Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 21, Issue 11, Pages -Publisher
MDPI
DOI: 10.3390/ijms21113971
Keywords
phosphopeptides; fractionation; acetonitrile; gradient; enrichment; mass spectrometry; titanium dioxide
Funding
- Ministry of Defense of the Czech Republic through the long-term organization development plan Medical Aspects ofWeapons of Mass Destruction of the Faculty of Military Health Sciences, University of Defense in Brno [DZRO-ZHN-2017]
- Ministry of Health of the Czech Republic [NV19-02-00297]
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Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.
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