Franco Basile

Pathogenic bacteria: Their detection and differentiation by rapid lipid profiling with pyrolysis mass spectrometry

Abstract Pyrolysis mass spectrometry (Py–MS) can be used to profile methylated fatty acids from bacterial pathogens without a chromatographic step. An in situ thermal hydrolysis and methylation (THM) step incorporated into the Py–MS analysis of whole bacteria reduces the sample preparation time from 60 min to less than 1 min. Detection of four bacterial pathogens with a field-portable aerosol-sampling Py–ion trap MS using FAME profiles from whole bacteria is demonstrated with a total analysis time of less than 10 min/lipid profile.

A rapid approach for the detection of dipicolinic acid in bacterial spores using pyrolysis/mass spectrometry.

Curie-point pyrolysis/triple quadrupole mass spectrometry and micro-tube furnace pyrolysis/quadrupole ion trap mass spectrometry have been used to detect dipicolinic acid (DPA) in sporulated whole bacteria. DPA in whole cells of sporulated Bacillus anthracis reacted in situ during pyrolysis with tetramethylammonium hydroxide to form the dimethyl ester derivative of DPA, dimethyl-2,6-dipicolinate (mDPA). The mDPA was identified by its positive-ion electron ionization fragmentation pattern and confirmed with tandem mass spectrometry. In an oxidative pyrolysis/quadrupole ion trap instrument, the mDPA mass spectrum showed characteristic positive-ion electron ionization fragmentation along with a significant [M+1]+ ion due to self-chemical ionization. The characteristic collision-induced dissociation fragments of mDPA were used to establish the presence of sporulation in B. anthracis whole cells at a concentration of 2.2 x 10(7) CFU (colony-forming units)/mL using the triple quadrupole instrument. The total time for analysis, including sample preparation, was less than 10 minutes for both instruments.

The use of biomarker compounds for the identification of bacteria by pyrolysis-mass spectrometry

Abstract Several recent advances in using biomarkers for bacteria characterization are discussed. A study using Gram-positive and Gram-negative bacteria has been conducted to compare both gas chromatographic and mass spectrometric analyses of the separately isolated and methylated fatty acids to an in situ reaction in which the saponification and methylation of the fatty acids is achieved using tetramethylammoninium hydroxide (TMAH). Principal components analysis of the three data sets showed the same three cluster patterns. In situ methylation using TMAH has also been conducted on free nucleotides, oligonucleotides, DNA, various amino acids, and oligopeptides. An increase in the volatility of important biomarkers was observed in all analyses which leads to an increase in the information content of the data. High resolution mass spectrometry has also been applied to the peaks corresponding to a series of biomarker compounds. The data show that most nominal masses are composed of several contributing species.

Rapid chemotaxonomy of pathogenic bacteria using in situ thermal hydrolysis and methylation as a sample preparation step coupled with a field–portable membrane-inlet quadrupole ion trap mass spectrometer

Abstract A field-portable, pyrolysis membrane-inlet quadrupole ion trap mass spectrometer has been used to characterize four pathogenic bacteria ( Bacillus anthracis, Brucella melitensis, Yersinia pestis , and Francisella tularensis ). Moreover, a variety of strains were included, prepared under various growth conditions and a range of growth stages. In these analyses, an in situ thermal hydrolysis-methylation procedure was used during pyrolysis with the reagent tetramethylammonium hydroxide. Mass spectra generated from the analysis of the four pathogens contained information related to the biochemical composition of the sample (i.e. biomarkers) including mass spectral peaks derived from methyl esters of fatty acids, DNA/RNA, and peptide/protein fragments. Using multivariate statistics, bacterial mass spectral fingerprints were analyzed to determine the variance in the data and the contribution of biomarker origin (i.e. lipid, protein, nucleic acid, etc.) for bacterial differentiation. An optimum 98.3% correct classification rate was obtained using cross validation with linear discriminant analysis (on four replicates each of 54 bacterial samples) using only biomarkers of lipid origin and the bacterial spore biomarker dipicolinic acid.

Electron Transfer Dissociation of Peptides Generated by Microwave D-Cleavage Digestion of Proteins

The nonenzymatic digestion of proteins by microwave D-cleavage is an effective technique for site-specific cleavage at aspartic acid (D). This specific cleavage C-terminal to D residues leads to inherently large peptides (15-25 amino acids) that are usually relatively highly charged (above +3) when ionized by electrospray ionization (ESI) due to the presence of several basic amino acids within their sequences. It is well-documented that highly charged peptide ions generated by ESI are well-suited for electron transfer dissociation (ETD), which produces c- and z-type fragment ions via gas-phase ion/ion reactions. In this paper, we describe the sequence analysis by ETD tandem mass spectrometry (MS/MS) of multiply charged peptides generated by microwave D-cleavage of several standard proteins. Results from ETD measurements are directly compared to CID MS/MS of the same multiply charged precursor ions. Our results demonstrate that the nonenzymatic microwave D-cleavage technique is a rapid (<6 min) and specific alternative to enzymatic cleavage with Lys-C or Asp-N to produce highly charged peptides that are amenable to informative ETD.

Online Microwave D-Cleavage LC-ESI-MS/MS of Intact Proteins: Site-Specific Cleavages at Aspartic Acid Residues and Disulfide Bonds

An online nonenzymatic digestion method utilizing a microwave-heated flow cell and mild acid hydrolysis at aspartic acid (D) for rapid protein identification is described. This methodology, here termed microwave D-cleavage, was tested with proteins ranging in size from 5 kDa (insulin) to 67 kDa (bovine serum albumin) and a bacterial cell lysate ( Escherichia coli). A microwave flow cell consisting of a 5 microL total volume reaction loop connected to a sealed reaction vessel was introduced into a research grade microwave oven. With this dynamic arrangement, the injected sample was subjected to microwave radiation as it flowed through the reaction loop and was digested in less than 5 min. Different digestion times can be achieved by varying the sample flow rate and/or length of the loop inside the microwave flow cell. The microwave flow cell can be operated individually with the output being collected for matrix assisted laser ionization/desorption (MALDI) mass spectrometry (MS) or connected online for liquid chromatography (LC) electrospray ionization (ESI)-MS. In the latter configuration, the microwave flow cell eluates containing digestion products were transferred online to a reversed phase liquid chromatography column for direct ESI-MS and ESI-MS/MS analyses (specifically, Collision Induced Dissociation, CID). Concurrently with the microwave D-cleavage step, disulfide bond reduction/cleavage was achieved by the coinjection of dithiothreitol (DTT) with the sample prior to online microwave heating and online LC-MS analysis and so eliminating the need for alkylation of the reduced protein. All protein standards, protein mixtures, and proteins in a bacterial cell lysate analyzed by this new online methodology were successfully identified via a SEQUEST database search of fragment ion mass spectra. Overall, online protein digestion and identification was achieved in less than 40 min total analysis time, including the chromatographic step.

Validation using sensitivity and target transform factor analyses of neural network models for classifying bacteria from mass spectra

Temperature constrained cascade correlation networks (TCCCNs) are computational neural networks that configure their own architecture, train rapidly, and give reproducible prediction results. TCCCN classification models were built using the Latin-partition method for five classes of pathogenic bacteria. Neural networks are problematic in that the relationships among the inputs (i.e., mass spectra) and the outputs (i.e., the bacterial identities) are not apparent. In this study, neural network models were constructed that successfully classified the targeted bacteria and the classification model was validated using sensitivity and target transformation factor analysis (TTFA). Without validation of the classification model, it is impossible to ascertain whether the bacteria are classified by peaks in the mass spectrum that have no causal relationships with the bacteria, but instead randomly correlate with the bacterial classes. Multiple single output network models did not offer any benefits when compared to single network models that had multiple outputs. A multiple output TCCCN model achieved classification accuracies of 96 ± 2% and exhibited improved performance over multiple single output TCCCN models. Chemical ionization mass spectra were obtained from in situ thermal hydrolysis methylation of freeze-dried bacteria. Mass spectral peaks that pertain to the neural network classification model of the pathogenic bacterial classes were obtained by sensitivity analysis. A significant number of mass spectral peaks that had high sensitivity corresponded to known biomarkers, which is the first time that the significant peaks used by a neural network model to classify mass spectra have been divulged. Furthermore, TTFA furnishes a useful visual target as to which peaks in the mass spectrum correlate with the bacterial identities.

Rapid online nonenzymatic protein digestion combining microwave heating acid hydrolysis and electrochemical oxidation.

We report an online nonenzymatic method for site-specific digestion of proteins to yield peptides that are well suited for collision-induced dissociation tandem mass spectrometry. The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochemical oxidation at tryptophan and tyrosine. The combined microwave/electrochemical digestion is reproducible and produces peptides with an average sequence length of 10 amino acids. This peptide length is similar to the average peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel nonenzymatic digestion method, when analyzed by electrospray ionization mass spectrometry, produce protonated molecules with mostly +1 and +2 charge states. The combination of these two nonenzymatic methods overcomes shortcomings with each individual method in that (i) peptides generated by the microwave-hydrolysis method have an average amino acid length of 16 amino acids and (ii) the electrochemical-cleavage method is unable to reproducibly digest proteins with molecular masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min of digestion time) on a series of standard peptides and proteins as well as an Escherichia coli protein extract.

Analysis of lipids from crude lung tissue extracts by desorption electrospray ionization mass spectrometry and pattern recognition

A method is described using desorption electrospray ionization (DESI) mass spectrometry (MS) to obtain phospholipid mass spectral profiles from crude lung tissue extracts. The measured DESI mass spectral lipid fingerprints were then analyzed by unsupervised learning principal components analysis (PCA). This combined approach was used to differentiate the effect(s) of two vaccination routes on lipid composition in mouse lungs. Specifically, the two vaccination routes compared were intranasal (i.n.) and intradermal (i.d.) inoculation of the Francisella tularensis live vaccine strain (Ft-LVS). Lung samples of control and LVS-inoculated mice were quickly extracted with a methanol/chloroform solution, and the crude extract was directly analyzed by DESI-MS, with a total turnaround time of less than 10 min/sample. All of the measured DESI mass spectra (in both positive and negative ion mode) were compared via PCA, resulting in clear differentiation of mass spectral profiles of i.n.-inoculated mouse lung tissues from those of i.d.-inoculated and control mouse lung tissues. Lipid biomarkers responsible for sample differentiation were identified via tandem MS (MS/MS) measurements or by comparison with mass spectra of lipid standards. The DESI-MS approach described here provided a practical and rapid means to analyze tissue samples without extensive extractions and solvent changes.

The effects of electron and chemical ionization modes on the MS profiling of whole bacteria.

Free fatty acid profiling of whole bacteria [Francisella tularensis, Brucella melitensis, Yersinia pestis, Bacillus anthracis (vegetative and sporulated), and Bacillus cereus] was carried out with direct probe mass spectrometry under 70-eV electron ionization (EI) and isobutane chemical ionization in both the positive (CI+) and negative modes (CI−). Electron ionization produced spectra that contained molecular ions and fragment ions from various free fatty acids. Spectra acquired with isobutane chemical ionization in the positive mode yielded molecular ions of free fatty acids as well as ions from other bacterial compounds not observed under EI conditions. Spectra obtained with negative chemical ionization did not contain as much taxonomic information as EI or CI+; however, some taxonomically significant compounds such as dipicolinic acid and poly(3-hydroxybutyrate) did produce negative ions. All ionization modes yielded spectra that could separate the bacteria by Gram-type when observed with principle components analysis (PCA). Chemical ionization in the positive ion mode produced the greatest amount of differentiation between the four genera of bacteria when the spectra where examined by PCA.