Mass spectrometry (Ms)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. to be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. this is necessary because of the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. recently, new alternative materials have emerged that allow excellent and robust enrichment of phosphopeptides. these monodisperse microsphere-based immobilized metal ion affinity chromatography (IMac) resins incorporate a flexible linker terminated with phosphonate groups that chelate either zirconium or titanium ions. the chelated zirconium or titanium ions bind specifically to phosphopeptides, with an affinity that is similar to that of other widely used metal oxide affinity chromatography materials (typically tio 2 ). Here we present a detailed protocol for the preparation of monodisperse microsphere-based ti 4 + -IMac adsorbents and the subsequent enrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samples before enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparation of the chromatographic system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed. Bioinformatics analysis specifically relating to site localization is also addressed. Finally, we demonstrate how the protocols provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes ~2 weeks to complete the protocol: 1 week to prepare the ti 4 + -IMac material, 2 d for sample preparation, 3 d for Ms analysis of the enriched sample and 2 d for data analysis.

Flavonoids have been recognised as one of the largest and most widespread groups of plant secondary metabolites, with marked antioxidant properties. The general name flavonoid refers to a class of more than 6500 molecules based upon a 15-carbon skeleton. In this paper a general overview of flavonoids, their classification, structures and analytical methods for their determination is presented.

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.