UniDecCD: Deconvolution of Charge Detection-Mass Spectrometry Data.

Native mass spectrometry (MS) has become a versatile tool for characterizing high-mass complexes and measuring biomolecular interactions. Native MS usually requires the resolution of different charge states produced by electrospray ionization to measure the mass, which is difficult for highly heterogeneous samples that have overlapping and unresolvable charge states. Charge detection-mass spectrometry (CD-MS) seeks to address this challenge by simultaneously measuring the charge and m/z for isolated ions. However, CD-MS often shows uncertainty in the charge measurement that limits the resolution. To overcome this charge state uncertainty, we developed UniDecCD (UCD) software for computational deconvolution of CD-MS data, which significantly improves the resolution of CD-MS data. Here, we describe the UCD algorithm and demonstrate its ability to improve the CD-MS resolution of proteins, megadalton viral capsids, and heterogeneous nanodiscs made from natural lipid extracts. UCD provides a user-friendly interface that will increase the accessibility of CD-MS technology and provide a valuable new computational tool for CD-MS data analysis.

Characterizing Novel Modifications of a Therapeutic Protein Using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry, Sedimentation Velocity Analytical Ultracentrifugation, and Structural Modeling.

Heterogeneity of biopharmaceutical products is common due to various co- and post-translational modifications and degradation events that occur during the biological production process and throughout the shelf life. Product-related variants resulting from these modifications potentially affect a product's biological activity and safety, and thus, their detailed structure characterization is of great importance for successful development of protein therapeutics. Specifically, in this study, two novel low-level product variants in a recombinant therapeutic protein were characterized via chromatographic enrichment followed by proteolytic digestion and analysis using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). One of the variants was identified to be the therapeutic protein missing a 61-amino-acid fragment from its N-terminus. Consequently, the other variant was found to be the therapeutic protein carrying the 61-amino-acid long peptide. Furthermore, detailed structure at the modification site of the latter variant was determined as that amino group from the protein's N-terminus linked to side chain carbonyl carbon at Asp 61 residue of the peptide, based on the complementary information from collision induced dissociation and electron transfer dissociation MS/MS analysis. Results from sedimentation velocity analytical ultracentrifugation and computational structural modeling supported the hypothesis that formation of these two variants was a result of protein self-association. In dimeric state, the head-to-toe stacking conformation of two therapeutic protein molecules allowed spatial closeness between the N-terminus of one molecule and the 61st amino acid of the other molecule, resulting in a novel peptide transfer  between the two protein molecules.

PrfA-like transcription factor gene lmo0753 contributes to L-rhamnose utilization in Listeria monocytogenes strains associated with human food-borne infections.

Listeria monocytogenes is a food-borne bacterial pathogen and the causative agent of human and animal listeriosis. Among the three major genetic lineages of L. monocytogenes (i.e., LI, LII, and LIII), LI and LII are predominantly associated with food-borne listeriosis outbreaks, whereas LIII is rarely implicated in human infections. In a previous study, we identified a Crp/Fnr family transcription factor gene, lmo0753, that was highly specific to outbreak-associated LI and LII but absent from LIII. Lmo0753 shares two conserved functional domains, including a DNA binding domain, with the well-characterized master virulence regulator PrfA in L. monocytogenes. In this study, we constructed lmo0753 deletion and complementation mutants in two fully sequenced L. monocytogenes LII strains, 10403S and EGDe, and compared the flagellar motility, phospholipase C production, hemolysis, and intracellular growth of the mutants and their respective wild types. Our results suggested that lmo0753 plays a role in hemolytic activity in both EGDe and 10403S. More interestingly, we found that deletion of lmo0753 led to the loss of l-rhamnose utilization in EGDe, but not in 10403S. RNA-seq analysis of EGDe Δ0753 incubated in phenol red medium containing l-rhamnose as the sole carbon source revealed that 126 (4.5%) and 546 (19.5%) out of 2,798 genes in the EGDe genome were up- and downregulated more than 2-fold, respectively, compared to the wild-type strain. Genes related to biotin biosynthesis, general stress response, and rhamnose metabolism were shown to be differentially regulated. Findings from this study collectively suggested varied functional roles of lmo0753 in different LII L. monocytogenes strain backgrounds associated with human listeriosis outbreaks.

Roles of a novel Crp/Fnr family transcription factor Lmo0753 in soil survival, biofilm production and surface attachment to fresh produce of Listeria monocytogenes.

Listeria monocytogenes is a foodborne bacterial pathogen and the causative agent of an infectious disease, listeriosis. L. monocytogenes is ubiquitous in nature and has the ability to persist in food processing environments for extended periods of time by forming biofilms and resisting industrial sanitization. Human listeriosis outbreaks are commonly linked to contaminated dairy products, ready-to-eat meats, and in recent years, fresh produce such as lettuce and cantaloupes. We identified a putative Crp/Fnr family transcription factor Lmo0753 that is highly specific to human-associated genetic lineages of L. monocytogenes. Lmo0753 possesses two conserved functional domains similar to the major virulence regulator PrfA in L. monocytogenes. To determine if Lmo0753 is involved in environmental persistence-related mechanisms, we compared lmo0753 deletion mutants with respective wild type and complementation mutants of two fully sequenced L. monocytogenes genetic lineage II strains 10403S and EGDe for the relative ability of growth under different nutrient availability and temperatures, soil survival, biofilm productivity and attachment to select fresh produce surfaces including romaine lettuce leaves and cantaloupe rinds. Our results collectively suggested that Lmo0753 plays an important role in L. monocytogenes biofilm production and attachment to fresh produce, which may contribute to the environmental persistence and recent emergence of this pathogen in human listeriosis outbreaks linked to fresh produce.

Characterization of the self-association of human interferon-α2b, albinterferon-α2b, and pegasys.

The self-association of human interferon-α2b (hIFN-α2b), albinterferon-α2b (a recombinant protein with human serum albumin and hIFN-α2b peptides fused together in a single polypeptide chain), and Pegasys (PEGylated hIFN-α2a) was characterized by analytical ultracentrifugation analyses. By examining the apparent sedimentation coefficient distribution profiles of each protein at different concentrations, it was concluded that the above three proteins are self-associating in albinterferon-α2b formulation buffer. By model fitting of sedimentation data using SEDANAL software, the stoichiometry and equilibrium constants of the self-association of these proteins were characterized. The self-association of hIFN-α2b results in the formation of stable dimers, fast-reversible tetramers, octamers, and hexadecamers. In contrast, although both albinterferon-α2b and Pegasys are self-associated, their self-association stoichiometries are significantly different from that of hIFN-α2b. The self-association of albinterferon-α2b results in the formation of reversible dimers and trimers, whereas the self-association of Pegasys gives only reversible dimers. The self-association behaviors of hIFN-α2b and albinterferon-α2b involves attractive electrostatic forces, which can be suppressed to a negligible level in low pH (pH 4.0-4.5) and high salt concentration (400 mM NaCl) buffer, allowing quantification of their size variant contents by sedimentation velocity analysis.