Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry.
Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.
Jin
Wen-Hai
WH
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China.
Dai
Jie
J
Zhou
Hu
H
Xia
Qi-Chang
QC
Zou
Han-Fa
HF
Zeng
Rong
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D005260:Female; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*
D010750:Phosphoproteins; D020543:Proteome