Preparation of monodisperse immobilized Ti4+ affinity chromatography microspheres for specific enrichment of phosphopeptides

Yu, Zhiyuan; Han, Guanghui; Sun, Shutao; Jiang, Xinning; Chen, Rui; Wang, Fangjun; Wu, Ren’an; Ye, Mingliang; Zou, Hanfa

doi:10.1016/j.aca.2009.01.033

Cited by 104 publications

(83 citation statements)

References 42 publications

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“…The Ti 4+ -IMAC microspheres were synthesized in our laboratory with the method described before [39]. The phosphopeptides were enriched by Ti 4+ -IMAC microspheres following the protocol previously reported [31].…”

Section: Phosphopeptides Enrichmentmentioning

confidence: 99%

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

Wang

Bian

Cheng

et al. 2013

Journal of Proteomics

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Section: Phosphopeptides Enrichmentmentioning

confidence: 99%

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

Wang

Bian

Cheng

et al. 2013

Journal of Proteomics

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“…The Ti 4+ -IMAC microspheres were prepared in our lab with the method described by Yu et al 26 The phosphopeptides were enriched by Ti 4+ -IMAC microspheres following the protocol reported by Zhou et al 27 Briefly, the Ti 4+ -IMAC microspheres were first suspended in loading buffer containing 80% ACN and 6% TFA and mixed with the peptide mixture with a microspheres-to-protein ratio of 10/1 (w/w). After violent vibration for 30 min, the supernatant was removed after centrifugation, then the microspheres were sequentially washed with washing buffer 1 containing 50% ACN, 6% TFA, 200 mM NaCl and washing buffer 2 containing 30% ACN, 0.1% TFA for 30 min.…”

Section: Phosphopeptide Enrichmentmentioning

confidence: 99%

Improve the Coverage for the Analysis of Phosphoproteome of HeLa Cells by a Tandem Digestion Approach

Bian

Song

et al. 2012

J. Proteome Res.

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Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.

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“…Though double enrichment by Ti (IV)-MCM-41 was performed, the overall selectivity was still quite low (less than 20%). Much high selectivity, typically over 60%, could be achieved for enrichment of phosphopeptides from protein digest by Ti (IV)-IMAC [18,28]. The poor selectivity achieved in this study was most probably due to the high complexity of the serum sample and the low abundance of endogenous phosphopeptides.…”

Section: 2mentioning

confidence: 79%

“…Ti (IV)-MCM-41 was prepared as reported previously [14]. Serum endogenous phosphopeptides were enriched with Ti (IV)-MCM-41 and the enrichment procedure was similar to that in our previous reports [14,18]. Briefly, 500 μL of human serum was diluted with 30% ACN/0.1% TFA to a final volume of 5 mL, protease inhibitor and phosphatase inhibitor were both added to 0.5% (v/v).…”

Section: Sample Preparationmentioning

confidence: 99%

“…Therefore, specific isolation of phosphopeptides is crucial for phosphorylation analysis. Ti (IV) based IMAC material was used for specific enrichment of phosphopeptides from protein digest [18,19,22,23]. Recently, an affinity material synthesized by modifying highly ordered mesoporous silica particles MCM-41 with titanium phosphonate (Ti (IV)-MCM-41) was demonstrated to have both size-exclusion capability of proteins and high specificity toward endogenous phosphopeptides [14].…”

Section: 2mentioning

confidence: 99%

See 1 more Smart Citation

Analysis of human serum phosphopeptidome by a focused database searching strategy

Zhu

Wang

Cheng

et al. 2013

Journal of Proteomics

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Preparation of monodisperse immobilized Ti4+ affinity chromatography microspheres for specific enrichment of phosphopeptides

Cited by 104 publications

References 42 publications

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

Improve the Coverage for the Analysis of Phosphoproteome of HeLa Cells by a Tandem Digestion Approach

Analysis of human serum phosphopeptidome by a focused database searching strategy

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