Leslie A. Harden

Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

ABSTRACT Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000- to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500- to 11,000-Da range were consistently high intensity. “Species-identifying” biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within ±5 Da with external instrument calibration. The highest-intensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) “strains” composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.

Web-Based Software for Rapid Top-Down Proteomic Identification of Protein Biomarkers, with Implications for Bacterial Identification

ABSTRACT We have developed web-based software for the rapid identification of protein biomarkers of bacterial microorganisms. Proteins from bacterial cell lysates were ionized by matrix-assisted laser desorption ionization (MALDI), mass isolated, and fragmented using a tandem time of flight (TOF-TOF) mass spectrometer. The sequence-specific fragment ions generated were compared to a database of in silico fragment ions derived from bacterial protein sequences whose molecular weights are the same as the nominal molecular weights of the protein biomarkers. A simple peak-matching and scoring algorithm was developed to compare tandem mass spectrometry (MS-MS) fragment ions to in silico fragment ions. In addition, a probability-based significance-testing algorithm (P value), developed previously by other researchers, was incorporated into the software for the purpose of comparison. The speed and accuracy of the software were tested by identification of 10 protein biomarkers from three Campylobacter strains that had been identified previously by bottom-up proteomics techniques. Protein biomarkers were identified using (i) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions with all possible in silico N and C terminus fragment ions (i.e., ions a, b, b-18, y, y-17, and y-18), (ii) their peak-matching scores and/or P values from a comparison of MS-MS fragment ions to residue-specific in silico fragment ions (i.e., in silico fragment ions resulting from polypeptide backbone fragmentation adjacent to specific residues [aspartic acid, glutamic acid, proline, etc.]), and (iii) fragment ion error analysis, which distinguished the systematic fragment ion error of a correct identification (caused by calibration drift of the second TOF mass analyzer) from the random fragment ion error of an incorrect identification.

Covalent attachment and dissociative loss of sinapinic acid to/from cysteine-containing proteins from bacterial cell lysates analyzed by MALDI-TOF-TOF mass spectrometry.

We report covalent attachment via a thiol ester linkage of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid or SA) to cysteine-containing protein biomarkers from bacterial cell lysates of E. coli analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry when using SA as the matrix. Evidence to support this conclusion is the appearance of additional peaks in the MS spectra when using SA, which are absent when using α-cyano-4-hydroxycinnamic acid (HCCA). The additional peaks appear at a mass-to-charge (m/z) ∼208 greater to the m/z of a more abundant protein ion peak. Protein biomarkers were identified by tandem mass spectrometry (MS/MS) using a MALDI time-of-flight/time-of-flight (TOF-TOF) mass spectrometer and top-down proteomics. Three protein biomarkers, HdeA, HdeB, and homeobox or YbgS (each containing two cysteine residues) were identified as having reactivity to SA. Non-cysteine-containing protein biomarkers showed no evidence of reactivity to SA. MS ions and MS/MS fragment ions were consistent with covalent attachment of SA via a thiol ester linkage to the side-chain of cysteine residues. MS/MS of a protein biomarker ion with a covalently attached SA revealed fragment ion peaks suggesting dissociative loss SA. We propose dissociative loss of SA is facilitated by a pentacyclic transition-state followed by proton abstraction of the β-hydrogen of the bound SA by a sulfur lone pair followed by dissociative loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal. The apparent reactivity of SA to cysteine/disulfide-containing proteins may complicate identification of such proteins, however the apparent differential reactivity of SA and HCCA toward cysteine/disulfide-containing proteins may be exploited for identification of unknown cysteine-containing proteins.

Quantification of Toxins in Soapberry (Sapindaceae) Arils: Hypoglycin A and Methylenecyclopropylglycine

Methylenecyclopropylglycine (MCPG) and hypoglycin A (HGA) are naturally occurring amino acids found in some soapberry fruits. Fatalities have been reported worldwide as a result of HGA ingestion, and exposure to MCPG has been implicated recently in the Asian outbreaks of hypoglycemic encephalopathy. In response to an outbreak linked to soapberry ingestion, the authors developed the first method to simultaneously quantify MCPG and HGA in soapberry fruits from 1 to 10 000 ppm of both toxins in dried fruit aril. Further, this is the first report of HGA in litchi, longan, and mamoncillo arils. This method is presented to specifically address the laboratory needs of public-health investigators in the hypoglycemic encephalitis outbreaks linked to soapberry fruit ingestion.

Expression, Purification, and Characterization of Almond (Prunus dulcis) Allergen Pru du 4

Biochemical characterizations of food allergens are required for understanding the allergenicity of food allergens. Such studies require a relatively large amount of highly purified allergens. The level of Pru du 4 in almond is low, and its expression in a soluble form in Escherichia coli required an expression tag. An MBP tag was used to enhance its expression and solubility. Sumo was used for the first time as a peptidase recognition site. The expression tag was removed with a sumo protease, and the resulting wild-type Pru du 4 was purified chromatographically. The stability of the allergen was investigated with chemical denaturation. The Gibbs free energy of Pru du 4 folding-unfolding transition was determined to be 5.4 ± 0.7 kcal/mol.

A family of small cyclic amphipathic peptides (SCAmpPs) genes in citrus.

BackgroundCitrus represents a crop of global importance both in economic impact and significance to nutrition. Citrus production worldwide is threatened by the disease Huanglongbing (HLB), caused by the phloem-limited pathogen Candidatus Liberibacter spp.. As a source of stable HLB-resistance has yet to be identified, there is considerable interest in characterization of novel disease-associated citrus genes.ResultsA gene family of Small Cyclic Amphipathic Peptides (SCAmpPs) in citrus is described. The citrus genomes contain 100–150 SCAmpPs genes, approximately 50 of which are represented in the citrus EST database. These genes encode small ~50 residue precursor proteins that are post-translationally processed, releasing 5–10 residue cyclic peptides. The structures of the SCAmpPs genes are highly conserved, with the small coding domains interrupted by a single intron and relatively extended untranslated regions. Some family members are very highly transcribed in specific citrus tissues, as determined by representation in tissue-specific cDNA libraries. Comparison of the ESTs of related SCAmpPs revealed an unexpected evolutionary profile, consistent with targeted mutagenesis of the predicted cyclic peptide domain. The SCAmpPs genes are displayed in clusters on the citrus chromosomes, with apparent association with receptor leucine-rich repeat protein arrays. This study focused on three SCAmpPs family members with high constitutive expression in citrus phloem. Unexpectedly high sequence conservation was observed in the promoter region of two phloem-expressed SCAmpPs that encode very distinct predicted cyclic products. The processed cyclic product of one of these phloem SCAmpPs was characterized by LC-MS-MS analysis of phloem tissue, revealing properties consistent with a K+ ionophore.ConclusionsThe SCAmpPs amino acid composition, protein structure, expression patterns, evolutionary profile and chromosomal distribution are consistent with designation as ribosomally synthesized defense-related peptides.