School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, UK.
Anal Chem. 2013 May 21;85(10):4836-43. doi: 10.1021/ac400646m. Epub 2013 May 6.
Large scale analysis of proteins by mass spectrometry is becoming increasingly routine; however, the presence of peptide isomers remains a significant challenge for both identification and quantitation in proteomics. Classes of isomers include sequence inversions, structural isomers, and localization variants. In many cases, liquid chromatography is inadequate for separation of peptide isomers. The resulting tandem mass spectra are composite, containing fragments from multiple precursor ions. The benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) for proteomics have been demonstrated by a number of groups, but previously work has focused on extending proteome coverage generally. Here, we present a systematic study of the benefits of FAIMS for a key challenge in proteomics, that of peptide isomers. We have applied FAIMS to the analysis of a phosphopeptide library comprising the sequences GPSGXVpSXAQLX(K/R) and SXPFKXpSPLXFG(K/R), where X = ADEFGLSTVY. The library has defined limits enabling us to make valid conclusions regarding FAIMS performance. The library contains numerous sequence inversions and structural isomers. In addition, there are large numbers of theoretical localization variants, allowing false localization rates to be determined. The FAIMS approach is compared with reversed-phase liquid chromatography and strong cation exchange chromatography. The FAIMS approach identified 35% of the peptide library, whereas LC-MS/MS alone identified 8% and LC-MS/MS with strong cation exchange chromatography prefractionation identified 17.3% of the library.
通过质谱法对蛋白质进行大规模分析正变得越来越普遍；然而，肽异构体的存在仍然是蛋白质组学中鉴定和定量的一个重大挑战。异构体的类别包括序列反转、结构异构体和定位变体。在许多情况下，液相色谱法不足以分离肽异构体。所得串联质谱是复合的，包含来自多个前体离子的片段。许多小组已经证明了高场非对称波形离子淌度谱（FAIMS）在蛋白质组学中的优势，但之前的工作主要集中在普遍扩展蛋白质组覆盖范围上。在这里，我们对 FAIMS 对蛋白质组学中的一个关键挑战——肽异构体的优势进行了系统研究。我们已经将 FAIMS 应用于由 GPSGXVpSXAQLX(K/R) 和 SXPFKXpSPLXFG(K/R) 序列组成的磷酸肽文库的分析，其中 X = ADEFGLSTVY。该文库具有定义的限制，使我们能够对 FAIMS 性能做出有效结论。该文库包含许多序列反转和结构异构体。此外，还有大量的理论定位变体，允许确定错误定位率。FAIMS 方法与反相液相色谱和强阳离子交换色谱进行了比较。FAIMS 方法鉴定了文库的 35%，而单独的 LC-MS/MS 鉴定了 8%，LC-MS/MS 与强阳离子交换色谱预分级鉴定了 17.3%。
