Fernández-Fernández Mario, Rodríguez-González Pablo, García Alonso J Ignacio
Department of Physical and Analytical Chemistry Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006, Oviedo, Spain.
J Mass Spectrom. 2016 Oct;51(10):980-987. doi: 10.1002/jms.3809.
We have developed a novel, rapid and easy calculation procedure for Mass Isotopomer Distribution Analysis based on multiple linear regression which allows the simultaneous calculation of the precursor pool enrichment and the fraction of newly synthesized labelled proteins (fractional synthesis) using linear algebra. To test this approach, we used the peptide RGGGLK as a model tryptic peptide containing three subunits of glycine. We selected glycine labelled in two C atoms ( C -glycine) as labelled amino acid to demonstrate that spectral overlap is not a problem in the proposed methodology. The developed methodology was tested first in vitro by changing the precursor pool enrichment from 10 to 40% of C -glycine. Secondly, a simulated in vivo synthesis of proteins was designed by combining the natural abundance RGGGLK peptide and 10 or 20% C -glycine at 1 : 1, 1 : 3 and 3 : 1 ratios. Precursor pool enrichments and fractional synthesis values were calculated with satisfactory precision and accuracy using a simple spreadsheet. This novel approach can provide a relatively rapid and easy means to measure protein turnover based on stable isotope tracers. Copyright © 2016 John Wiley & Sons, Ltd.
我们基于多元线性回归开发了一种新颖、快速且简便的质量同位素异构体分布分析计算程序，该程序利用线性代数可同时计算前体库丰度和新合成标记蛋白质的比例（合成分数）。为测试此方法，我们使用肽RGGGLK作为含三个甘氨酸亚基的胰蛋白酶解肽模型。我们选择在两个碳原子上标记的甘氨酸（¹³C-甘氨酸）作为标记氨基酸，以证明在所提出的方法中光谱重叠不是问题。首先通过将¹³C-甘氨酸的前体库丰度从10%改变至40%，在体外对所开发的方法进行测试。其次，通过将天然丰度的RGGGLK肽与10%或20%的¹³C-甘氨酸按1∶1、1∶3和3∶1的比例组合，设计了一种蛋白质的模拟体内合成。使用简单的电子表格以令人满意的精度和准确度计算前体库丰度和合成分数值。这种新方法能够提供一种基于稳定同位素示踪剂相对快速且简便的测量蛋白质周转率的手段。版权所有© 2016约翰·威利父子有限公司。
