Multiple phosphorylated protein selective analysis via fluorous derivatization and liquid chromatography-electrospray ionization-mass spectrometry analysis
| Autor: | Yohei Sakaguchi, Hitoshi Nohta, Shimba Kawasue, Tadashi Hayama, Reiko Koga, Hideyuki Yoshida |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
inorganic chemicals
Spectrometry Mass Electrospray Ionization Halogenation Electrospray ionization Biophysics Proteomics Mass spectrometry 01 natural sciences Biochemistry 03 medical and health sciences chemistry.chemical_compound Liquid chromatography–mass spectrometry Humans Phosphorylation Derivatization Molecular Biology 030304 developmental biology 0303 health sciences Chromatography Chemistry 010401 analytical chemistry Caseins Cell Biology Peptide Fragments 0104 chemical sciences Reagent Michael reaction Chromatography Liquid |
| Zdroj: | Analytical Biochemistry. 628:114247 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2021.114247 |
| Popis: | Post-translational modification of proteins is involved in protein function and higher-order structure. Among such modification, phosphorylation is an important intracellular signal transduction pathway. Many studies on phosphorylated protein analysis using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) have been developed. However, there are few reports on the analysis of highly phosphorylated proteins because of their handling difficulty. Hence, we developed an analytical method that converts multiple phosphate groups contained in the peptides into perfluoroalkyl groups for selective analysis using fluorous affinity. Here, tryptic digested β-casein fragment peptides [RELEELNVPGEIVE(pSer)L(pSer)(pSer)(pSer)EESITR and FQ(pSer)EEQQQTEDELQDK] were used as model phosphorylated peptides. 1H,1H,2H,2H-Perfluorooctanethiol (PFOT) and 2,2,2-trifluoroethanethiol (TFET) were used as derivatization reagents for mono-phosphorylated peptides and multi-phosphorylated peptides, respectively, to derivatize via β-elimination/Michael addition. The derivatives were analyzed by LC-ESI-MS. A fluorous LC column is typically used to selectively retain the fluorous-derivatized peptides, which are expected to be separated from contaminants and non-phosphorylated peptides. When this method was applied to β-casein, TFET- and PFOT-derivatized peptides were strongly retained in the fluorous LC column and clearly separated from non-phosphorylated peptides on the chromatogram. Therefore, the developed method enables quantification of mono- and multi-phosphorylated peptides and is suitable for application in proteomics. |
| Databáze: | OpenAIRE |
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