Semi-automated chromatographic procedure for the isolation of acetylated N-terminal fragments from protein digests.

摘要: Abstract Several published procedures have been combined to develop a general strategy for the specific identification and isolation of acetylated-N-terminal fragment from all other proteolytic fragments. This ruse can be divided into four steps: (i) succinylation substrate block lysine NH2 groups; (ii) enzymatic digestion modified protein; (iii) automated phenylisothiocyanate derivatization protease derived fragments newly generated “free” N-termini; (iv) reversed-phase high-performance liquid chromatography with on-line photodiode array spectroscopy. The individual phenylthiocarbamyl-peptide species exhibit an increased reversed phase retention time greater UV (210–297 nm) profile compared corresponding control (-phenylisothiocyanate) digest. N-terminal acetylated shows neither shift nor augmented profile. To validate each process step, synthetic peptides proteins known sequence were used as test samples. desired was isolated three positively identified by electrospray mass spectrometry amino acid composition, Proteins blocking groups should amenable this procedure.