Naslov
Protein identification by MALDI MS/MS using de novo sequencing of derivatized tryptic peptides

Autori
Horvatić, Anita ; Dodig, Ivana ; Marković, Ana ; Bačun-Družina, Višnja ; Butorac, Ana ; Pavoković, Dubravko ; Krsnik-Rasol, Marijana ; Cindrić, Mario

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
4th Summer School of Mass Spectrometry in Biotechnology and Medicine, Book of Abstracts / Jamal Souady - , 2010

Skup
4th Summer School of Mass Spectrometry in Biotechnology and Medicine

Mjesto i datum
Dubrovnik, Hrvatska, 04.07.2010. - 10.07.2010

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
de novo sequencing; derivatization; MALDI

Sažetak
Gel-based matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) followed by peptide mass fingerprinting (PMF) and MALDI MS/MS ion database searches are frequently used methods for high-throughput identification of proteins. Protein identification is directly related to ion intensities, mass measurement accuracy, protein sequence coverage and tryptic peptide fragmentation efficiency. To avoid low ion intensities and spectra complexity due to backbone fragmentation (mixture of internal fragments, a-, b- and y-ions, and ammonia and water lost and adduct ions etc.) different chemical modifications of peptides have been introduced into MALDI MS proteomics analyses. Derivatization procedures increase structural information and result in complete peptide sequence by influencing positively on the formation of single series of fragment ions. N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) has been routinely used for proteome analyses. Herein we report a novel derivatization method applied on in-gel tryptic digests of bacterial (L. brevis) and plant (A. thaliana) samples by a reagent containing two negatively charged groups. Derivatization reaction introduced negative groups at N-terminus of a peptide. MS/MS fragmentation of derivatized tryptic peptides was monitored in both positive and negative ion mode. While in negative ion mode single series of derivatized b-fragment ions were selectively detected, single series of y-ions in positive ion mode were used as a control. De novo sequencing of derivatized peptides resulted in unambiguous peptide identification yielding higher database protein search scores. Described approach of de novo sequencing of derivatized peptides is important not only for identification of known proteins, but also for identification of proteins not available in databases (reverse translation of protein to DNA followed by genome-based fingerprint scanning).

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA