Highly Multiplexed Targeted Proteomics Acquisition on a TIMS-QTOF,Analytical Chemistry

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Highly Multiplexed Targeted Proteomics Acquisition on a TIMS-QTOF
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-12-17 , DOI: 10.1021/acs.analchem.0c03180
Antoine Lesur ₁ , Pierre-Olivier Schmit ₂ , François Bernardin ₁ , Elisabeth Letellier ₃ , Sven Brehmer ₄ , Jens Decker ₄ , Gunnar Dittmar _{1,

3}

Affiliation

Targeted proteomics allows the highly sensitive detection of specific peptides and proteins in complex biological samples. Here, we describe a methodology for targeted peptide quantification using a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF Pro). The prm-PASEF method exploits the multiplexing capability provided by the trapped ion mobility separation, allowing more than 200 peptides to be monitored over a 30 min liquid chromatography separation. Compared to conventional parallel reaction monitoring (PRM), precursor ions are accumulated in the trapped ion mobility spectrometry (TIMS) cells and separated according to their shape and charge before eluting into the quadrupole time-of-flight (QTOF) part of the mass spectrometer. The ion mobility trap allows measuring up to six peptides from a single 100 ms ion mobility separation with the current setup. Using these improved mass spectrometric capabilities, we detected and quantified 216 isotope-labeled synthetic peptides (AQUA peptides) spiked in HeLa human cell extract with limits of quantification of 17.2 amol for some peptides. The acquisition method is highly reproducible between injections and enables accurate quantification in biological samples, as demonstrated by quantifying KRas, NRas, and HRas as well as several Ras mutations in lung and colon cancer cell lines on fast 10 min gradient separations.

中文翻译：

在TIMS-QTOF上高度多重靶向蛋白质组学的采集

靶向蛋白质组学可以高度灵敏地检测复杂生物样品中的特定肽和蛋白质。在这里，我们描述了一种使用捕获的离子迁移率四极杆飞行时间质谱仪（timsTOF Pro）进行靶向肽定量的方法。prm-PASEF方法利用了由捕获的离子迁移率分离提供的多路复用功能，可以在30分钟的液相色谱分离中监测200多种肽。与常规的平行反应监测（PRM）相比，前驱物离子在被捕获的离子迁移谱（TIMS）池中积累，并根据其形状和电荷进行分离，然后洗脱到质谱仪的四极杆飞行时间（QTOF）部分中。离子淌度阱可以在当前设置下通过一次100 ms的离子淌度分离来测量多达6种肽。使用这些改进的质谱功能，我们检测并定量了掺入HeLa人细胞提取物中的216种同位素标记的合成肽（AQUA肽），其中某些肽的定量限为17.2 amol。采集方法在两次进样之间具有很高的重现性，并且能够对生物样品进行准确的定量，这可以通过快速10分钟梯度分离对KRas，NRas和HRas以及肺和结肠癌细胞系中的Ras突变进行定量来证明。我们检测并定量了掺入HeLa人细胞提取物中的216种同位素标记的合成肽（AQUA肽），某些肽的定量限为17.2 amol。采集方法在两次进样之间具有很高的重现性，并且能够对生物样品进行准确的定量，如通过在10分钟快速梯度分离中定量KRas，NRas和HRas以及肺和结肠癌细胞系中的若干Ras突变所证明的那样。我们检测并定量了掺入HeLa人细胞提取物中的216种同位素标记的合成肽（AQUA肽），某些肽的定量限为17.2 amol。采集方法在两次进样之间具有很高的重现性，并且能够对生物样品进行准确的定量，如通过在10分钟快速梯度分离中定量KRas，NRas和HRas以及肺和结肠癌细胞系中的若干Ras突变所证明的那样。

更新日期：2021-01-26

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