David M. Schieltz

Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry

The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity.

Mass Spectrometric Analysis of Multiple Pertussis Toxins and Toxoids

Bordetella pertussis (Bp) is the causative agent of pertussis, a vaccine preventable disease occurring primarily in children. In recent years, there has been increased reporting of pertussis. Current pertussis vaccines are acellular and consist of Bp proteins including the major virulence factor pertussis toxin (Ptx), a 5-subunit exotoxin. Variation in Ptx subunit amino acid (AA) sequence could possibly affect the immune response. A blind comparative mass spectrometric (MS) analysis of commercially available Ptx as well as the chemically modified toxoid (Ptxd) from licensed vaccines was performed to assess peptide sequence and AA coverage variability as well as relative amounts of Ptx subunits. Qualitatively, there are similarities among the various sources based on AA percent coverages and MS/MS fragmentation profiles. Additionally, based on a label-free mass spectrometry-based quantification method there is differential relative abundance of the subunits among the sources.

A historical and proteomic analysis of botulinum neurotoxin type/G

BackgroundClostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex.ResultsAn in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs--nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison.ConclusionsThe in silico protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MSE data analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin.

On-column trypsin digestion coupled with LC-MS/MS for quantification of apolipoproteins

Apolipoproteins measured in plasma or serum are potential biomarkers for assessing metabolic irregularities that are associated with the development of cardiovascular disease (CVD). LC-MS/MS allows quantitative measurement of multiple apolipoproteins in the same sample run. However, the accuracy and precision of the LC-MS/MS measurement depends on the reproducibility of the enzymatic protein digestion step. With the application of an immobilized enzyme reactor (IMER), the reproducibility of the trypsin digestion can be controlled with high precision via flow rate, column volume and temperature. In this report, we demonstrate the application of an integrated IMER-LC-MS/MS platform for the simultaneous quantitative analysis of eight apolipoproteins. Using a dilution series of a characterized serum pool as calibrator, the method was validated by repeated analysis of pooled sera and individual serum samples with a wide range of lipid profiles, all showing intra-assay CV<4.4% and inter-assay CV<8%. In addition, the method was compared with traditional homogeneous digestion coupled LC-MS/MS for the quantification of apoA-I and apoB-100. Applied in large scale human population studies, this method can serve the translation of a wider panel of apolipoprotein biomarkers from research to clinical application. SIGNIFICANCE Currently, the translation of apolipoprotein biomarkers to clinical application is impaired because of the high cost of large cohort studies using traditional single-analyte immunoassays. The application of on-line tryptic digestion coupled with LC-MS/MS analysis is an effective way to address this problem. In this work we demonstrate a high throughput, multiplexed, automated proteomics workflow for the simultaneous analysis of multiple proteins.

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: A fast-track strategy applicable to other microorganisms

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (>95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.

High throughput quantification of apolipoproteins A-I and B-100 by isotope dilution MS targeting fast trypsin releasable peptides without reduction and alkylation

Apolipoprotein A‐I (ApoA‐I) and apolipoprotein B‐100 (ApoB‐100) are amphipathic proteins that are strong predictors of cardiovascular disease risk. The traceable calibration of apolipoprotein assays is a persistent challenge, especially for ApoB‐100, which cannot be solubilized in purified form.

Complete Genome Sequences of Four Bordetella pertussis Vaccine Reference Strains from Serum Institute of India

ABSTRACT Serum Institute of India is among the worlds largest vaccine producers. Here, we report the complete genome sequences for four Bordetella pertussis strains used by Serum Institute of India in the production of whole-cell pertussis vaccines.

Surfaceome Analysis Protocol for the Identification of Novel Bordetella pertussis Antigens

The bacterial surfaceome, comprising outer membrane-sorted and/or associated (i.e., cell transporters), cell surface-exposed (i.e., adhesins) and extracellularly secreted proteins (i.e., toxins), has been characterized in bacterial pathogens, such as Bordetella pertussis (Bp) to provide information for use in development of diagnostic and prevention strategies. This protein subset has clinical significance, as these bacterial proteins are often associated with attachment to host cells, microbial pathogenesis and antibody-mediated immunity. Here we describe classical surface membrane protein enrichment techniques, followed by proteomic methodologies, such as gel-free protein separation and antibody-affinity capture technologies in combination with nano-liquid chromatography mass spectrometry, for the identification and characterization of Bp surfaceome proteins.