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Mass spectrometric analysis of intact glycopeptides can reveal detailed information about glycosite, glycan structural features, and their heterogeneity. Sialyl glycopeptides can be positively, negatively, or neutrally charged depending on pH of their buffer solution and ionization conditions. To detect sialoglycopeptides, a negative-ion mode mass spectrometry may be applied with a minimal loss of sialic acids, although the positively charged or neutral glycopeptides may be excluded. Alternatively, the sialyl glycopeptides can be identified using positive-ion mode analysis by doping a high concentration of sodium salts to the analytes. Although manipulation of unmodified sialoglycopeptides can be useful for analysis of samples, less than optimal ionization, facile loss of sialyl and unfavorable ionization of accompanying non-sialyl peptides make such strategies suboptimal. Currently available chemical derivatization methods, while stabilizing for sialic acid, mask sialic acid linkage configuration. Here, we report the development of a novel approach to neutralize sialic acids via sequentially chemical modification that also reveals their linkage configuration, often an important determinant in biological function. This method utilizes several components to facilitate glycopeptide identification. These include the following: solid phase derivatization, enhanced ionization of sialoglycopeptides, differentiation of sialic acid linkage, and enrichment of the modified glycopeptides by hydrophilic interaction liquid chromatography. This technology can be used as a tool for quantitative analysis of protein sialylation in diseases with determination of sialic acid linkage configuration. Graphical Abstract ᅟ

Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein–protein and protein–ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli . Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). Graphical Abstract ᅟ

Many software solutions are available for proteomics and glycomics studies, but none are ideal for the structural analysis of peptidoglycan (PG), the essential and major component of bacterial cell envelopes. It icomprises glycan chains and peptide stems, both containing unusual amino acids and sugars. This has forced the field to rely on manual analysis approaches, which are time-consuming, labour-intensive, and prone to error. The lack of automated tools has hampered the ability to perform high-throughput analyses and prevented the adoption of a standard methodology. Here, we describe a novel tool called PGFinder for the analysis of PG structure and demonstrate that it represents a powerful tool to quantify PG fragments and discover novel structural features. Our analysis workflow, which relies on open-access tools, is a breakthrough towards a consistent and reproducible analysis of bacterial PGs. It represents a significant advance towards peptidoglycomics as a full-fledged discipline.

The validation of candidate biomarkers often is hampered by the lack of a reliable means of assessing and comparing performance. We present here a reference set of serum and plasma samples to facilitate the validation of biomarkers for resectable pancreatic cancer. The reference set includes a large cohort of stage I-II pancreatic cancer patients, recruited from 5 different institutions, and relevant control groups. We characterized the performance of the current best serological biomarker for pancreatic cancer, CA 19-9, using plasma samples from the reference set to provide a benchmark for future biomarker studies and to further our knowledge of CA 19-9 in early-stage pancreatic cancer and the control groups. CA 19-9 distinguished pancreatic cancers from the healthy and chronic pancreatitis groups with an average sensitivity and specificity of 70-74%, similar to previous studies using all stages of pancreatic cancer. Chronic pancreatitis patients did not show CA 19-9 elevations, but patients with benign biliary obstruction had elevations nearly as high as the cancer patients. We gained additional information about the biomarker by comparing two distinct assays. The two CA 9-9 assays agreed well in overall performance but diverged in measurements of individual samples, potentially due to subtle differences in antibody specificity as revealed by glycan array analysis. Thus, the reference set promises be a valuable resource for biomarker validation and comparison, and the CA 19-9 data presented here will be useful for benchmarking and for exploring relationships to CA 19-9.

Applications of antibody de novo sequencing in the biopharmaceutical industry range from the discovery of new antibody drug candidates to identifying reagents for research and determining the primary structure of innovator products for biosimilar development. When murine, phage display, or patient-derived monoclonal antibodies against a target of interest are available, but the cDNA or the original cell line is not, de novo protein sequencing is required to humanize and recombinantly express these antibodies, followed by in vitro and in vivo testing for functional validation. Availability of fully automated software tools for monoclonal antibody de novo sequencing enables efficient and routine analysis. Here, we present a novel method to automatically de novo sequence antibodies using mass spectrometry and the Supernovo software. The robustness of the algorithm is demonstrated through a series of stress tests. Graphical Abstract ᅟ

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM) 1 . The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins 2 , 3 . In Escherichia coli , Braun’s lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability 4 ; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present 5 . We used a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii , Agrobacterium tumefaciens and Legionella pneumophila . In C. burnetii , we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG. β-barrel outer-membrane proteins are covalently attached to peptidoglycan in Gram-negative bacteria including Coxiella burnetii , Agrobacterium tumefaciens and Legionella pneumophila .

A cone snail venom peptide, μO§-conotoxin GVIIJ from Conus geographus , has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. μO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJ SSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) Na V1.2. A mutant channel of rNa V1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10 ³-fold less sensitive to GVIIJ SSG than was wild-type rNa V1.2. In contrast, although rNa V1.5 was >10 ⁴-fold less sensitive to GVIIJ SSG than Na V1.2, an rNa V1.5 mutant with a cysteine in the homologous location, rNa V1.5[L869C], was >10 ³-fold more sensitive than wild-type rNa V1.5. The susceptibility of rNa V1.2 to GVIIJ SSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNa Vβ2 or rNa Vβ4, but not that of rNa Vβ1 or rNa Vβ3, protected rNa V1.1 to -1.7 (excluding Na V1.5) against block by GVIIJ SSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which Na Vβ- and Na Vα-subunits are present in native neurons.

Peptidoglycan is an essential component of the bacterial cell envelope—a mesh-like macromolecule that protects the bacterium from osmotic stress and its internal turgor pressure. The composition and architecture of peptidoglycan is heterogeneous and changes as bacteria grow, divide, and respond to their environment. Though peptidoglycan has long been studied via LC-MS/MS, the analysis of this data remains challenging as peptidoglycan’s unusual composition and branching can’t be handled by proteomics software. Here we describe user-friendly open-source tools and a web interface for building peptidoglycan databases, performing MS searches, and predicting the MS/MS fragmentation of muropeptides. We then use Rhizobium leguminosarum to describe a step-by-step strategy for the high-resolution analysis of peptidoglycan. The unprecedented detail of R. leguminosarum ’s peptidoglycan composition (>250 muropeptides) reveals even the subtlest remodelling between growth conditions. These new and easier to use tools enable more systematic analyses of peptidoglycan dynamics. Peptidoglycan (PG) is an essential component of the bacterial cell envelope, however, the structural characterization of PG via mass spectrometry remains challenging. Here, the authors develop PGFinder, an open-source web application for the LC-MS/MS analysis of peptidoglycan, and describe a step-by-step analysis strategy using Rhizobium leguminosarum as a model.

Biomolecular surface mapping methods offer an important alternative method for characterizing protein–protein and protein–ligand interactions in cases in which it is not possible to determine high-resolution three-dimensional (3D) structures of complexes. Hydroxyl radical footprinting offers a significant advance in footprint resolution compared with traditional chemical derivatization. Here we present results of footprinting performed with hydroxyl radicals generated on the nanosecond time scale by laser-induced photodissociation of hydrogen peroxide. We applied this emerging method to a carbohydrate-binding protein, galectin-1. Since galectin-1 occurs as a homodimer, footprinting was employed to characterize the interface of the monomeric subunits. Efficient analysis of the mass spectrometry data for the oxidized protein was achieved with the recently developed ByOnic (Palo Alto, CA) software that was altered to handle the large number of modifications arising from side-chain oxidation. Quantification of the level of oxidation has been achieved by employing spectral intensities for all of the observed oxidation states on a per-residue basis. The level of accuracy achievable from spectral intensities was determined by examination of mixtures of synthetic peptides related to those present after oxidation and tryptic digestion of galectin-1. A direct relationship between side-chain solvent accessibility and level of oxidation emerged, which enabled the prediction of the level of oxidation given the 3D structure of the protein. The precision of this relationship was enhanced through the use of average solvent accessibilities computed from 10 ns molecular dynamics simulations of the protein. A combination of oxidative surface footprinting and molecular dynamics simulation allows identification of the interfacial residues in dimeric galectin-1.