[Phosphorylated membrane proteome analysis of human neuroblastoma SH-SY5Y cell line].
A strategy with the combination of multiprotease digestion and the selective enrichment of phosphopeptides by silica hybrid monolith based immobilized Ti4+ affinity chromatography (Ti4+ -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 Ti4+ -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.
CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physic Chinese Academy of Sciences, National Chromatographic R. & A. Center, Dalian 116023, China.
Chunyan
C
Hou
Junfeng
J
Ma
Dingyin
D
Tao
Lihua
L
Zhang
Zhen
Z
Liang
Yukui
Y
Zhang
D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D009447:Neuroblastoma / Q000473:pathology*; D010748:Phosphopeptides / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D014025:Titanium / Q000737:chemistry
D008565:Membrane Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; D014025:Titanium