Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix-assisted laser desorption/ionizationde novosequencing
| Autor: | Ana Butorac, Dragan Gamberger, Mario Cindrić, Marija Nišavić, Janko Diminic, Meliha Solak Mekić, Amela Hozić |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
chemistry.chemical_classification Chromatography Sequence analysis 010401 analytical chemistry Organic Chemistry Lysine Peptide Mass spectrometry Proteomics 01 natural sciences 0104 chemical sciences Analytical Chemistry Matrix (chemical analysis) 03 medical and health sciences chemistry.chemical_compound Matrix-assisted laser desorption/ionization 030104 developmental biology chemistry Derivatization Spectroscopy |
| Zdroj: | Rapid Communications in Mass Spectrometry. 30:1687-1694 |
| ISSN: | 0951-4198 |
| DOI: | 10.1002/rcm.7594 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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