Naslov
Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix-assisted laser desorption/ionization de novo sequencing
(Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix- assisted laser desorption/ionization de novo sequencing)

Autori
Butorac, Ana ; Solak Mekić, Meliha ; Hozić, Amela ; Diminić, Janko ; Gamberger, Dragan ; Nišavić, Marija ; Cindrić, Mario

Izvornik
RCM. Rapid communications in mass spectrometry (0951-4198) 30 (2016), 14; 1687-1694

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
de novo sequencing ; peptide derivatization ; MALDI-TOF/TOF

Sažetak
RATIONALE One of the most challenging tasks of proteomics is peptide de novo sequencing. 4- Sulfophenyl isothiocyanate (SPITC) peptide derivatization enables acquisition of high- quality tandem mass spectra (MS/MS) for de novo sequencing, but unwanted non-specific reactions and reduced mass spectra (MS) signal intensities still represent the obstacles in high-throughput de novo sequencing. METHODS We developed a SPITC peptide derivatization procedure under acidic conditions (pH ≤5). Derivatized peptides were analyzed by matrix- assisted laser desorption/ionization (MALDI-MS) in negative ion mode followed by MS/MS in positive ion mode. A de novo sequencing tool, named DUST, adjusted to SPITC chemistry, was designed for successful high-throughput peptide de novo sequencing. This high-throughput peptide de novo sequencing was tested on Fusarium delphinoides, an organism with an uncharacterized genome. RESULTS The SPITC derivatization procedure under acidic conditions produced a significantly improved MS dataset in comparison to commonly used derivatization under basic conditions. Signal intensities were 6 to 10 times greater and the over-sulfonation effect measured on lysine- containing peptides was significantly decreased. Furthermore, development of a novel DUST algorithm enabled automated de novo sequencing with the calculated accuracy of 70.6%. CONCLUSIONS The SPITC derivatization and de novo sequencing approach outlined here provides a reliable method for high-throughput peptide de novo sequencing. High-throughput peptide de novo sequencing enabled protein mutation identification and identification of proteins from organisms with non-sequenced genomes.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

POVEZANOST RADA