Mass-spectrometry-based methods have made significant progress in the characterization of post-translational modifications (PTMs) in peptides and proteins; however, room remains to improve fragmentation methods. Ideal MS/MS methods are expected to simultaneously provide extensive sequence information and localization of PTM sites and retain labile PTM groups. This collection of criteria is difficult to meet, and the various activation methods available today offer different capabilities. In order to examine the specific case of phosphorylation on peptides, we investigate electron transfer dissociation (ETD), electron-activated dissociation (EAD), and 193 nm ultraviolet photodissociation (UVPD) and compare all three methods with classical collision-induced dissociation (CID). EAD and UVPD show extensive backbone fragmentation, comparable in scope to that of CID. These methods provide diverse backbone fragmentation, producing a/x, b/y, and c/z ions with substantial sequence coverages. EAD displays a high retention efficiency of the phosphate modification, attributed to its electron-mediated fragmentation mechanisms, as observed in ETD. UVPD offers reasonable retention efficiency, also allowing localization of the PTM site. EAD experiments were also performed in an LC-MS/MS workflow by analyzing phosphopeptides spiked in human plasma, and spectra allow accurate identification of the modified sites and discrimination of isomers. Based on the overall performance, EAD and 193 nm UVPD offer alternative options to CID and ETD for phosphoproteomics.

ADP-ribosylation is a posttranslational modification (PTM) that has crucial functions in a wide range of cellular processes. Although mass spectrometry (MS) in recent years has emerged as a valuable tool for profiling ADP-ribosylation on a system level, the use of conventional MS methods to profile ADP-ribosylation sites in an unbiased way remains a challenge. Here, we describe a protocol for identification of ADP-ribosylated proteins in vivo on a proteome-wide level, and localization of the amino acid side chains modified with this PTM. The method relies on the enrichment of ADP-ribosylated peptides using the Af1521 macrodomain (Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911-1920, 2005), followed by liquid chromatography-high-resolution tandem MS (LC-MS/MS) with electron transfer dissociation-based peptide fragmentation methods, resulting in accurate localization of ADP-ribosylation sites. This protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture to data processing using the MaxQuant software suite.

Design-of-experiment (DOE) approaches, originally conceived by Fischer, are widely applied in industry, particularly in the context of production for which they have been greatly expended. In a research and development context, DOE can be of great use for method development. Specifically, DOE can greatly speed up instrument parameter optimization by first identifying parameters that are critical to a given outcome, showing parameter interdependency where it occurs and accelerating optimization of said parameters using matrices of experimental conditions. While DOE approaches have been applied in mass spectrometry experiments, they have so far failed to gain widespread adoption. This could be attributed to the fact that DOE can get quite complex and daunting to the everyday user. Here we make the case that a subset of DOE tools, hereafter called SimpleDOE (sDOE), can make DOE accessible and useful to the Mass Spectrometry community at large. We illustrate the progressive gains from a purely manual approach to sDOE through a stepwise optimization of parameters affecting the efficiency of top-down ETD fragmentation of proteins on a high-resolution Q-TOF mass spectrometer, where the aim is to maximize sequence coverage of fragmentation events.

Obtaining extensive sequencing of an intact protein is essential in order to simultaneously determine both the nature and exact localization of chemical and genetic modifications which distinguish different proteoforms arising from the same gene. To effectively achieve such characterization, it is necessary to take advantage of the analytical potential offered by the top-down mass spectrometry approach to protein sequence analysis. However, as a protein increases in size, its gas-phase dissociation produces overlapping, low signal-to-noise fragments. The application of advanced ion dissociation techniques such as electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD) can improve the sequencing results compared to slow-heating techniques such as collisional dissociation; nonetheless, even ETD- and UVPD-based approaches have thus far fallen short in their capacity to reliably enable extensive sequencing of proteoforms ≥30 kDa. To overcome this issue, we have applied proton transfer charge reduction (PTCR) to limit signal overlap in tandem mass spectra (MS2) produced by ETD (alone or with supplemental ion activation, EThcD). Compared to conventional MS2 experiments, following ETD/EThcD MS2 with PTCR MS3 prior to m/z analysis of deprotonated product ions in the Orbitrap mass analyzer proved beneficial for the identification of additional large protein fragments (≥10 kDa), thus improving the overall sequencing and in particular the coverage of the central portion of all four analyzed proteins spanning from 29 to 56 kDa. Specifically, PTCR-based data acquisition led to 39% sequence coverage for the 56 kDa glutamate dehydrogenase, which was further increased to 44% by combining fragments obtained via HCD followed by PTCR MS3.

Mucin-type O-glycosylation is among the most complex post-translational modifications. Despite mediating many physiological processes, O-glycosylation remains understudied compared to other modifications, simply because the right analytical tools are lacking. In particular, analysis of intact O-glycopeptides by mass spectrometry is challenging for several reasons; O-glycosylation lacks a consensus motif, glycopeptides have low charge density which impairs ETD fragmentation, and the glycan structures modifying the peptides are unpredictable. Recently, we introduced chemically modified monosaccharide analogues that allowed selective tracking and characterization of mucin-type O-glycans after bioorthogonal derivatization with biotin-based enrichment handles. In doing so, we realized that the chemical modifications used in these studies have additional benefits that allow for improved analysis by tandem mass spectrometry. In this work, we built on this discovery by generating a series of new GalNAc analogue glycopeptides. We characterized the mass spectrometric signatures of these modified glycopeptides and their signature residues left by bioorthogonal reporter reagents. Our data indicate that chemical methods for glycopeptide profiling offer opportunities to optimize attributes such as increased charge state, higher charge density, and predictable fragmentation behavior.

Proteomics has exposed a plethora of posttranslational modifications, but demonstrating functional relevance requires new approaches. Top-down proteomics of intact proteins has the potential to fully characterize protein modifications in terms of amount, site(s), and the order in which they are deposited on the protein; information that so far has been elusive to extract by shotgun proteomics. Data acquisition and analysis of intact multimodified proteins have however been a major challenge, in particular for positional isomers that carry the same number of modifications at different sites. Solutions were previously proposed to extract this information from fragmentation spectra, but these have so far mainly been limited to peptides and have entailed a large degree of manual interpretation. Here, we apply high-resolution Orbitrap fusion top-down analyses in combination with bioinformatics approaches to attempt to characterize multiple modified proteins and quantify positional isomers. Automated covalent fragment ion type definition, detection of mass precision and accuracy, and extensive use of replicate spectra increase sequence coverage and drive down false fragment assignments from 10% to 1.5%. Such improved performance in fragment assignment is key to localize and quantify modifications from fragment spectra. The method is tested by investigating positional isomers of Ubiquitin mixed in known concentrations, which results in quantification of high ratios at very low standard errors of the mean (<5%), as well as with synthetic phosphorylated peptides. Application to multiphosphorylated Bora provides an estimation of the so far unknown stoichiometry of the known set of phosphosites and uncovers new sites from hyperphosphorylated Bora.

Standardized diagnostic criteria for Eustachian tube (ET) dysfunction (ETD) have not been established. The purpose of this study was to characterize the relationship between ET inflammation and ETD symptoms and to determine the diagnostic performance of a quantitative score.
Patients were enrolled in a rhinology clinic between October 2018 and June 2019. Patients underwent nasal endoscopy and completed the 7-item Eustachian Tube Dysfunction Questionnaire (ETDQ-7). Nasopharyngeal inflammation identified on endoscopy was quantified using the Endoscopic Evaluation of the Eustachian Tube (3ET) score. Tympanometry was performed as indicated. Comorbid conditions were assigned during the patient encounter.
A total of 414 patients were included in the study. Patients with clinically significant ETD symptoms (ETDQ-7 ≥2.1) had higher 3ET scores than those without symptoms. A 1-point increase in 3ET score was associated with a 1.7-fold increase in odds of clinically significant ETD symptoms (adjusted OR [aOR], 1.72; 95% CI, 1.46 to 2.05). The 3ET scores were correlated with ETDQ-7 scores (ρ = 0.54) and 22-item Sino-Nasal Outcome Test (SNOT-22) scores (ρ = 0.52). 3ET scores were not associated with tympanometric peak pressures. Patients with ETD symptoms were more likely to have laryngopharyngeal reflux (aOR, 2.71; 95% CI, 1.24 to 6.18). A 3ET score of 4 predicted symptomatic state in 80% of cases with a specificity of 97.8% and positive predictive value of 96.6%.
Inflammatory findings at the nasopharyngeal ET orifice are associated with clinically significant ETD symptoms. The 3ET score is specific for a symptomatic state and has potential clinical utility in the evaluation of suspected ETD. ©2021 ARSAAOA, LLC.

Chronic eustachian tube dysfunction is a condition that affects a large number of adults every year. While many cases can be managed pharmacologically, there is a significant portion that does not respond to medical treatment alone. Invasive methods from the aural side like grommet or tympanic tube insertion have complications associated with it and often cannot address the underlying condition of the nasopharyngeal side of the eustachian tube. Balloon eustachian tuboplasty (BET) is a minimally invasive intervention that has been used successfully in countries like Germany and USA in the past decade. This paper presents the experience of a new clinical user of BET in India by retrospectively looking at the outcomes of the first 10 patients on which the procedure was performed. A marketed product EustaCare was used for the cases. There was a 90% procedural success rate within 4 weeks with one patient showing no improvement in symptoms even after 8 weeks. There were no adverse events associated with the procedure nor technical issues with the product. The product is easy to learn and use. Further study is needed to draw any conclusions on long term safety and efficacy, though historical records and preliminary results for this procedure described in this paper seem promising.

Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area of N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods in terms of identifications, indicating that ETD-based methods are not required for routine N-glycoproteomics even if they can generate higher quality spectra. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.

The advancement of viral glycomics has paralleled that of the mass spectrometry glycomics toolbox. In some regard the glycoproteins studied have provided the impetus for this advancement. Viral proteins are often highly glycosylated, especially those targeted by the host immune system. Glycosylation tends to be dynamic over time as viruses propagate in host populations leading to increased number of and/or \"movement\" of glycosylation sites in response to the immune system and other pressures. This relationship can lead to highly glycosylated, difficult to analyze glycoproteins that challenge the capabilities of modern mass spectrometry. In this review, we briefly discuss five general areas where glycosylation is important in the viral niche and how mass spectrometry has been used to reveal key information regarding structure-function relationships between viral glycoproteins and host cells. We describe the recent past and current glycomics toolbox used in these analyses and give examples of how the requirement to analyze these complex glycoproteins has provided the incentive for some advances seen in glycomics mass spectrometry. A general overview of viral glycomics, special cases, mass spectrometry methods and work-flows, informatics and complementary chemical techniques currently used are discussed. © 2020 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd. Mass Spec Rev.