Strategy For Protein Mixture Identification By Immobilized PH Gradients Isoelectric Focusing And Mass Spectrometry

A method based on isoelectric focusing (IEF) and CapLC-Nanoflow-MS/MS, MALDI-TOF-MS and MALDI-TOF-TOF-MS was founded. Sample loading, staining, reproducibility and in-gel digestion about 1D IPG-IEF were investigated. In 5 staining methods, method B2 has the advantage of dyeing acid end of strip, faster destaining and it is suitable for mass spectrometry detection. It would have some application foreground on functional proteomics. An optimal in-gel digestion procedure for separated proteins following IEF was acquired. Compared to 1D SDS PAGE, IPG IEF could have some advantage in separating protein mixtures on isoelectric point, especially protein isomers, and the separated proteins could be directly identified without in-gel digestion.By the mentioned method we had successfully identified 33 proteins from extracts of K562 cell line, 12 identified proteins were identical with the literature for K562 cell research. 18 proteins were identified by MALDI-TOF-TOF-MS. 16 proteins were identified by LC-MS/MS which can identify a protein by 3-5 peptides on average (at most 7 peptides), it showed that the results were believable, and It is suitable for the method to analyzing complex mixtureThe apoptosis was induced by Ara-c in HL-60 cell line, and the ultrastructural pathologic characters of HL-60 cell apoptosis were observed with transmission electron microscope. HL-60 cells treated by 6×10~-5 mol/L Ara-c appeared the early stage character of apoptosis after 6 hours, then appeared the later stage of apoptosis after 24 hours. The differential protein expression map about 1D IPG-IEF was built. The HL-60 control group detected 49 IEF bands, meanwhile HL-60 treated by Ara-c only detected 30 bands after 6 h, 29 bands after 12 h, 29 bands after 24 h and 28 bands after 36 h, about less 40% bands than the control group. The number of alkali proteins bands was decreasing among treated groups. 6 significant proteins were identified in the differential IEF bands by De novo sequencing. The protein H2B was only detected at 6 h, and its relative quantity level was much higher. It may be one of the early stage characters of apoptosis. Heterogeneous nuclear ribonucleoprotein A1 appeared in both leukaemia cells, as a potential diagnosis marker. EF1a-like protein may involve in biological synthesis, protein turnover, and signal transporter. The changing curves of proteins amount with different time were showed mainly for 4 types. The apex of "mountain type" appeared after 6 hours and it maybe associated with apoptosis initiation; the milestone of "crag type" appeared after 24 hours showed these proteins maybe have the function of resisting apoptosis.