Invasion and
metastasis of
hepatocellular carcinoma (HCC) is a major cause for lethal
liver cancer. Signaling pathways associated with
cancer progression are frequently reconfigured by aberrant phosphorylation of key
proteins. To capture the key phosphorylation events in HCC
metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step
IMAC and LC-MS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high
metastasis). In total, 6593
phosphopeptides with 6420 phosphorylation sites (p-sites) of 2930
phosphoproteins were identified. Statistical analysis of gene ontology (GO) categories for the identified
phosphoproteins showed that several of the biological processes, such as transcriptional regulation,
mRNA processing and RNA splicing, were over-represented. Further analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations demonstrated that
phosphoproteins in multiple pathways, such as spliceosome, the
insulin signaling pathway and the cell cycle, were significantly enriched. In particular, we compared our dataset with a previously published phosphoproteome in a normal liver sample, and the results revealed that a number of
proteins in the spliceosome pathway, such as
U2 small nuclear RNA Auxiliary Factor 2 (U2AF2), Eukaryotic
Initiation Factor 4A-III (EIF4A3), Cell Division Cycle 5-Like (CDC5L) and Survival Motor Neuron Domain Containing 1 (SMNDC1), were exclusively identified as
phosphoproteins only in the MHCC97-H cell line. These results indicated that the phosphorylation of spliceosome
proteins may participate in the
metastasis of HCC by regulating
mRNA processing and RNA splicing.