期刊
TALANTA
卷 204, 期 -, 页码 670-676出版社
ELSEVIER
DOI: 10.1016/j.talanta.2019.06.008
关键词
Titanium ion; 4-Armed Poly(ethylene oxide); Phosphopeptides; Polymer IMAC
资金
- National Natural Science Foundation of China (NSFC) [81572833, 21574022]
- Chinese National Key Program on Basic Research (973) Grant [2011CB910702, 2013CB911202]
Protein phosphorylation is a reversible and important post-translational modification. Identification of phosphopeptides without enrichment is difficult for the low-abundance of phosphopeptides in real complex biological samples. Therefore, the effective and selective concentration of phosphopeptides prior to proteomic identification by mass spectrometer is necessary. In this study, we synthesized a novel titanium-based immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides. To improve material hydrophilia to the maximum extent, titanium ions were immobilized on the 4-armed Poly(ethylene oxide)(4 mu-PEO-Ti4+), a totally soluble polymer with large molecular weight (20000 g/mol). The 4 mu-PEO-Ti4+ was used to enrich phosphopeptides from tryptic digests of standard proteins and real complex biological samples, followed by MALDI-TOF MS analysis. In enrichment of phosphopeptides from 4 pmol beta-casein, the 4 mu-PEO-Ti4+ performed the best property with starting material of 99-132 mu g, loading buffer of 50% ACN/5% TFA (v/v), elution buffer of 10% NH3 center dot H2O (v/v) and elution time of 30 min. The 4 mu-PEO-Ti4+ has a superior detection sensitivity as low as 2 fmol for phosphopeptides. The high selectivity of 4 mu-PEO-Ti4+ allows a deep enrichment of phosphopeptides of beta-casein from a mixture with BSA of 1000-fold abundant. The 4 mu-PEO-Ti4+ shows great stability and endurability and can be recycled up to at least 5 times. In addition, 4 mu-PEO-Ti4+ could detect 10 and 15 phosphopeptides from non-fat milk and nonenzymatic human saliva, respectively. In total, 4 mu-PEO-Ti4+ is a novel excellent material which shows great sensitive and selective enrichment of low-abundance phosphopeptides in real complex biological samples.
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