Phosphoproteomic Analysis of Rat Liver by High Capacity IMAC and LC−MS/MS

Abstract

Proper liver function is crucial for metabolism control and to clear toxic substances from the bloodstream. Many small-molecule therapeutics accumulate in the liver, negatively impacting liver function and often resulting in hepatotoxicity and cell death. Several analytical methods are currently utilized to evaluate hepatotoxicity and monitor liver function. To date, none of these methods have specifically targeted protein phosphorylation-mediated signal transduction pathways which should be altered in response to toxic effects of small molecule therapeutics. To develop novel assays to probe specific signaling pathways in the liver, identification and quantification of specific protein phosphorylation sites in this complex organ is necessary. Here, we have utilized an optimized immobilized metal affinity chromatography (IMAC) protocol to enrich phosphorylated peptides from a tryptic digest of proteins isolated from whole liver lysate. LC-MS/MS analysis of IMAC-enriched peptides resulted in the identification of more than 300 phosphorylation sites from over 200 proteins in rat liver, a significant advance over previously published analyses of the liver phosphoproteome. Previously characterized phosphorylation sites and potentially novel sites were identified in the current study, including sites on proteins implicated in metabolism regulation, transcription, translation, and canonical signaling pathways. Moreover, protein phosphorylation analysis was performed without prior fractionation of the sample, enabling analysis of small sample amounts while minimizing analysis time, potentially allowing for high-throughput assays to be performed with this methodology. From these data, it appears that this methodology can be used to identify new phosphorylation sites and, in combination with a stable isotope-labeling step, to investigate the effects of liver diseases, cancer and evaluate potential toxicology of new drug substances.

Quantitative comparison of IMAC and TiO2 surfaces used in the study of regulated, dynamic protein phosphorylation

“…Moreover, the neutral loss of phosphoric acid derived was observed as the most intense fragment ions in the MS/MS spectra of many phosphopeptides, leaving the majority of the spectrum difficult to interpret. White et al utilized an optimized IMAC protocol to enrich phosphorylated peptides from a tryptic digest of proteins isolated from liver lysate, reporting the identification of more than 300 phosphorylation sites when including Mascot Score 15 or above [53]. Although we have identified numerous putative phosphopeptides with Mascot Score 15 or above, we have not included those alignments in our report because the confidence of protein identification is insufficient.…”

Large‐scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography

“…The first one is chemical reaction-based methods which include beta-elimination of the phosphate group and subsequent introduction of an affinity tag [11], and then captured by beads and released [12]. The second one is chromatography based methods which include affinity chromatography with immobilized metal ions (IMAC) [13][14][15][16][17][18][19][20][21][22][23], metal oxides such as titanium dioxide (TiO 2 ) and zirconium dioxide (ZrO 2 ) [24][25][26][27][28][29][30][31], and strong cation exchange chromatography (SCX) [32][33][34][35]. Among all of these methods, IMAC with Fe 31 is the most popular technique for the enrichment of phosphorylated peptides.…”

Large-scale phosphorylation analysis of mouse liver

“…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.…”