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.