Implementation of UV-photodissociation on a benchtop Q Exactive mass spectrometer and its application to phospho-proteomics

Abstract

Proteomics applications performed on the popular Q Exactive Orbitrap benchtop mass analyzer have so far relied exclusively on 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 UVPD fragmentation, complete with synchronization triggering to make it compatible with LC-MS/MS workflows. We show that UVPD is not only directly compatible with LC-MS workflows, but 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 on the 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 PTMs and is a powerful and complementary addition to the proteomic toolbox.