Large-scale phosphorylation analysis of mouse liver

Abstract

Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. Our understanding of the nature of this network as a whole remains limited, largely because of an array of technical challenges in the isolation and high-throughput sequencing of phosphorylated species. In the present work, we demonstrate that a combination of tandem phosphopeptide enrichment methods, high performance MS, and optimized database search/data filtering strategies is a powerful tool for surveying the phosphoproteome. Using our integrated analytical platform, we report the identification of 5,635 nonredundant phosphorylation sites from 2,328 proteins from mouse liver. From this list of sites, we extracted both novel and known motifs for specific Ser/Thr kinases including a ''dipolar'' motif. We also found that C-terminal phosphorylation was more frequent than at any other location and that the distribution of potential kinases for these sites was unique. Finally, we identified double phosphorylation motifs that may be involved in ordered phosphorylation.mass spectrometry ͉ proteomics I n eukaryotes, protein phosphorylation is among the most important regulatory events in cells, guiding primary biological processes, such as cell division, growth, migration, differentiation, and intercellular communication (1, 2). Numerous efforts to study protein phosphorylation continue to provide the scientific community with an expanding phosphorylation database resource. Sequence alignment studies (3) have defined which genes encode protein kinases. In addition, in vitro reactions of kinases with synthetic peptide libraries have been used to define kinase specificities (4, 5). Finally, protein targets for individual kinases are being defined through elegant systematic studies (6-8). Notwithstanding, only MS-based proteomics currently provides the capability to identify at once and on a massive scale kinase substrates and the specific positions of their modification (9).A major goal of systems biology is to integrate all in vivo phosphorylation events into the context of an organism. Phosphorylation analysis from primary tissue, in contrast to immortalized cell lines, best represents events that are occurring in the basal physiological state of an organism even though tissues often contain heterogeneous populations of cells. In the present study we chose mouse liver as a model tissue for establishing a pipeline for large-scale phosphorylation analysis. In addition, protein phosphorylation plays a critical role in normal liver development and function; therefore, the sites obtained here could be further characterized into physiological context. Several phosphorylation-related (PI3K and Akt signaling) liver phenotypes have been reported to be related to altered lipid and glucose metabolism via insulin control (10, 11) and liver regeneration (ribosomal protein S6) (12). Previously, only two studies have examined phosphorylation sites from liver tissue with 26 (13) and 339 (14) sites.As the fie...

Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma

“…MS/MS spectra were evaluated using SEQUEST and the Core platform from Harvard University 35–37 . Files were searched against the NCBI rattus norvegicus FASTA database updated on May 22, 2015.…”

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

“…To graphically display the identified motifs, logo-like representations were generated with Weblogo [51]. These motifs were associated with specific kinases by searching in literatures [9,21] as well as in the Arabidopsis phosphorylation site database (PhosPhAt) [15] and Human protein reference database (HPRD) [52,53].…”

“…In total, 26 Ser motifs and 5 Thr motifs were significantly enriched (Supplemental Table 5). After associating these motifs with specific protein kinases in the literature and databases [9,15,21,52,53] The enrichment of these kinases indicates that they play crucial roles in early development and growth of Arabidopsis seedlings. 14 phosphorylation motifs (Fig.…”

“…Typically, a low-pH offline strong cation exchange (SCX) LC is used for LC-based phosphopeptide enrichment and fractionation. The principle is that under the acidic pH condition (pH 2.7), the negatively charged phosphopeptides tend to elute earlier and are thus separated from the majority of nonphosphopeptides [9,14,21]. However, there are some clear drawbacks for these offline LC separation methods such as time-consuming procedures and big sample loss caused by repeated desalting and lyophilization steps.…”

“…The most commonly used affinity-based methods are the (Fe 3+ )-based immobilized metal affinity chromatography (Fe 3+ -IMAC) [6,21,[26][27][28] and titanium dioxide (TiO 2 ) affinity chromatography [9,14,29,30]. Recently, on the basis of these methods, some new enrichment strategies have been developed by adopting different metal oxides (ZrO 2 and Nb 2 O 5 ) or IMAC with alternative metal ions (Ga 3+ , Zr 4+ , and Ti 4+ ) [19,20].…”