Quantitative proteomics targeting classes of motif-containing peptides using immunoaffinity-based mass spectrometry.
Quantitative proteomics targeting classes of motif-containing peptides using immunoaffinity-based mass spectrometry.
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DOI：
10.1074/mcp.m111.016238
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发表时间：
2012-08
期刊：
影响因子：
--
通讯作者：
Wingren C
中科院分区：
文献类型：
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作者：
Olsson N;James P;Borrebaeck CA;Wingren C
开发高性能的技术平台来生成详细的蛋白质表达谱或蛋白质图谱是必不可少的。最近，我们提出了一个新的平台，我们称之为全球蛋白质组调查，其中我们结合亲和蛋白质组学和质谱的最佳特性，以一种独立的方式探测任何蛋白质组，同时仍然使用有限的抗体集。我们使用了所谓的上下文无关基序特异性抗体，针对短氨基酸基序。这使得从消化的蛋白质组中富集含有基序的肽，然后通过质谱法检测和鉴定。在这项研究中，我们已经证明了全球蛋白质组调查技术的定量能力、可重复性、灵敏度和覆盖范围，通过在葡萄糖或乙醇中培养的细胞培养物中标记酵母培养物的氨基酸进行稳定同位素标记。数据表明，可以以高度可重复性的方式检测，鉴定和定量广泛的含有基序的肽（蛋白质）。平均而言，发现六种不同基序特异性抗体中的每一种都针对大约75种不同的含基序的蛋白质。此外，从蛋白质中产生的肽，从每细胞100多万个拷贝到不到50个拷贝，都可能成为靶标。值得注意的是，之前在PeptideAtlas数据库中未报道的一组重要肽是分析目标之一。定量数据与同一样品常规强阳离子交换分馏得到的相应数据相吻合。最后，在葡萄糖和乙醇培养的酵母中可以检测到几种具有已知和未知功能的差异表达蛋白，其中许多与中心碳代谢有关。综上所述，该研究证明了我们基于免疫亲和的质谱平台在针对含基序肽类的可重复定量蛋白质组学方面的潜力。
The development of high-performance technology platforms for generating detailed protein expression profiles, or protein atlases, is essential. Recently, we presented a novel platform that we termed global proteome survey, where we combined the best features of affinity proteomics and mass spectrometry, to probe any proteome in a species independent manner while still using a limited set of antibodies. We used so called context-independent-motif-specific antibodies, directed against short amino acid motifs. This enabled enrichment of motif-containing peptides from a digested proteome, which then were detected and identified by mass spectrometry. In this study, we have demonstrated the quantitative capability, reproducibility, sensitivity, and coverage of the global proteome survey technology by targeting stable isotope labeling with amino acids in cell culture-labeled yeast cultures cultivated in glucose or ethanol. The data showed that a wide range of motif-containing peptides (proteins) could be detected, identified, and quantified in a highly reproducible manner. On average, each of six different motif-specific antibodies was found to target about 75 different motif-containing proteins. Furthermore, peptides originating from proteins spanning in abundance from over a million down to less than 50 copies per cell, could be targeted. It is worth noting that a significant set of peptides previously not reported in the PeptideAtlas database was among the profiled targets. The quantitative data corroborated well with the corresponding data generated after conventional strong cation exchange fractionation of the same samples. Finally, several differentially expressed proteins, with both known and unknown functions, many relevant for the central carbon metabolism, could be detected in the glucose- versus ethanol-cultivated yeast. Taken together, the study demonstrated the potential of our immunoaffinity-based mass spectrometry platform for reproducible quantitative proteomics targeting classes of motif-containing peptides.