Comprehensive Analysis of in Vivo Phosphoproteome of Mouse Liver Microsomes.

Protein phosphorylation at serine, threonine, and tyrosine residues are some of the most widespread reversible post-translational modifications. Microsomes are vesicle-like bodies, not ordinarily present within living cells, which form from pieces of the endoplasmic reticulum (ER), plasma membrane, mitochondria, or Golgi apparatus of broken eukaryotic cells. Here we investigated the total phosphoproteome of mouse liver microsomes (MLMs) using TiO2 enrichment of phosphopeptides coupled to on-line 2D-LC-MS/MS. In total, 699 phosphorylation sites in 527 proteins were identified in MLMs. When compared with the current phosphoSitePlus database, 155 novel phosphoproteins were identified in MLM. The distributions of phosphosites were 89.4, 8.0, and 2.6% for phosphoserine, phosphotheronine, and phosphotyrosine, respectively. By Motif-X analysis, eight Ser motifs and one Thr motif were found, and five acidic, two basophilic-, and two proline-directed motifs were assigned. The potential functions of phosphoproteins in MLM were assigned by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In GO annotation, phosphorylated microsomal proteins were involved in mRNA processing, mRNA metabolic processes, and RNA splicing. In the KEGG pathway analysis, phosphorylated microsomal proteins were highly enriched in ribosome protein processing in ER and ribosomes and in RNA transport. Furthermore, we determined that 52 and 23 phosphoproteins were potential substrates of cAMP-dependent protein kinase A and casein kinase II, respectively, many of which are 40S/60S ribosomal proteins. Overall, our results provide an overview of features of protein phosphorylation in MLMs that should be a valuable resource for the future understanding of protein synthesis or translation involving phosphorylation.