Divergent role of Mitochondrial Amidoxime Reducing Component 1 (MARC1) in human and mouse.

Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.

High throughput and high confidence sequence variant analysis in therapeutic antibodies using Evosep One liquid chromatography tandem mass spectrometry with synthetic heavy peptides.

Sequence variants are anomalous misincorporations of amino acids into the primary structure of therapeutic antibodies during DNA replication and protein biosynthesis. As these low abundance variants contribute to molecular heterogeneity and could negatively impact the safety and efficacy of a protein therapeutic, analytical methods like liquid chromatography tandem mass spectrometry (LC-MS2) are used to monitor them with the goal of establishing control strategies that limit their occurrence. Current LC-MS2 strategies depend on relatively long gradients that minimize coelution between abundant non-variant peptide peaks and trace-level variants to limit ion suppression that can potentially conceal the latter. However, lengthy LC gradients reduce the number of samples that can be analyzed per day, limiting the practicality of LC-MS2 when analyzing large sample sets. Furthermore, confident variant identification partly depends on capturing rich MS2 spectra that localize any amino acid misincorporations, which can be challenging due to the low abundance of this class of analyte. This work drastically reduces the cycle time to run each therapeutic antibody sample with roughly the same or even more variant identifications, compared to traditional LC-MS2 analysis, by integrating an Evosep One LC platform with an Orbitrap Fusion Lumos mass spectrometer. It also introduces a novel strategy using synthetic peptides that contain heavy isotopes placed near both termini to validate lower confidence variants in one targeted LC-MS2 run according to retention time, precursor mass signal, and MS2 fragment patterns shared with the heavy peptide variant. Taken together, this approach enables high-throughput sequence variant analysis at 30 samples per day as well as validation for lower confidence variants that can be integrated into therapeutic antibody process development and characterization.

Electron Transfer Dissociation Parameter Optimization Using Design of Experiments Increases Sequence Coverage of Monoclonal Antibodies.

Middle-down analysis of monoclonal antibodies (mAbs) by tandem mass spectrometry (MS2) can provide detailed insight into their primary structure with minimal sample preparation. The middle-down approach uses an enzyme to cleave mAbs into Fc/2, LC, and Fd subunits that are then analyzed by reversed phase liquid chromatography tandem mass spectrometry (RPLC-MS2). As maximum sequence coverage is desired to obtain meaningful structural information at the subunit level, a host of dissociation methods have been developed, and sometimes combined, to bolster fragmentation and increase the number of identified fragments. Here, we present a design of experiments (DOE) approach to optimize MS2 parameters, in particular those that may influence electron transfer dissociation (ETD) efficiency to increase the sequence coverage of antibody subunits. Applying this approach to the NIST monoclonal antibody standard (NISTmAb) using three RPLC-MS2 runs resulted in high sequence coverages of 67%, 67%, and 52% for Fc/2, LC, and Fd subunits, respectively. In addition, we apply this DOE strategy to model the parameters required to maximize the number of fragments produced in "low", "medium", and "high" mass ranges, which ultimately resulted in even higher sequence coverages of NISTmAb subunits (75%, 78%, and 64% for Fc/2, LC, and Fd subunits, respectively). The DOE approach provides high sequence coverage percentages utilizing only one fragmentation method, ETD, and could be extended to other state-of-the-art techniques that combine multiple fragmentation mechanisms to increase coverage.

Combination of FAIMS, Protein A Depletion, and Native Digest Conditions Enables Deep Proteomic Profiling of Host Cell Proteins in Monoclonal Antibodies.

Host cell proteins (HCPs) are residual impurities generated by the expression cell line during the production of biopharmaceuticals. Although the majority of these contaminants are removed during purification, HCPs can represent a considerable risk to the efficacy and safety of a therapeutic protein if not actively monitored. The enzyme-linked immunosorbent assay (ELISA) is commonly used throughout production to monitor HCP levels but has limited ability to identify novel HCPs or provide detailed quantification. Liquid chromatography tandem mass spectrometry (LC-MS2) methods are increasingly being used in conjunction with established ELISA techniques to provide rapid adaptability to increasingly complex samples as well as highly quantitative and informative results. However, MS-based methods are still hindered by the large dynamic range between high abundance biopharmaceutical proteins and low abundance HCPs. Here, we propose a multifactorial approach designed to optimize HCP detection in purified monoclonal antibody samples with LC-MS2. By first depleting the sample of antibody on a protein A column, then specifically digesting HCPs while precipitating remaining antibody, and finally reducing spectral complexity through compensation voltage (CV) switching using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we identified multiple-fold more HCPs in the NIST monoclonal antibody standard than any single established mass spectrometry technique reported in the literature. Our analyses consistently identified over 600 high confidence mouse HCPs, a multifold increase over established methods, while maintaining high reproducibility.

Effects of cryogenic preservation and storage on the molecular characteristics of microorganisms in sediments.

Sediment samples from a large physical-model aquifer and laboratory-generated samples were used to systematically assess the effects of whole-sample freezing on the integrity of biomolecules relevant to bioremediation. Impacts of freezing on DNA and RNA were assessed using quantitative polymerase chain reaction (PCR) as well as the community fingerprinting method, PCR single-strand conformation polymorphism (PCR-SSCP). We did not observe any significant degradation of a suite of genes and gene transcripts, including short-lived mRNA transcripts, from P. putida F1 or from B. subtilis JH642 in single-species samples, or from archaea in enrichment culture samples that also contained members of diverse bacterial phyla. Similarly, freezing did not change the relative abundance of dominant phylotypes in enrichment culture samples as measured by PCR-SSCP of bacterial 16S rDNA. Additionally, freezing and storage for 5 months at -80 °C did not affect the microbial community composition of samples from the model aquifer. Of even greater significance is that freezing and storage did not affect the relative abundance of 16S rRNA phylotypes, since in vivo rRNA content is often correlated with cellular growth rate. Thus, we conclude that cryogenic preservation and storage of intact sediment samples can be used for accurate molecular characterization of microbial populations and may facilitate high-resolution capture of biogeochemical interfaces important to bioremediation.

Persulfate persistence under thermal activation conditions.

Contaminant destruction with in situ chemical oxidation (ISCO) using persulfate (peroxydisulfate, S2O8(2-)) can be enhanced by activation, which increases the rate of persulfate decomposition to sulfate radicals (SO4*-). This step initiates a chain of radical reactions involving species (including SO4*- and OH*) that oxidize contaminants more rapidly than persulfate does directly. Among current activation methods, thermal activation is the least well studied. Combining new data for environmentally relevant conditions with previously published data, we have computed three sets of Arrhenius parameters (In A and Eact) that describe the rate of persulfate decomposition in homogeneous solutions over a wide range of temperature and pH. The addition of soil increases the decomposition rate of persulfate due to reactions with organic matter and possibly mineral surfaces, but the kinetics are still pseudo-first-order in persulfate and conform to the Arrhenius model. A series of respike experiments with soil at 70 degrees C demonstrate that once the oxidant demand is met, reaction rates return to values near those observed in the homogeneous solution case. However, even after the oxidant demand is met, the relatively short lifetime of the persulfate at elevated temperatures (e.g., >50 degrees C) will limit the delivery time over which persulfate can be effective.