Radical Fragment Ions in Collision-Induced Dissociation Mass Spectrometry

Collision-induced dissociation (CID) is a common fragmentation strategy in mass spectrometry (MS) analysis. A conventional understanding is that fragment ions generated in low-energy CID should follow the even-electron rule. As such, (de)protonated precursor ions should predominately generate (de)protonated fragment ions with very few radical fragment ions (RFIs). However, the extent to which RFIs present in MS2 spectra has not been comprehensively investigated. This work uses the latest NIST 20 tandem mass spectral library to investigate of the occurrence of RFIs in CID MS2 experiments. In particular, RFIs were recognized using their integer double bond equivalent (DBE) values calculated from their annotated molecular formulas. Our study shows unexpected results as 65.4% and 68.8% of MS2 spectra contain at least 10% RFIs by ion-count (total number of ions) in positive and negative electrospray ionization (ESI) modes, respectively. Furthermore, we classified chemicals based on their compound classes and chemical substructures, and calculated the percentages of RFIs in each class. Results show that “Organic 1,3-dipolar compounds” and “Lignans, neolignans and related compounds” are the top 2 compound superclasses which tend to produce RFIs in their CID MS2 spectra. Moreover, aromatic, arylbromide, heteroaromatic, alkylarylether, phenol, and conjugated double bond-containing chemicals are more likely to produce RFIs. We also found four possible patterns of change in RFI percentages as a function of CID collision energy. Finally, we demonstrate that the inadequate consideration of RFIs in most conventional bioinformatic tools might cause problems during in silico fragmentation and de novo annotation of MS2 spectra. This work provides a further understanding of CID MS2 mechanism, and the unexpectedly large percentage of RFIs suggests a need for consideration in the development of bioinformatic software for MS2 interpretation.

O-Glycosylation Landscapes of SARS-CoV-2 Spike Proteins

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized through high-resolution mass spectrometry. Following digestion with trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the GalNAc-type O-glycosylation pattern of S proteins, including O-glycosites and the six most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 255 intact O-glycopeptides composed of 50 peptides sequences and 43 O-glycosites were discovered by higher energy collision-induced dissociation (HCD), and three O-glycosites were confidently identified by electron transfer/higher energy collision-induced dissociation (EThcD) in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex (1). In contrast, in the human cell-expressed S protein S1 subunit, 407 intact O-glycopeptides composed of 34 peptides sequences and 30 O-glycosites were discovered by HCD, and 11 O-glycosites were unambiguously assigned by EThcD. However, the measurement of O-glycosylation occupancy hasn’t been made. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex (1)NeuAc (1) and HexNAc(1)Hex (1)NeuAc (2). Our results reveal that the SARS-CoV-2 S protein is an O-glycoprotein; the O-glycosites and O-glycan compositions vary with the host cell type. These comprehensive O-glycosylation landscapes of the S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.

Structural identification of N-glycan isomers using logically derived sequence tandem mass spectrometry

N-linked glycosylation is one of the most important protein post-translational modifications. Despite the importance of N-glycans, the structural determination of N-glycan isomers remains challenging. Here we develop a mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MSn), to determine the structures of N-glycan isomers that cannot be determined using conventional mass spectrometry. In LODES/MSn, the sequences of successive collision-induced dissociation are derived from carbohydrate dissociation mechanisms and apply to N-glycans in an ion trap for structural determination. We validate LODES/MSn using synthesized N-glycans and subsequently applied this method to N-glycans extracted from soybean, ovalbumin, and IgY. Our method does not require permethylation, reduction, and labeling of N-glycans, or the mass spectrum databases of oligosaccharides and N-glycan standards. Moreover, it can be applied to all types of N-glycans (high-mannose, hybrid, and complex), as well as the N-glycans degraded from larger N-glycans by any enzyme or acid hydrolysis.

Chemical Conversion of Hardly Ionizable Rhenium Aryl Chlorocomplexes with p-Substituted Anilines

Fast and selective analytical methods help to ensure the chemical identity and desired purity of the prepared complexes before their medical application, and play an indispensable role in clinical practice. Mass spectrometry, despite some limitations, is an integral part of these methods. In the context of mass spectrometry, specific problems arise with the low ionization efficiency of particular analytes. Chemical derivatization was used as one of the most effective methods to improve the analyte’s response and separation characteristics. The Schotten–Baumann reaction was successfully adapted for the derivatization of ESI hardly ionizable Re(VII) bis(catechol) oxochlorocomplex. Various alkyl and halogen p-substituted anilines as possible derivatization agents were tested. Unlike the starting complex, the reaction products were easily ionizable in electrospray, providing structurally characteristic molecular and fragment anions. DFT computer modeling, which proposed significant conformation changes of prepared complexes within their deprotonation, proved to have a close link to MS spectra. High-resolution MS and MS/MS measurements complemented with collision-induced dissociation experiments for detailed specification of prepared complexes’ fragmentation pathways were used. The specified fragmentation schemes were analogous for all studied derivatives, with an exception for [Re(O)(Cat)2PIPA].