Denis Andrzejewski

Experimental Factors Affecting the Quality and Reproducibility of MALDI TOF Mass Spectra Obtained from Whole Bacteria Cells

Numerous experimental factors are shown to significantly influence the spectra obtained when bacteria are analyzed by MALDI TOF/MS. Detailed investigation of the instrument parameters and sample preparation are all shown to influence the spectra. Of these, the preanalysis sample preparation steps incorporate the most important elements influencing the quality and reproducibility of the spectra. Some of the most important sample preparation factors include the method employed for sterilization, the type of matrix, the matrix solvent and concentration of cells in the matrix, as well as the type and concentration of acid added to the matrix. The effects of these parameters, as well as other aspects of sample preparation and the effects of several instrumental parameters on spectra are presented. Optimization and control of all experimental variables leads to a stable protocol for analysis of bacteria. The protocol employs a Nd:Yag laser and describes both sample handling and instrument conditions which consistently yield reproducible MALDI TOF mass spectra with greater than 25 peaks from both gram-positive and gram-negative bacteria.

Platform for Identification of Salmonella Serovar Differentiating Bacterial Proteins by Top-Down Mass Spectrometry: S. Typhimurium vs S. Heidelberg

Intact protein expression profiling has proven to be a powerful tool for bacterial subspecies differentiation. To facilitate typing, epidemiology, and trace-back of Salmonella contamination in the food supply, a minimum of serovar level differentiation is required. Subsequent identification and validation of marker proteins is integral to rapid screening development and to determining which proteins are subject to environmental pressure. Bacterial sequencing efforts have expanded the number of sequenced genomes available for single-nucleotide polymorphism (SNP) analyses, but annotation is often missing, start site errors are not uncommon, and the likelihood of expression is not known. In this work we show that the combination of intact protein expression profiles and top-down liquid chromatography-mass spectrometry (LC-MS/MS) facilitates the identification of proteins that result from expressed serovar specific nonsynonymous SNPs. Combinations of these marker proteins can be used in assays for rapid differentiation of bacteria. LC-MS generated intact protein expression profiles establish which bacterial protein masses differ across samples and can be determined without prior knowledge of the sample. Subsequent top-down LC-MS/MS is used to identify expressed proteins and their post-translational modifications (PTM), identify serovar specific markers, and validate genomic predicted orthologues as expressed biomarkers.

Gas chromatographic determination of toxic quinolizidine alkaloids in blue cohosh Caulophyllum thalictroides (L.) Michx

Blue cohosh (Caulophyllum thalictroides (L.) Michx., Berberidaceae) is a North American perennial herb which is found as an ingredient in dietary supplement products in the United States. The plant contains the alkaloids N-methylcytisine, baptifoline, anagyrine and magnoflorine. Some of the alkaloids, including the quinolizidine alkaloid anagyrine, are toxic to range animals and have been implicated as teratogens in higher animals. Since the traditional use of the herb involves administration to women of reproductive age to treat menstrual cramps, and to pregnant women in the last 3–4 weeks of pregnancy to ease parturition, therefore the safety of these products to the fetus is of concern. Three of these alkaloids have been determined in authentic blue cohosh and several dietary supplements. Levels found were: 5–850 ppm for N-methylcytisine, 2–390 ppm for anagyrine, and 9–900 ppm for baptifoline. The lower alkaloid concentrations were found in products containing liquid extracts. Alkaloid identities were confirmed by mass spectrometry.

Identification of 2-bromo-3,4,5,6-tetrachloroaniline and its quantification in the color additives D&C Red Nos. 27 and 28 (phloxine B) using solid-phase microextraction and gas chromatography–mass spectrometry

The present work describes (a) the identification and characterization of a contaminant, 2-bromo-3,4,5,6-tetrachloroaniline (2BTCA), in the color additives D&C Red Nos. 27 and 28 (phloxine B) and (b) the determination of the extent and level of 2BTCA contamination in certified lots of these colors. For these purposes, 2BTCA (a compound not previously reported in the literature) and its positional isomer 4-bromo-2,3,5,6-tetrachloroaniline (4BTCA) were synthetically prepared. 4BTCA was used as the internal standard for the quantification of 2BTCA in the colors. Test portions from 35 certified lots of D&C Red Nos. 27 and 28 were analyzed for 2BTCA using a solid-phase microextraction-GC-MS method. Those lots were submitted for certification by both domestic (seven) and foreign (four) manufacturers during the past 4 years. Of the test portions analyzed, 22 (62.9%) contained 2BTCA in amounts ranging from 0.15 to 435.7 ppm with an average value of approximately 131.7 ppm. The remaining 13 (37.1%) test portions contained no detectable 2BTCA or less than 0.01 ppm, which is the limit of quantification of the present method. The analyses revealed substantial differences in the level of 2BTCA across lots from the same manufacturer as well as among different manufacturers. The wide range of 2BTCA levels found in the analyzed lots suggests that the presence of 2BTCA in D&C Red Nos. 27 and 28 may be avoided or significantly reduced during the manufacturing process. A direct correlation was observed between the presence of 2BTCA and that of 3,4,5,6-tetrachlorophthalic acid in analyzed batches of D&C Red Nos. 27 and 28. A chemical pathway that could explain the presence of 2BTCA in these color additives, and ways to avoid its formation, are also proposed.

Draft Genome Sequences of Two O104:H21 Escherichia coli Isolates Causing Hemorrhagic Colitis during a 1994 Montana Outbreak Provide Insight into Their Pathogenicity

ABSTRACT We sequenced the genomes of two strains of O104:H21 enterohemorrhagic Escherichia coli (EHEC) isolated during an outbreak of hemorrhagic colitis in Montana in 1994. These strains carried a plasmid that contains several virulence genes not present in pO157. The genome sequences will improve phylogenetic analysis of other non-O157 E. coli strains in the future.

A discriminant based charge deconvolution analysis pipeline for protein profiling of whole cell extracts using liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry.

A discriminant based charge deconvolution analysis pipeline is proposed. The molecular weight determination (MoWeD) charge deconvolution method was applied directly to the discrimination rules obtained by the fuzzy rule-building expert system (FuRES) pattern classifier. This approach was demonstrated with synthetic electrospray ionization-mass spectra. Identification of the tentative protein biomarkers by bacterial cell extracts of Salmonella enterica serovar typhimurium strains A1 and A19 by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was also demonstrated. The data analysis time was reduced by applying this approach. In addition, this method was less affected by noise and baseline drift.

Structural identification of an FD&C red no. 40 contaminant

Abstract NMR and mass spectral experiments have resulted in the determination of structure and assignment of carbon NMR signals of a manufacturing impurity of FD&C Red No. 40. Mass spectrometry showed its molecular weight to he 270, with a base peak of 228. A variety of two-dimensional NMR techniques demonstrated that this compound is a benzonaphthobicyclo-[2.2.2]octanone, which may arise from the Diels-Alder reaction of a naphthyl-benzyne species with 2-naphthol.

Bacterial Identification at the Serovar Level by Top-Down Mass Spectrometry

Food safety efforts require serovar and strain-level specificity of Salmonella contamination to trace back bacterial contamination to its source. Detection methods that require selection of probe-based assays are limited by probe selection. In this work we show that the combination of intact protein expression profiles and top-down liquid chromatography-tandem mass spectrometry (LC-MS/MS) facilitates the identification of proteins that result from expressed, serovar-specific non-synonymous single-nucleotide polymorphisms (SNPs). Intact protein expression profiles provide a nontargeted mass spectrometry-based method that facilitates a relatively unbiased snapshot of the expressed proteins in a wide range of bacterial samples and is amenable to both screening and targeted analysis. Such an inherently multiplexed technique facilitates differentiation of closely related bacteria, as well as the detection of un-sequenced or newly acquired SNPs and plasmid proteins that may be specific to a given strain without prior knowledge of the sample. Subsequent identification of expressed proteins, serovar-specific biomarkers, and post-translational modifications by top-down LC-MS/MS is integral to rapid screening development and facilitates collaboration with genome-based methods.