Cysteinyl peptide capture for shotgun proteomics: global assessment of chemoselective fractionation.
Cysteinyl peptide capture for shotgun proteomics: global assessment of chemoselective fractionation.
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DOI：
10.1021/pr1007015
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发表时间：
2010-10-01
影响因子：
4.4
通讯作者：
Liebler, Daniel C.
中科院分区：
文献类型：
--
作者：
Lin, De;Li, Jing;Slebos, Robbert J. C.;Liebler, Daniel C.
细胞和组织蛋白质组的复杂性是蛋白质组学生物标志物发现中最重要的技术挑战之一。基于多维液相色谱-串联质谱（LC-MS/MS）的鸟枪蛋白质组学可以与半胱氨酸肽（Cys-肽）的选择性富集相结合，以降低样品复杂性并增加蛋白质组覆盖率。在这里，我们评估了Cys-肽富集对全球蛋白质组库存的影响。我们采用了一种新的可切割的巯基反应性生物素化探针，N-（2-（2-（2-（2-（3-（1-羟基-2-氧代-2-苯乙基）苯氧基）乙酰胺基）乙氧基）-乙氧基）乙基）-5-（2-氧代六氢-1H-噻吩并[3，4-d]咪唑-4-基）戊酰胺（伊布），以捕获消化后的Cys-肽。用伊布试剂处理胰蛋白酶解酶，然后进行链霉亲和素捕获和温和的碱性水解，释放出高度纯化的具有残留S-羧甲基标签的Cys-肽群。等电聚焦（IEF）后，Cys-肽的LC-MS/MS显着扩大了酿酒酵母（酵母）和人结肠癌RKO细胞中蛋白质组的覆盖范围。基于IBB的分级分离增强了Cys-蛋白的检测，与其半胱氨酸含量成正比。富集程度通常为2 - 8倍，但对于少数蛋白质，范围高达近20倍。已发表的酵母蛋白质组的拷贝数注释使MS/MS谱计数数据与酵母蛋白质丰度的基准化成为可能，并揭示了富含半胱氨酸的较低丰度蛋白质的选择性富集。光谱计数数据进一步建立了RKO细胞中的这种关系。低丰度蛋白的增强检测是由于Cys-肽捕获的化学选择性，而不是通过分级分离简化肽混合物。半胱氨酰肽（Cys-肽）的化学选择性富集已用于蛋白质组分析以降低样品复杂性并增加蛋白质组覆盖率。我们通过基于多维液相色谱-串联质谱（LC-MS/MS）的鸟枪蛋白质组学评估了Cys-肽富集对全球蛋白质组学库存的影响。低丰度蛋白的增强检测是由于Cys-肽捕获的化学选择性，而不是通过分级分离简化肽混合物。
The complexity of cell and tissue proteomes presents one of the most significant technical challenges in proteomic biomarker discovery. Multidimensional liquid chromatography−tandem mass spectrometry (LC−MS/MS)-based shotgun proteomics can be coupled with selective enrichment of cysteinyl peptides (Cys-peptides) to reduce sample complexity and increase proteome coverage. Here we evaluated the impact of Cys-peptide enrichment on global proteomic inventories. We employed a new cleavable thiol-reactive biotinylating probe, N-(2-(2-(2-(2-(3-(1-hydroxy-2-oxo-2-phenylethyl)phenoxy)acetamido)ethoxy)-ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (IBB), to capture Cys-peptides after digestion. Treatment of tryptic digests with the IBB reagent followed by streptavidin capture and mild alkaline hydrolysis releases a highly purified population of Cys-peptides with a residual S-carboxymethyl tag. Isoelectric focusing (IEF) followed by LC−MS/MS of Cys-peptides significantly expanded proteome coverage in Saccharomyces cerevisiae (yeast) and in human colon carcinoma RKO cells. IBB-based fractionation enhanced detection of Cys-proteins in direct proportion to their cysteine content. The degree of enrichment typically was 2−8-fold but ranged up to almost 20-fold for a few proteins. Published copy number annotation for the yeast proteome enabled benchmarking of MS/MS spectral count data to yeast protein abundance and revealed selective enrichment of cysteine-rich, lower abundance proteins. Spectral count data further established this relationship in RKO cells. Enhanced detection of low abundance proteins was due to the chemoselectivity of Cys-peptide capture, rather than simplification of the peptide mixture through fractionation. Chemoselective enrichment of cysteinyl peptides (Cys-peptides) has been used in proteome analyses to reduce sample complexity and increase proteome coverage. We evaluated the impact of Cys-peptide enrichment on global proteomic inventories by multidimensional liquid chromatography−tandem mass spectrometry (LC−MS/MS)-based shotgun proteomics. Enhanced detection of low abundance proteins was due to the chemoselectivity of Cys-peptide capture, rather than simplification of the peptide mixture through fractionation.
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影响因子：
15
作者：
Hansen, KC;Rock, RS;Chan, SI
通讯作者：
Chan, SI
影响因子：
4.1
作者：
Foettinger, A;Leitner, A;Lindner, W
通讯作者：
Lindner, W
影响因子：
7.4
作者：
Licklider, LJ;Thoreen, CC;Gygi, SP
通讯作者：
Gygi, SP
影响因子：
7.4
作者：
Liu, HB;Sadygov, RG;Yates, JR
通讯作者：
Yates, JR
影响因子：
4.1
作者：
Dennehy, MK;Richards, KAM;Liebler, DC
通讯作者：
Liebler, DC