Impact of Charge Reduction on Sequence Coverage of Proteins by 193 nm Ultraviolet Photodissociation.

An ongoing goal of top-down mass spectrometry is to increase the performance for larger proteins. Using higher energy activation methods, like 193 nm ultraviolet photodissociation (UVPD), offers the potential to cause more extensive fragmentation of large proteins and thereby yield greater sequence coverage. Obtaining high sequence coverage requires confident identification and assignment of fragment ions, and this process is hampered by spectral congestion and low signal-to-noise ratio (S/N) of the fragment ions.

Leveraging Complementary Ion Activation Methods with Proton Transfer Charge Reduction Reactions for Comprehensive Characterization of Monoclonal Antibody Heavy Chain Subunits.

Characterization of large proteins by top-down mass spectrometry is challenged by low S/N of fragment ions and spectral congestion. Proton transfer charge reduction (PTCR) is one strategy that has shown great potential for addressing spectral congestion and enhancing sequence coverage of large proteins, but low S/N remains an obstacle, requiring extensive spectral averaging. Here we advance the characterization of large proteins, including an antibody (mAb) and an antibody drug conjugate (ADC), on a liquid chromatography time scale by implementing a hybrid strategy that combines ultraviolet photodissociation (UVPD), electron transfer higher collision energy dissociation (EThcD), PTCR, and gas-phase fractionation.

Proteo-SAFARI: An R Application for Identification of Fragment Ions in Top-Down MS/MS Spectra of Proteins.

Proteo-SAFARI is a shiny application for fragment assignment by relative isotopes, an R-based software application designed for identification of protein fragment ions directly in the / domain. This program provides an open-source, user-friendly application for identification of fragment ions from a candidate protein sequence with support for custom covalent modifications and various visualizations of identified fragments. Additionally, Proteo-SAFARI includes a nonnegative least-squares fitting approach to determine the contributions of various hydrogen shifted fragment ions ( + 1, + 1, - 1, - 2) observed in UVPD mass spectra which exhibit overlapping isotopic distributions.

Hydrophilic Interaction Chromatography Coupled to Ultraviolet Photodissociation Affords Identification, Localization, and Relative Quantitation of Glycans on Intact Glycoproteins.

Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans, including microheterogeneity of some of the glycosites (amino acid residues). Various mass spectrometry (MS) strategies have proven tremendously successful for localizing and identifying glycans, typically utilizing a bottom-up workflow in which glycoproteins are digested to create glycopeptides to facilitate analysis. An emerging alternative is top-down MS that aims to characterize intact glycoproteins to allow precise identification and localization of glycans.

Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation.

As the development of new biotherapeutics advances, increasingly sophisticated tandem mass spectrometry methods are needed to characterize the most complex molecules, including antibody drug conjugates (ADCs). Lysine-linked ADCs, such as trastuzumab-emtansine (T-DM1), are among the most heterogeneous biotherapeutics. Here, we implement a workflow that combines limited proteolysis with HCD-triggered EThcD and UVPD mass spectrometry for the characterization of the resulting middle-down large-sized peptides of T-DM1.

Enhancing Top-Down Analysis of Proteins by Combining Ultraviolet Photodissociation (UVPD), Proton-Transfer Charge Reduction (PTCR), and Gas-Phase Fractionation to Alleviate the Impact of Nondissociated Precursor Ions.

Recent advances in top-down mass spectrometry strategies continue to improve the analysis of intact proteins. 193 nm ultraviolet photodissociation (UVPD) is one method well-suited for top-down analysis. UVPD is often performed using relatively low photon flux in order to limit multiple-generation dissociation of fragment ions and maximize sequence coverage.

Characterization of Unbranched Ubiquitin Tetramers by Combining Ultraviolet Photodissociation with Proton Transfer Charge Reduction Reactions.

Polyubiquitination is an important post-translational modification (PTM) that regulates various biological functions. The linkage sites and topologies of polyubiquitination chains are important factors in determining the fate of polyubiquitinated proteins. Characterization of polyubiquitin chains is the first step in understanding the biological functions of protein ubiquitination, but it is challenging owing to the repeating nature of the ubiquitin chains and the difficulty in deciphering linkage positions.

Impact of Internal Fragments on Top-Down Analysis of Intact Proteins by 193 nm UVPD.

193 nm ultraviolet photodissociation (UVPD) allows high sequence coverage to be obtained for intact proteins using terminal fragments alone. However, internal fragments, those that contain neither N- nor C- terminus, are typically ignored, neglecting their potential to bolster characterization of intact proteins. Here, we explore internal fragments generated by 193 nm UVPD for proteins ranging in size from 17-47 kDa and using the ClipsMS algorithm to facilitate searches for internal fragments.