Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry that provides ready access to protein oxidative footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine a single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated by mapping the interface of a RAS-monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map the protein structure with single amino acid residue resolution.

Charge deconvolution infers the mass from mass over charge (m/z) measurements in electrospray ionization mass spectra. When applied over a wide input m/z or broad target mass range, charge-deconvolution algorithms can produce artifacts, such as false masses at one-half or one-third of the correct mass. Indeed, a maximum entropy term in the objective function of MaxEnt, the most commonly used charge deconvolution algorithm, favors a deconvolved spectrum with many peaks over one with fewer peaks. Here we describe a new “parsimonious” charge deconvolution algorithm that produces fewer artifacts. The algorithm is especially well-suited to high-resolution native mass spectrometry of intact glycoproteins and protein complexes. Deconvolution of native mass spectra poses special challenges due to salt and small molecule adducts, multimers, wide mass ranges, and fewer and lower charge states. We demonstrate the performance of the new deconvolution algorithm on a range of samples. On the heavily glycosylated plasma properdin glycoprotein, the new algorithm could deconvolve monomer and dimer simultaneously and, when focused on the m/z range of the monomer, gave accurate and interpretable masses for glycoforms that had previously been analyzed manually using m/z peaks rather than deconvolved masses. On therapeutic antibodies, the new algorithm facilitated the analysis of extensions, truncations, and Fab glycosylation. The algorithm facilitates the use of native mass spectrometry for the qualitative and quantitative analysis of protein and protein assemblies.

Several recent reports suggest that HNO may be produced endogenously by reaction of H2S and S-nitrosoglutathione (GSNO). This hypothesis was tested using deoxymyoglobin (MbFeII) to trap the expected HNO released from the target reaction, which should generate the stable HNO adduct, HNO-Mb, under anaerobic conditions. Under numerous experimental conditions, the sole globin product was NO-Mb, as characterized by absorbance, EPR, and NMR spectroscopies. Analogous reactions of GSNO with other biological reductants such as ascorbic acid, dithiothreitol, glutathione, and dithionite also yielded NO-Mb as the sole globin product; however, whereas analogous reduction of GSNO using NaBH4 generates HNO-Mb in high yield. Quantitative GC/MS analyses of reactions of GS15NO with H2S showed that the main reaction product was 15NO, with 15N2 produced at a comparable level to 15N2O. Overall yield of N2O is unchanged by the presence of MbFeII, discounting the intermediacy of either NO or HNO in its formation. Taken together, these results argue against the generation of free HNO as a major pathway in the reactions of GSNO with H2S, and instead imply some as yet uncharacterized intermediates generate the nitrogenic gases.

To extend proteome coverage obtained from bottom-up mass spectrometry approaches, three complementary ion activation methods, higher energy collision dissociation (HCD), ultraviolet photodissociation (UVPD), and negative mode UVPD (NUVPD), are used to interrogate the tryptic peptides in a human hepatocyte lysate using a high performance Orbitrap mass spectrometer. The utility of combining results from multiple activation techniques (HCD+UVPD+NUVPD) is analyzed for total depth and breadth of proteome coverage. This study also benchmarks a new version of the Byonic algorithm, which has been customized for database searches of UVPD and NUVPD data. Searches utilizing the customized algorithm resulted in over 50% more peptide identifications for UVPD and NUVPD tryptic peptide data sets compared to other search algorithms. Inclusion of UVPD and NUVPD spectra resulted in over 600 additional protein identifications relative to HCD alone.

In April 2016, the Food and Drug Administration approved the first biosimilar monoclonal antibody (mAb), Inflectra/Remsima (Celltrion), based off the original product Remicade (infliximab, Janssen). Biosimilars promise significant cost savings for patients, but the unavoidable differences between innovator and copycat biologics raise questions regarding product interchangeability. In this study, Remicade and Remsima were examined by native mass spectrometry, ion mobility, and quantitative peptide mapping. The levels of oxidation, deamidation, and mutation of individual amino acids were remarkably similar. We found different levels of C-terminal truncation, soluble protein aggregates, and glycation that all likely have a limited clinical impact. Importantly, we identified more than 25 glycoforms for each product and observed glycoform population differences, with afucosylated glycans accounting for 19.7% of Remicade and 13.2% of Remsima glycoforms, which translated into a 2-fold reduction in the level of FcγIIIa receptor binding for Remsima. While this difference was acknowledged in Remsima regulatory filings, our glycoform analysis and receptor binding results appear to be somewhat different from the published values, likely because of methodological differences between laboratories and improved glycoform identification by our laboratory using a peptide map-based method. Our mass spectrometry-based analysis provides rapid and robust analytical information vital for biosimilar development. We have demonstrated the utility of our multiple-attribute monitoring workflow using the model mAbs Remicade and Remsima and have provided a template for analysis of future mAb biosimilars.

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.

The authors performed a multiple-pulsed atrazine experiment to measure responses of autotrophic endpoints in outdoor stream mesocosms. The experiment was designed to synthetically simulate worst-case atrazine chemographs from streams in agricultural catchments to achieve 60-d mean concentrations of 0g/L (control), 10g/L, 20g/L, and 30g/L. The authors dosed triplicate streams with pulses of 0g/L, 50g/L, 100g/L, and 150g/L atrazine for 4d, followed by 7d without dosing. This 11-d cycle occurred 3 times, followed by a recovery (untreated) period from day 34 to day 60. Mean +/- standard error 60-d atrazine concentrations were 0.07 +/- 0.03g/L, 10.7 +/- 0.05g/L, 20.9 +/- 0.24g/L, and 31.0 +/- 0.17g/L for the control, 10-g/L, 20-g/L, and 30-g/L treatments, respectively. Multivariate analyses revealed that periphyton and phytoplankton community structure did not differ among treatments on any day of the experiment, including during the atrazine pulses. Control periphyton biomass in riffles was higher immediately following the peak of the first atrazine pulse and remained slightly higher than some of the atrazine treatments on most days through the peak of the last pulse. However, periphyton biomass was not different among treatments at the end of the present study. Phytoplankton biomass was not affected by atrazine. Metaphyton biomass in pools was higher in the controls near the midpoint of the present study and remained higher on most days for the remainder of the study. Ceratophyllum demersum, a submersed macrophyte, biomass was higher in controls than in 20-g/L and 30-g/L treatments before pulse 3 but was not different subsequent to pulse 3 through the end of the present study. Maximum daily dissolved oxygen (DO, percentage of saturation) declined during each pulse in approximate proportion to magnitude of dose but rapidly converged among treatments after the third pulse. However, DO increased in controls relative to all atrazine treatments during the last 17d of the experiment, likely a result of metaphyton cover in the pools. Finally, atrazine significantly limited uptake of PO(4)(3-)and uptake and/or denitrification of NO3- but only during pulses; percentage of dose removed from the water column was >85% for P and >95% for N after pulse 3 through the end of the present study. Collectively, only DO and metaphyton biomass differed at the end of the present study and only slightly. Some other endpoints were affected but only during pulses, if at all. The high levels of primary production and accumulation of algal biomass in all streams suggest that effects of pulses of atrazine at the concentrations used in the present study appear transient and likely do not represent ecologically significant adverse outcomes to periphyton, phytoplankton, and aquatic macrophytes, particularly in agricultural streams subjected to high nutrient loads. (c) 2015 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Mass-mobility correlations were investigated by ion mobility-mass spectrometry (IM-MS) for 11 structurally-unique homologous series composed of commercially-available compounds. Structural variation involved the inclusion of different repeat units (i.e., glucose, propylene glycol, or ethylene glycol) in oligomeric series and different terminal groups in CH2-homologous series. Terminal group structures included both aliphatic and aromatic acids, as well as aliphatic and aromatic amines. Mass-mobility correlations were also investigated for select CH2-homologous series identified in a pyrolysis bio-oil and compared with results observed for commercial series. A linear mass-mobility correlation (R 2 ≥ 0.996) was established for all series except those in which a substantial change in the gas-phase conformation of ions was probable. Slopes observed for CH2-homologous series with a single terminal group were significantly steeper than slopes observed for series containing two terminal groups. Additionally, a correlation between slope and double bond equivalents (DBE) suggested that the CH2-homologous series identified in bio-oil were structurally similar to commercial series containing two terminal groups.

Bio-oils produced from biomass pyrolysis are an attractive fuel source that requires significant upgrading. Before upgrade strategies can be developed, the molecular composition of bio-oils needs to be better understood. In this work, oily and aqueous fractions of bio-oils produced by slow pyrolysis of two feedstocks, pine shavings (PS) and corn stover (CS), were analyzed by negative electrospray ionization (ESI)-Orbitrap and ion mobility-time-of-flight mass spectrometry (IM-TOF-MS). Analyte ion signal was observed primarily between m/z 80 and 450 in the mass spectra of these samples. Mass defect analysis and collision-induced dissociation (CID) experiments performed on mobility-separated ions indicated a high degree of homology among bio-oil samples produced from both feedstocks. Oxygen-rich species having between 1 and 9 oxygen atoms and with double bond equivalents (DBEs) ranging from 1 to 15 were identified, indicating that catalytic upgrading will likely be required if slow-pyrolysis bio-oils are to be utilized as fuel. IM-MS and IM-MS/MS analysis of ions belonging to select CH2-homologous series suggest that mass-mobility correlations and post-ion mobility CID mass spectra may be useful in defining structural relationships among members of a given Kendrick mass defect series.

An emerging need in the biopharmaceutical industry is for new bioinformatics tools that have been developed for comprehensively characterizing therapeutic proteins and efficiently analyzing large sets of liquid chromatography-tandem mass spectrometry and UV data. The focus of this chapter is the demonstration of new software that integrates complementary data and returns results with a high degree of confidence aided by advanced inspection tools to manually verify any result and avoid risk of false or missing information. These software tools (Byologice and Byomap (TM)) have been applied to the NIST reference mAb for high and low concentration sequences, variants and modifications and associated report elements are presented. They can read any mass spectrometer vendor format, offering a single set of tools to compare information between groups and departments.

We report unexpected mass spectrometric observations of glycosylated human leukocyte antigen (HLA) class I-bound peptides. Complemented by molecular modeling, in vitro enzymatic assays, and oxonium ion patterns, we propose that the observed O-linked glycans carrying up to five monosaccharides are extended O-GlcNAc’s rather than GalNAc-initiated O-glycans. A cytosolic O-GlcNAc modification is normally terminal and does not extend to produce a polysaccharide, but O-GlcNAc on an HLA peptide presents a special case because the loaded HLA class I complex traffics through the endoplasmic reticulum and Golgi apparatus on its way to the cell membrane and is hence exposed to glycosyltransferases. We also report for the first time natural HLA class I presentation of O- and N-linked glycopeptides derived from membrane proteins. HLA class I peptides with centrally located oligosaccharides have been shown to be immunogenic and may thus be important targets for immune surveillance.

Mass spectrometry (MS) characterization of recombinant monoclonal antibody (mAb) drugs and their degraded and/or post-translationally modified counterparts, drug-product-related impurities and variants, is critical for successful development of biotherapeutics. Specifically in this study, drug-product-related impurities of an anti-Clostridium difficile IgG1 mAb drug substance were profiled by cation-exchange liquid chromatography (CEX) followed by the CEX peaks being fraction-collected for MS characterization. A reversed-phase liquid chromatography/mass spectrometry (LC/MS) methodology was developed on a Thermo Q-Exactive orbitrap mass spectrometer for (1) accurate mass measurements of the mAb, its CEX fractionated impurities, and their respective heavy chains and light chains and (2) middle-down LC/MS/MS of the light chains and the heavy chains using higher energy C-trap dissociation (HCD). The accurate mass measurements and the HCD middle-down MS/MS experiments identify that major impurities and variants of the anti-C. difficile mAb are degradation species of the heavy chains at residue Asn101 as well as at the hinge region amino acids, including Cys222, Lys224, His226, and Thr227, with levels ranging from 0.3% to 6.2% of the total drug substance. Additional impurities were identified as light chain C-terminal truncation at Gly93 and oxidized heavy chains at Met40, Met93, and Met430. Our impurity characterization results demonstrate that the middle-down MS method allows direct and accurate identification of drug-product-related impurities of therapeutic mAbs. It is particularly useful for those low-level impurities and variants that are not suitable for further fractionation and characterization by bottom-up MS.

Sequential extraction of an asphaltene sample with magnesium oxide nanoparticles provides an approach to remove selectively molecules from a complex fraction. This method of extraction relies, in part, on adsorption preferences for differing heteroatoms to leave weakly adsorbing sample constituents in a toluene solution. The extracted sample exhibits reduced complexity enabling more reliable identification of the remaining molecules in solution. Mass spectrometry (MS) data indicate a general bias for preferential removal of higher molecular weight species. This is supported by a shift in the average m/z ratio of the asphaltene distribution to lower m/z, as well as a decrease in the intensity observed for higher m/z ion signals, for higher extraction numbers. UV-vis absorption data corroborate MS data to provide an appreciable visual means to quantify sample uptake after several sequential extraction steps with MgO. Furthermore, both UV and MS data indicate a point of diminishing returns, after which subsequent extraction with MgO nanoparticles results in limited adsorption of remaining asphaltene constituents. The remaining asphaltene constituents can then be treated with NiO nanoparticles in order to identify molecules containing pyridyl functional groups. Implementation of a more exhaustive MgO extraction, prior to treatment with NiO, resulted in an improved method for profiling pyridyl-containing structures in a complex asphaltene mixture relative to previous work.

In this study, water hyacinth was hydrolyzed to sugars with dilute sulphuric acid (0.25 M) under subcritical water conditions (P = 20 bar, T = 130 °C) for 2 h. The sugar solution was then carbonized under subcritical conditions to produce carbon microsphere. The subcritical water carbonization was conducted at 40 bar and various temperatures (160–200 °C) and times (6–10 h). The highest yield of carbon microspheres was 0.1019 g/g dry water hyacinth at the temperature of 200 °C for 10 h. The carbon microsphere was activated using a combination of chemical (KOH solution) and physical (microwave) treatments to increase the specific surface area and porosity of carbon microsphere. Electrocapacitive study of carbon microspheres showed that the carbon microsphere activated at impregnation ratio of 1:1 and microwave power of 630 W has the highest specific capacitance and excellent electrochemical stability.

Byonic is a new proteomics search engine that can identify peptides carrying N- and O-linked glycans. Byonic offers a number of ways to search for glycopeptides, including preset glycan tables and manually entered glycan masses, and the search strategy affects the quality and quantity of spectrum assignments. Here we show how a progression of searches, from wider to narrower in both proteins and glycans, can improve sensitivity and specificity for glycopeptide identification. We obtained data from the following samples: Glycophorin-A, PSA, human blood serum enriched for glycoproteins, and secreted proteins from human endothelial cells. All data were acquired on various Thermo Orbitrap instruments and included both HCD and ETD fragmentation. We first searched the data with a full human protein database with contaminants and decoys, and later with smaller databases produced by Byonic's “focused database” option. We started with Byonic's preset glycan search, which allows only one glycan per peptide, and then, guided by prior search results, augmented or replaced these tables with user-defined glycan modifications with appropriate limits on each type of modification. We found that focused protein databases containing 10 – 200 proteins greatly improve the sensitivity of glycopeptide search relative to full-database searches. We found a database of likely glycoproteins, determined by PNG-ase release of N-glycans in O18 water, helpful for identifying glycopeptides carrying single N-linked glycans in the endothelial secretome. Focused glycan lists also improve sensitivity, and make possible still more complex searches. We have identified glycopeptides carrying up to two N-glycans, one N-glycan and one O-glycan, and up to four O-glycans, with only minor ambiguities in modification placement and mass distribution. More complex searches, for example, five or more O-glycans, will require improvements in completeness of fragmentation and computational methods.

Although structural isomers may yield indistinguishable ion mobility (IM) arrival times and similar fragment ions in tandem mass spectrometry (MS), it is demonstrated that post-IM/collision-induced dissociation MS (post-IM/CID MS) combined with chemometrics can enable independent study of the IM-overlapped isomers. The new approach allowed us to investigate the propensity of selected b type fragment ions from AlaAlaAlaHisAlaAlaAla-NH 2 (AAA(His)AAA) heptapeptide to form different isomers. Principle component analysis (PCA) of the unresolved post-IM/CID profiles indicated the presence of two different isomer types for b 4 + , b 5 + , and b 6 + and a single isomer type for b 7 + fragments of AAA(His)AAA. We employed a simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) to calculate the total IM profiles and CID mass spectra of b fragment isomers. The deconvoluted CID mass spectra showed discernible fragmentation patterns for the two isomers of b 4 + , b 5 + , and b 6 + fragments. Under our experimental conditions, calculated percentages of the “cyclic” isomers (at the 95 % confidence level for n = 3) for b 4 + , b 5 + , and b 6 + were 61 (± 5) %, 36 (± 5) %, and 48 (± 2) %, respectively. Results from the SIMPLISMA deconvolution of b 5 + species resembled the CID MS patterns of fully resolved IM profiles for the two b 5 + isomers. The “cyclic” isomers for each of the two-component b fragment ions were less susceptible to ion fragmentation than their “linear” counterparts. ᅟ

Extraction efficiencies for a series of model compounds representing heteroatom functional groups believed to be present in asphaltenes were determined in batch extractions with a variety of metal oxide nanoparticles (Fe3O4, TiO2, NiO, Co3O4, and MgO). Extraction efficiencies from toluene solution varied depending upon both the adsorbate and the type of metal oxide used for extraction. However, the adsorbate was found to be the most important factor governing selectivity, which generally followed the trend: benzoic acid >> pyridine approximate to phenol > pyrrole > thiophene approximate to diphenylsulfide approximate to benzophenone. An important exception to this trend was that MgO did not appreciably adsorb pyridine. The divergent adsorption behavior of pyridine on NiO (extraction efficiency = 82 +/- 1%) and MgO (extraction efficiency = 0 +/- 2%) was subsequently exploited to demonstrate a novel approach for profiling pyridine-containing molecules in an authentic asphaltene sample. Specifically, mass spectra of the asphaltene mixture were obtained before and after treatment with NiO or MgO and compared to identify peaks exhibiting reduced intensity after treatment with NiO but no appreciable change in intensity after treatment with MgO. Results of batch extraction studies with model compounds and elemental composition data deduced from accurate mass measurements support that these peaks likely correspond to (or minimally contain) a molecule(s) possessing a pyridyl functional group.