Intact protein characterization using mass spectrometry thus far has been achieved at the cost of throughput. Presented here is the application of 193 nm ultraviolet photodissociation (UVPD) for top down identification and characterization of proteins in complex mixtures in an online fashion. Liquid chromatographic separation at the intact protein level coupled with fast UVPD and high-resolution detection resulted in confident identification of 46 unique sequences compared to 44 using HCD from prepared Escherichia coli ribosomes. Importantly, nearly all proteins identified in both the UVPD and optimized HCD analyses demonstrated a substantial increase in confidence in identification (as defined by an average decrease in E value of ∼40 orders of magnitude) due to the higher number of matched fragment ions. Also shown is the potential for high-throughput characterization of intact proteins via liquid chromatography (LC)–UVPD-MS of molecular weight-based fractions of a Saccharomyces cerevisiae lysate. In total, protein products from 215 genes were identified and found in 292 distinct proteoforms, 168 of which contained some type of post-translational modification.

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Bacterial selenocysteine incorporation occurs in response to opal stop codons and is dependent on the presence of a selenocysteine insertion sequence (SECIS) element, which recruits the selenocysteine specific elongation factor and tRNASec needed to reassign the UGA codon. The SECIS element is a stem-loop RNA structure immediately following the UGA codon and forms part of the coding sequence in bacterial selenoproteins. Although the site specific incorporation of selenocysteine is of great interest for protein engineering, the sequence constraints imposed by the adjoining SECIS element severely limit its use. We have evolved an E. coli tRNASec that is compatible with the canonical translation machinery and can suppress amber stop codons to incorporate selenocysteine with high efficiency. This evolved tRNASec allows the production of new recombinant selenoproteins containing structural motifs such as selenyl-sulfhydryl and diselenide bonds.

O -glycopeptides are often acidic owing to the frequent occurrence of acidic saccharides in the glycan, rendering traditional proteomic workflows that rely on positive mode tandem mass spectrometry (MS/MS) less effective. In this report, we demonstrate the utility of negative mode ultraviolet photodissociation (UVPD) MS for the characterization of acidic O-linked glycopeptide anions. This method was evaluated for a series of singly- and multiply-deprotonated glycopeptides from the model glycoprotein kappa casein, resulting in production of both peptide and glycan product ions that afforded 100% sequence coverage of the peptide and glycan moieties from a single MS/MS event. The most abundant and frequent peptide sequence ions were a/x-type products, which, importantly, were found to retain the labile glycan modifications. The glycan-specific ions mainly arose from glycosidic bond cleavages (B, Y, C, and Z ions) in addition to some less common cross-ring cleavages. Based on the UVPD fragmentation patterns, an automated database searching strategy (based on the MassMatrix algorithm) was designed that is specific for the analysis of glycopeptide anions by UVPD. This algorithm was used to identify glycopeptides from mixtures of glycosylated and non-glycosylated peptides, sequence both glycan and peptide moieties simultaneously, and pinpoint the correct site(s) of glycosylation. This methodology was applied to uncover novel site-specificity of the O-linked glycosylated OmpA/MotB from the “superbug” A. baumannii to help aid in the elucidation of the functional role that protein glycosylation plays in pathogenesis.

Protein ubiquitin modifications present a vexing analytical challenge, because of the dynamic changes in the site of modification on the substrate, the number of ubiquitin moieties attached, and the diversity of linkage patterns in which they are attached. Presented here is a method to confidently assign size and linkage type of polyubiquitin modifications. The method combines intact mass measurement to determine the number of ubiquitin moieties in the chain with backbone fragmentation by 193-nm ultraviolet photodissociation (UVPD) to determine the linkage pattern. UVPD fragmentation of proteins leads to reproducible backbone cleavage at almost every inter-residue position, and in polyubiquitin chains, the N-terminally derived fragments from each constituent monomer are identical, up to the site of conjugation. The N-terminal ubiquitin fragment ions are superimposed to create a diagnostic pattern that allows easy recognition of the dominant chain linkages. The method is demonstrated by achieving almost-complete fragmentation of monoubiquitin and then, subsequently, fragmentation of dimeric, tetrameric, and longer Lys48- and Lys63-linked ubiquitin chains. The utility of the method for the analysis of mixed linkage chains is confirmed for mixtures of Lys48 and Lys63 tetramers with known relative concentrations and for an in vitro-formulated ubiquitin chain attached to a substrate protein.