Quantitative Proteomics Study For Preparing SILAC Internal Standard And Phosphoproteomics Of A Hepatocellular Carcinoma Cell Line With High Metastasis Ability

Proteomics is a new area of life science which is widely used for identifying andquantifying proteins on a large scale and is also used to study the function of proteins.As technology progresses, proteomics has been developed and popularized quickly,which has become the mainstay of life science. In the study of quantitative proteomics,SILAC（stable isotope labeling with amino acids in cell culture）has become the goldstandard due to its high accuracy and precision. The premise of success for SILACbased quantification is the preparation of completely SILAC labeled proteome internalstandard, which always needs7cell doublings internationally, which is also time andcost consuming. Besides, the labeling efficiency always depends on the type and statusof the cultured cell that mays result in incompletely labeling. In this study, we studiedand established a method to store MHCC97-L with high labeling efficiency in liquid,by which we got the SILAC internal standard with high labeling efficiency rapidly andeffectively with a little cost. Two day after thawed and propagated, the SILAC cellwhose labeling efficiency was over97%become the same with MHCC97-L culturedunder normal condition. The cell cultured in SILAC medium showed high similaritywith that in regular medium as for cell morphology, cell size and doubling time.Quantitative proteomics indicated that no difference existed between SILAC MHCC 97-L before cryopreserved and after cryopreserved. This study was helpful for theSILAC based quantitative proteomics.Phosphorylation is one of the most extensive and important post translationalmodifications, which plays significant roles in normal physiological activities andduring the process of disease occurrence and development. Althoughphosphoproteomics is the most developed and high-throughput among the posttranslational modification, it is still restricted by the enrichment of phosphopeptides. Sofar, no phosphoproteomics study can reveal the phosphorylation state of the cellcompletely. Here, we firstly optimized the ratio for IMAC beads and starting amount ofpeptides. We found10μL5%IMAC beads could enrich as much as80-93μg peptides.Under this condition, we identified more than2000phosphopeptides and the enrichmentratio was reached90%. Further study indicated that mono-phosphopeptides andmulti-phosphopeptides competed with each other, which reduced the number ofmulti-phosphopeptides that was identified. We tested the consecutive incubation step,which reduced the complexity and resulted in more phosphopeptides that was identified,especially the multi-phosphopeptides. Finally, we identified35,032phosphopeptides ina HCC cell line, HCCLM6by combining of high pH reverse phase chromatography,consecutive incubation and LC-MS/MS (Liquid Chromatography-Mass Spectrometry)method, which was the largest dataset with one sample in the world. This studypromoted further development of phosphoproteome and paved the way for the functionstudy of phosphoproteome.