The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer’s disease and in cancer pathogenesis. The 5 nanosecond, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision-based and electron-based dissociation methods.

How chromatin shapes pathways that promote genome-epigenome integrity in response to DNA damage is an issue of crucial importance. We report that human bromodomain (BRD)-containing proteins, the primary ''readers'' of acetylated chromatin, are vital for the DNA damage response (DDR). We discovered that more than one-third of all human BRD proteins change localization in response to DNA damage. We identified ZMYND8 (zinc finger and MYND [myeloid, Nervy, and DEAF-1] domain containing 8) as a novel DDR factor that recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to damaged chromatin. Our data define a transcription-associated DDR pathway mediated by ZMYND8 and the NuRD complex that targets DNA damage, including when it occurs within transcriptionally active chromatin, to repress transcription and promote repair by homologous recombination. Thus, our data identify human BRD proteins as key chromatin modulators of the DDR and provide novel insights into how DNA damage within actively transcribed regions requires chromatin-binding proteins to orchestrate the appropriate response in concordance with the damage-associated chromatin context.

Photodissociation mass spectrometry combines the ability to activate and fragment ions using photons with the sensitive detection of the resulting product ions by mass spectrometry. The resulting combination affords a versatile tool for characterization of biological molecules. The scope and breadth of photodissociation mass spectrometry have increased substantially over the past decade as new research groups have entered the field and developed a number of innovative applications that illustrate the ability of photodissociation to produce rich fragmentation patterns, to cleave bonds selectively, and to target specific molecules based on incorporation of chromophores. This review focuses on many of the key developments in photodissociation mass spectrometry over the past decade with a particular emphasis on its applications to biological molecules.

A sulfated peptide activates a rice immune receptor.

Complete structural characterization of complex lipids, such as glycerophospholipids, by tandem mass spectrometry (MS/MS) continues to present a major challenge. Conventional activation methods do not generate fragmentation patterns that permit the simultaneous discernment of isomers which differ in both the positions of acyl chains on the glycerol backbone and the double bonds within the acyl chains. Herein we describe a hybrid collisional activation/UVPD workflow that yields near-complete structural information for glycerophospholipids. This hybrid MS3 strategy affords the lipid’s sum composition based on the accurate mass measured for the intact lipid as well as highly specific diagnostic product ions that reveal both the acyl chain assignment (i.e. sn-position) and the site-specific location of double bonds in the acyl chains. This approach is demonstrated to differentiate sn-positional and double bond positional isomers, such as the regioisomeric phosphatidylcholines PC 16:0/18:1(n-9) and PC 18:1(n-9)/16:0 and has been integrated into an LC-MS3 workflow.

The development of new ion-activation/dissociation methods continues to be one of the most active areas of mass spectrometry owing to the broad applications of tandem mass spectrometry in the identification and structural characterization of molecules. This Review will showcase the impact of ultraviolet photodissociation (UVPD) as a frontier strategy for generating informative fragmentation patterns of ions, especially for biological molecules whose complicated structures, subtle modifications, and large sizes often impede molecular characterization. UVPD energizes ions via absorption of high-energy photons, which allows access to new dissociation pathways relative to more conventional ion-activation methods. Applications of UVPD for the analysis of peptides, proteins, lipids, and other classes of biologically relevant molecules are emphasized in this Review. CONTENTS 1. Introduction 3328 1.1. Scope of Review 3328 1.2. Instrumentation 3330 2. UVPD for Peptides 3330 2.1. Mechanistic Studies of Peptides 3330 2.2. UVPD for Peptide Sequencing and Bottom-Up Proteomics 3332 2.3. UVPD for Identification of Post-Translational Modifications 3333 2.4. PTM Analysis Using Other Wavelengths 3336 2.5. Improving S/N of UVPD 3336 2.6. UVPD and Derivatization Strategies 3337 2.6.1. Radical-Directed Dissociation 3337 2.6.2. Photoelectron-Transfer Dissociation 3340 2.6.3. UVPD (266 nm) for Selective Bond Cleavages 3340 2.6.4. UVPD Using 351/355 nm Photons 3340 2.6.5. UVPD with Online Reactions and Hydrogen/Deuterium Exchange 3342 2.6.6. Derivatization and Photodissociation Using Visible Wavelengths 3343 2.7. UVPD for Middle-Down Proteomics 3343 3. UVPD for Intact Proteins 3345 3.1. Fundamental Aspects 3347 3.2. Hybrid Methods and New Concepts for Top-Down Analysis 3347 3.3. Other Wavelengths for Top-Down Analysis 3351 4. UVPD for Native Mass Spectrometry and Structural Biology Applications 3352 4.1. Chemical-Probe Methods 3352 4.2. Native MS 3354 4.2.1. Protein−Ligand Complexes 3355 4.2.2. UVPD for Multimeric Protein Complexes 3357 4.3. UVPD and Ion Mobility 3359 5. UVPD for Lipids 3360 5.1. Radical-Directed Dissociation 3360 5.2. UVPD (193 nm) 3361 5.3. UVPD (213 nm) 3362 5.4. UVPD and DESI 3364 5.5. Lipid A and Lipopolysaccharides 3364 6.

Graphical Abstract

Ultraviolet photodissociation (UVPD) mass spectrometry (MS) was used to characterize the sequences of proteins in native protein–ligand and protein–protein complexes and to provide auxiliary information about the binding sites of the ligands and protein–protein interfaces. UVPD outperformed collisional induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) in terms of yielding the most comprehensive diagnostic primary sequence information about the proteins in the complexes. UVPD also generated noncovalent fragment ions containing a portion of the protein still bound to the ligand which revealed some insight into the nature of the binding sites of myoglobin/heme, eIF4E/m7GTP, and human peptidyl-prolyl cis–trans isomerase 1 (Pin1) in complex with the peptide derived from the C-terminal domain of RNA polymerase II (CTD). Noncovalently bound protein–protein fragment ions from oligomeric β-lactoglobulin dimers and hexameric insulin complexes were also produced upon UVPD, providing some illumination of tertiary and quaternary protein structural features.