Abstract
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.
Highlights
Reversible protein phosphorylation, one of the crucial post-translational modifications, alters the structural conformation of proteins, affecting their role in cell signaling networks
Due to the relatively low stoichiometry of phosphoproteins in the whole proteome, specific methods leading to the enrichment for phosphopeptides in the bottom-up phosphoproteomics are necessary for a successful analysis
Other enrichment approaches in an optimized setup were successfully introduced for phosphoproteomic research [11], TiO2-based metal oxide affinity chromatography (MOAC) is probably the most widespread enrichment method
Summary
Reversible protein phosphorylation, one of the crucial post-translational modifications, alters the structural conformation of proteins, affecting their role in cell signaling networks. Phosphorylation is the most frequent post-translational modification of proteins, the number of phosphoproteins to the whole proteome is still relatively low [3]. Phosphoproteomics is a branch of proteomics that identifies, characterizes, and quantifies proteins possessing phosphate groups in their structure. It often uses liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) as a sensitive and specific detection method that is robust both for identification and quantification of phosphoproteins, including localization of present phosphorylation in their primary structure [4,5]. Due to the relatively low stoichiometry of phosphoproteins in the whole proteome, specific methods leading to the enrichment for phosphopeptides in the bottom-up phosphoproteomics are necessary for a successful analysis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
