Abstract
A strategy with the combination of multiprotease digestion and the selective enrichment of phosphopeptides by silica hybrid monolith based immobilized Ti 4+affinity chromatography (Ti 4+-IMAC) was proposed, and applied in the global profiling of phosphorylated membrane proteome of neuroblastoma SH-SY5Y cells. The fraction of membrane proteins was extracted by ultra speed centrifuge, followed by washing with 1 mol/L sodium chloride and 0.1 mol/L sodium carbonate. For digestion, chymotrypsin and pepsin with broader specificity were used as complementary enzymes to trypsin. The phosphopeptides were then selectively enriched by monolithic Ti 4+-IMAC column, and analyzed by nanoflow high performance liquid chromatography and mass spectrometry. A total of 43 phosphoproteins were identified, among which 14 proteins were located on the membrane. All these results demonstrated that the proposed strategy might be promising to promote the in-depth study of neuroblastoma and discover the candidate biomarkers.
Original language | English (US) |
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Pages (from-to) | 851-856 |
Number of pages | 6 |
Journal | Chinese Journal of Chromatography (Se Pu) |
Volume | 29 |
Issue number | 9 |
DOIs | |
State | Published - Sep 28 2011 |
Externally published | Yes |
Keywords
- Human neuroblastoma
- Immobilized Ti affinity chromatography
- Membrane protein
- Multi-enzyme digestion
- Organic-inorganic hybrid silica monolith
- Phosphopeptide enrichment
ASJC Scopus subject areas
- General Medicine