Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry

Abstract

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 manud 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.

Lc/Ms:an Essential Tool in Drug Development

“…The second type of system shown is the time-off light (TOF) mass spectrometer, which has the added capability of providing a higher mass resolution spectrum from each component that is assayed. The third system shown is the triple quadruple MS-MS system, which is most often used for bio analytical assays but can also be used for metabolite identification assays 3 . The fourth MS system is called an ion-trap mass spectrometer and has the unique capability of producing MS data that are important when performing structural elucidation assays.…”

Amino acid sequence and D/L‐configuration determination methods for D‐amino acid‐containing peptides in living organisms

“…To enhance the ionization efficiency, 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate (PETAPITC; Aebersold et al, 1992) and 4-(3-pyridinylmethylaminocarboxypropyl)phenyl isothiocyanate (PITC 311; Bures et al, 1995) were developed as an Edman reagent. The ladder sequencing by the mass spectrometric detection was reported using PITC containing 5% (v/v) phenylisocyanate (PIC) without the separation of PTH amino acids (Chait et al, 1993).…”

Applications of electrospray ionization mass spectrometry to neutral organic molecules including fullerenes

“…Reagent 12 was prepared according to the procedure for phenyl isothiocynate (PITC) [8] from 4amino-benzo-15-crown-5 (Parish Chemical Co., Odem, UT). Tagging of amino acids was carried out in the usual way.…”