Benefits of selective peptide derivatization with sulfonating reagent at acidic pH for facile matrix-assisted laser desorption/ionization de novo sequencing
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Mekic, Meliha Solak
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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 highthroughput de novo sequencing. METHODS: We developed a SPITC peptide derivatization procedure under acidic conditions (pH LT = 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 signifi...cantly 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. Copyright (C) 2016 John Wiley and Sons, Ltd.
Source:Rapid Communications in Mass Spectrometry, 2016, 30, 14, 1687-1694
- INNOMOL - Enhancement of the Innovation Potential in SEE through new Molecular Solutions in Research and Development (EU-316289)
- European Fund for Regional Development [STRIP/Aplimetafarma/RC.2.2.08-0046]