"Proteotyping": Population proteomics of human leukocytes using top down mass spectrometry
"Proteotyping": Population proteomics of human leukocytes using top down mass spectrometry
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DOI：
10.1021/ac800141g
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发表时间：
2008-04-15
影响因子：
7.4
通讯作者：
Kelleher, Neil L.
中科院分区：
文献类型：
--
作者：
Roth, Michael J.;Parks, Bryan A.;Kelleher, Neil L.
通过标准的基于肽的蛋白质组学来表征单个蛋白质上的编码多态性（cSNP）、选择性剪接和翻译后修饰（PTM）的组合是具有挑战性的，这是由于< 100% sequence coverage and the uncoupling effect of proteolysis on such variations >10-20个残基分开。由于自上而下的MS测量整个蛋白质，因此可以检测到影响一级序列的所有变异的组合，因为它们组合出现。由所有类型的变异产生的蛋白质形式在这里被称为“蛋白质型”，类似于DNA水平上的单倍型。使用双重在线/离线自上而下MS策略分析从去白细胞过滤器收获的人原代白细胞的蛋白质，产生> 600个独特的完整质量，其中133个是从67个独特基因中鉴定的。利用一个二维平台，称为多维蛋白质表征的自动化自上而下（MudCAT），108上述蛋白质形式，随后确定在MS/MS的情况下，在4天。此外，MudCAT能够定量杂合子的等位基因比率和磷酸化物质的PTM占位率。人类蛋白质组的多样性体现在以下事实中：32种鉴定的蛋白质含有cSNP、PTM或被检测为蛋白水解产物。在这些信息中，有三种部分磷酸化的蛋白质和三种在已知cSNP基因座杂合的蛋白质，有证据表明不同等位基因的表达比例为非1：1。
Characterizing combinations of coding polymorphisms (cSNPs), alternative splicing and post-translational modifications (PTMs) on a single protein by standard peptide-based proteomics is challenging owing to < 100% sequence coverage and the uncoupling effect of proteolysis on such variations > 10-20 residues apart. Because top down MS measures the whole protein, combinations of all the variations affecting primary sequence can be detected as they occur in combination. The protein form generated by all types of variation is here termed the "proteotype", akin to a haplotype at the DNA level. Analysis of proteins from human primary leukocytes harvested from leukoreduction filters using a dual on-fine/ off-line top down MS strategy produced > 600 unique intact masses, 133 of which were identified from 67 unique genes. Utilizing a two-dimensional platform, termed multidimensional protein characterization by automated top down (MudCAT), 108 of the above protein forms were subsequently identified in the absence of MS/MS in 4 days. Additionally, MudCAT enables the quantitation of allele ratios for heterozygotes and PTM occupancies for phosphorylated species. The diversity of the human proteome is embodied in the fact that 32 of the identified proteins harbored cSNPs, PTMs, or were detected as proteolysis products. Among the information were three partially phosphorylated proteins and three proteins heterozygous at known cSNP loci, with evidence for non-1: 1 expression ratios obtained for different alleles.