyanates; D010455:Peptides; D011506:Proteins; D000644:Quaternary Ammonium Compounds; D013861:Thiocyanates; C081492:3-(4'-(ethylene-N,N,N-trimethylamino)phenyl)-2-isothiocyanate