当前位置： SCI文献检索 > INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE期刊下所有文献 > SILAM for quantitative proteomics of liver Akt1/PKBα after burn injury.

SILAM for quantitative proteomics of liver Akt1/PKBα after burn injury.

Abstract：

:Akt1/protein kinase Bα (Akt1/PKBα) is a downstream mediator of the insulin signaling system. In this study we explored mechanism(s) for its role in burn injury. Akt1/PKBα in liver extracts from mice with burn injury fed with (2H7)-L-Leu was immunoprecipitated and isolated with SDS-PAGE. Two tryptic peptides, one in the kinase loop and a control peptide just outside of the loop were sequenced via nano-LC interfaced with quadruple time-of-flight tandem mass spectrometry (Q-TOF tandem MS). Their relative isotopologue abundances were determined by stable isotope labeling by amino acids in mammalians (SILAM). Relative quantifications based on paired heavy/light peptides were obtained in 3 steps. The first step included homogenization of mixtures of equal amounts of tissue from burned and sham-treated animals (i.e., isotope dilution) and acquisition of uncorrected data based on parent monoisotopic MS ion ratios. The second step included determination of isotopic enrichment of the kinase from burned mice on Day 7 and the third step enrichment correction of partially labeled heavy and light monoisotopic MS ion ratios for relative quantification of bioactivity (loop peptide) and expression level (control peptide). Protein synthesis and enrichment after injury were found to be dependent on tissue and turnover of individual proteins. Three heavy and light monoisotopic ion ratios for albumin peptides from burned mice indicated ~55% enrichment and ~16.7-fold downregulation. In contract, serum amyloid P had ~66% enrichment and was significantly upregulated. Akt1/PKBα had ~56% enrichment and kinase level in response to the burn injury was upregulated compared with the control peptide. However, kinase bioactivity, represented by the Cys296 peptide, was significantly reduced. Overall, we demonstrated that i) quantitative proteomics can be performed without completely labeled mice; ii) measurement of enrichment of acyl-tRNAs is unnecessary and iii) Cys296 plays an important role in kinase activity after burn injury.

journal_name

Int J Mol Med

journal_title

International journal of molecular medicine

authors

Lu XM,Tompkins RG,Fischman AJ

doi

10.3892/ijmm.2011.861

subject

Has Abstract

pub_date

2012-03-01 00:00:00

pages

461-71

issue

eissn

1107-3756

issn

1791-244X

journal_volume

pub_type

杂志文章

在线工具

SILAM for quantitative proteomics of liver Akt1/PKBα after burn injury.