High throughput proteome-wide precision measurements of protein expression using mass spectrometry
High throughput proteome-wide precision measurements of protein expression using mass spectrometry
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DOI：
10.1021/ja991063o
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发表时间：
1999-09-01
影响因子：
15
通讯作者：
Smith, RD
中科院分区：
文献类型：
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作者：
Pasa-Tolic, L;Jensen, PK;Smith, RD
与细胞几乎静态的基因组相反，蛋白质组（生物体表达的蛋白质补体）会随着外部刺激和内部过程而不断变化。基于基因表达和寡核苷酸微阵列分析的系列分析，mRNA 水平（即转录组）的全局基因表达分析最近已变得可行。这些技术允许同时轮询组织或细胞群的数千个基因的激活状态。然而，测量 mRNA 丰度而不是功能基因产物（即蛋白质）的测定在蛋白质修饰方面缺乏信息，并且由于 mRNA 和蛋白质的稳定性、表达率等差异，不能很好地反映蛋白质丰度。作者开发了一种方法，利用在稳定同位素标记培养基（例如稀有同位素耗尽和正常）中培养的生物体为所有检测到的蛋白质提供有效的内部校准物，从而能够在整个蛋白质组范围内精确测量细胞扰动引起的蛋白质丰度变化。在样品处理步骤之前将两种（或更多）同位素独特的细胞群混合，消除与细胞裂解、分离和质谱分析相关的所有实验变量。因此，相对蛋白质丰度的变化通过每种蛋白质的两种同位素不同且可解析版本的比率精确地反映。
In contrast to a cell's virtually static genome, the proteome, the protein complement expressed by an organism, continually changes in response to external stimuli and internal processes. Global gene expression analysis at the mRNA level (i.e., transcriptome) has recently become feasible based on the serial analysis of gene expression and oligonucleotide micro-array assays. These techniques allow the activation states of thousands of genes to be polled simultaneously for a tissue or cell population. However, assays that measure mRNA abundances rather than the functional gene products (i.e., proteins) are uninformative with regard to protein modifications, and can poorly reflect protein abundances due to differences in stabilities, expression rates, etc., for both the mRNAs and proteins. The authors have developed an approach utilizing organisms cultured in stable-isotope labeled media (e.g., rare-isotope depleted and normal) to provide effective internal calibrants for all detected proteins, thus enabling precise proteome-wide measurement of changes in protein abundances resulting from cellular perturbations. The two (or more) isotopically distinctive cell populations are mixed prior to sample processing steps, eliminating all experimental variables associated with cell lysis, separation, and mass spectrometric analysis. Changes in relative protein abundances are thus precisely reflected by the ratio of two isotopically different and resolvable versions of each protein.