De novo sequencing of tryptic peptides sulfonated by 4-sulfophenyl isothiocyanate for unambiguous protein identification using post-source decay matrix-assisted laser desorption/ionization mass spectrometry.
A simple method of solid-phase derivatization and sequencing of tryptic peptides has been developed for rapid and unambiguous identification of spots on two-dimensional gels using post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The proteolytic digests of proteins are chemically modified by 4-sulfophenyl isothiocyanate. The derivatization reaction introduces a negative sulfonic acid group at the N-terminus of a peptide, which can increase the efficiency of PSD fragmentation and enable the selective detection of only a single series of fragment ions (y-ions). This chemically assisted method avoids the limitation of high background normally observed in MALDI-PSD spectra, and makes the spectra easier to interpret and facilitates de novo sequencing of internal fragment. The modification reaction is conducted in C(18) microZipTips to decrease the background and to enhance the signal/noise. Derivatization procedures were optimized for MALDI-PSD to increase the structural information and to obtain a complete peptide sequence even in critical cases. The MALDI-PSD mass spectra of two model peptides and their sulfonated derivatives are compared. For some proteins unambiguous identification could be achieved by MALDI-PSD sequencing of derivatized peptides obtained from in-gel digests of phosphorylase B and proteins of hepatic satellite cells (HSC).
Chen
Ping
P
College of Life Sciences, Hunan Normal University, Changsha, 410081 China.
Nie
Song
S
Mi
Wei
W
Wang
Xian-Chun
XC
Liang
Song-Ping
SP
D000595:Amino Acid Sequence; D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry*; D002417:Cattle; D002460:Cell Line; D017879:Isothiocyanates / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D020539:Sequence Analysis, Protein / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D001557:Benzenesulfonates; D017879:Isothiocyanates; D010446:Peptide Fragments; C015308:4-isothiocyanatobenzenesulfonate; D014357:Trypsin