Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.
J Proteome Res. 2012 Oct 5;11(10):4972-82. doi: 10.1021/pr300514u. Epub 2012 Aug 31.
We present a novel analytical platform for peptides quantitative assays in biological matrices based on microscale liquid chromatography fractionation and matrix-assisted laser desorption/ionization mass spectrometric detection using the selected reaction monitoring (SRM) mode. The MALDI source was equipped with a high frequency Nd:YAG laser (1000 Hz) and mounted on a triple quadrupole/linear ion trap mass spectrometer (MALDI-QqQ(LIT)). Compared to conventional LC-ESI-SRM/MS, the separated analytes are "time-frozen" onto the MALDI plate in fractions, and navigation through the LC chromatogram makes it possible to perform SRM experiments as well as enhanced product ion spectra acquisition for confirmatory analyses without time constraints. The LC spots were analyzed using different rastering speeds ranging from 0.25 to 4 mm/sec with the shortest analysis time of 425 ms/spot. Since the LC runs can be multiplexed and do not need a comprehensive investigation, the present platform offers a valuable alternative to LC-ESI-SRM/MS for high throughput proteomic analyses. In addition, the derivatization of the N-terminal α-amino group by sulfonation was found to be key for the fragmentation of singly charged peptides under low collision energy regime. Under such conditions, y-ion series were observed in the MS/MS spectra, and thus the design of SRM experiments was greatly simplified. The quantitative performance of the platform was compared to that of LC-ESI-SRM/MS by spiking yeast tryptic peptides in human plasma digests. Both platforms exhibited similar sensitivities, accuracy (within ±20%) and precision (under 20%) in the relative quantification mode. As a proof of principle, the relative and absolute quantification of proteins associated with glycolysis, glyoxylate and tricarboxylic acid (TCA) cycles over a growth time course of Saccharomyces cerevisiae on glucose media was successfully performed using isotopic dilution.
我们提出了一种基于微尺度液相色谱分离和基质辅助激光解吸/电离串联质谱（MALDI-QqQ（LIT））检测的新型分析平台，用于在生物基质中定量测定肽。MALDI 源配备了高频 Nd:YAG 激光（1000 Hz），并安装在三重四极杆/线性离子阱质谱仪（MALDI-QqQ（LIT））上。与传统的 LC-ESI-SRM/MS 相比，分离的分析物在 LC 色谱图中“时间冻结”成分数，并通过导航进行 SRM 实验以及增强产物离子谱采集，用于无需时间限制的确认分析。使用不同的扫描速度（0.25 至 4mm/sec）对 LC 斑点进行分析，最短分析时间为 425ms/spot。由于 LC 运行可以进行多路复用，并且不需要全面调查，因此该平台为高通量蛋白质组学分析提供了 LC-ESI-SRM/MS 的有价值替代方案。此外，通过磺化对 N-末端α-氨基基团进行衍生化被发现是在低碰撞能条件下使单电荷肽碎片化的关键。在这种条件下，在 MS/MS 谱中观察到 y-离子系列，从而大大简化了 SRM 实验的设计。通过向人血浆消化物中的酵母胰蛋白酶肽中添加样品来比较平台的定量性能与 LC-ESI-SRM/MS。在相对定量模式下，两种平台均表现出相似的灵敏度、准确性（在±20%范围内）和精密度（在 20%以下）。作为原理验证，使用同位素稀释成功地对与糖酵解、乙醛酸和三羧酸（TCA）循环相关的蛋白质进行了相对和绝对定量，这些蛋白质在酿酒酵母的葡萄糖培养基生长时间过程中。
