Massachusetts General Hospital, Boston, MA, USA.
Int J Mol Med. 2012 Mar;29(3):461-71. doi: 10.3892/ijmm.2011.861. Epub 2011 Dec 14.
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.
Akt1/蛋白激酶 Bα (Akt1/PKBα) 是胰岛素信号系统的下游介质。在这项研究中,我们探讨了其在烧伤中的作用机制。用(2H7)-L-Leu 喂养烧伤小鼠的肝提取物中的 Akt1/PKBα 用 SDS-PAGE 进行免疫沉淀和分离。两个胰蛋白酶肽段,一个在激酶环内,一个在环外的对照肽段,通过纳米 LC 与四极飞行时间串联质谱 (Q-TOF 串联 MS) 进行测序。通过稳定同位素标记哺乳动物中的氨基酸 (SILAM) 确定它们的相对同量异位体丰度。基于配对重/轻肽的相对定量在 3 个步骤中获得。第一步包括将等量的烧伤和假处理动物组织的混合物匀浆(即同位素稀释),并根据母体单同位素 MS 离子比获得未经校正的数据。第二步包括确定第 7 天烧伤小鼠激酶的同位素丰度,第三步是对部分标记的重和轻单同位素 MS 离子比进行富集校正,以进行生物活性(环肽)和表达水平(对照肽)的相对定量。发现蛋白质合成和损伤后的富集依赖于组织和个体蛋白质的周转率。来自烧伤小鼠的白蛋白肽的三个重和轻单同位素离子比表明约 55%的富集和约 16.7 倍的下调。相比之下,血清淀粉样蛋白 P 有~66%的富集并且显著上调。Akt1/PKBα 的富集程度约为 56%,与对照肽相比,烧伤后的激酶水平上调。然而,激酶的生物活性,代表 Cys296 肽,显著降低。总的来说,我们证明了 i)无需完全标记小鼠即可进行定量蛋白质组学;ii)测量酰基-tRNA 的丰度是不必要的;iii)Cys296 在烧伤后激酶活性中起重要作用。