Implementation of Ultraviolet Photodissociation on a Benchtop Q Exactive Mass Spectrometer and Its Application to Phosphoproteomics
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- 作者：Kyle L. Fort ; Andrey Dyachenko ; Clement M. Potel ; Eleonora Corradini ; Fabio Marino ; Arjan Barendregt ; Alexander A. Makarov ; Richard A. Scheltema ; Albert J. R. Heck
- 刊名：Analytical Chemistry
- 出版年：2016
- 出版时间：February 16, 2016
- 年：2016
- 卷：88
- 期：4
- 页码：2303-2310
- 全文大小：574K
- ISSN：1520-6882
文摘
Proteomics applications performed on the popular benchtop Q Exactive Orbitrap mass spectrometer have so far relied exclusively on higher collision-energy dissociation (HCD) fragmentation for peptide sequencing. While this fragmentation technique is applicable to a wide range of biological questions, it also has limitations, and all questions cannot be addressed equally well. Here, we demonstrate that the fragmentation capabilities of the Q Exactive mass spectrometer can be extended with ultraviolet photodissociation (UVPD) fragmentation, complete with synchronization triggering to make it compatible with liquid chromatography (LC)/tandem mass spectrometry (MS/MS) workflows. We show that UVPD not only is directly compatible with LC/MS workflows but also, when combined with these workflows, can result in higher database scores and increased identification rates for complex samples as compared to HCD methods. UVPD as a fragmentation technique offers prompt, high-energy fragmentation, which can potentially lead to improved analyses of labile post-translational modifications. Techniques like HCD result in substantial amounts of modification losses, competing with fragmentation pathways that provide information-rich ion fragments. We investigate here the utility of UVPD for identification of phosphorylated peptides and find that UVPD fragmentation reduces the extent of labile modification loss by up to ∼60%. Collectively, when integrated into a complete workflow on the Q Exactive Orbitrap, UVPD provides distinct advantages to the analysis of post-translational modifications and is a powerful and complementary addition to the proteomic toolbox.