Article pubs.acs.org/ac
Rapid Discrimination of Bacteria by Paper Spray Mass Spectrometry Ahmed M. Hamid,† Alan K. Jarmusch,† Valentina Pirro,‡ David H. Pincus,§ Bradford G. Clay,§ Gaspard Gervasi,∥ and R. Graham Cooks*,† †
Department of Chemistry and Center for Analytical Instrumentation Development, Purdue University, West Lafayette, Indiana 47907, United States ‡ Department of Chemistry, University of Turin, Turin 10124, Italy § bioMérieux, Inc., Hazelwood, Missouri 63042, United States ∥ bioMérieux, Marcy l’Etoile 69280, France S Supporting Information *
ABSTRACT: Paper spray mass spectrometry ambient ionization is utilized for rapid discrimination of bacteria without sample preparation. Bacterial colonies were smeared onto filter paper precut to a sharp point, then wetted with solvent and held at a high potential. Charged droplets released by field emission were sucked into the mass spectrometer inlet and mass spectra were recorded. Sixteen different species representing eight different genera from Gram-positive and Gram-negative bacteria were investigated. Phospholipids were the predominant species observed in the mass spectra in both the negative and positive ion modes. Multivariate data analysis based on principal component analysis, followed by linear discriminant analysis, allowed bacterial discrimination. The lipid information in the negative ion mass spectra proved useful for species level differentiation of the investigated Gram-positive bacteria. Gram-negative bacteria were differentiated at the species level by using a numerical data fusion strategy of positive and negative ion mass spectra.
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applications of the method.20−23 Subsequently, fast atom bombardment was used as a rapid method of discrimination.17,24−28 Thermal degradation combined with in situ chemical derivatization is a powerful discriminatory method that has been widely applied in the context of biological security monitoring.29,30 Subsequently, measurements of protein profiles have formed the basis of discrimination in electrospray ionization methods.31−33 Finally and most recently, matrixassisted laser desorption ionization-time-of-flight (MALDITOF), also based on protein profiles, has been developed as an important MS method in microbiology.34,35 Bacterial identification by MALDI-MS offers rapid analysis and accurate identification of bacteria based on comparing a protein fingerprint of the organism tested to a database.6,34,36−41 Ambient ionization methods generate ions outside the vacuum of the mass spectrometer and require little or no sample preparation.42,43 In the exploration of the capabilities of several ambient ionization methods including low temperature plasma (LTP), 44 desorption electrospray ionization (DESI),45−47 nanospray desorption electrospray ionization (nanoDESI),2,48−52 direct analysis in real time (DART),53
he rapid and accurate identification of microorganisms is relevant to numerous fields, including clinical medicine, public health, biotechnology, and food safety.1−4 Bacteria discrimination and subsequent identification rely on the observation of endogenous biochemical and/or genetic differences. Biochemical identification of bacteria is commonly performed by integrating information on culture morphology, effects of physical alteration of culture media (e.g., hemolysis), and specific enzymatic reactions (e.g., tryptophanase). The simplicity of these experiments is advantageous; however, the complexity of the biological systems limits the usefulness of simple tests in distinguishing closely related organisms. Enzyme-linked immunosorbent assay (ELISA),5 polymerase chain reaction (PCR),6,7 and nucleic acid hybridization8 are each capable of detecting more subtle genetic differences between bacteria. Thus, the identification of bacteria, sometimes down to the strain level can be achieved by genomic methods but they are often labor-intensive, time-consuming, and expensive.9−13 The combined advantages of sensitivity, specificity, and speed make mass spectrometry (MS) an attractive analytical approach for the analysis of intact bacteria.14−19 Some decades ago, Curie-point pyrolysis MS was shown to be highly discriminatory, but the reduction in molecular complexity at high temperature and the need for pure samples limited the © 2014 American Chemical Society
Received: April 7, 2014 Accepted: July 11, 2014 Published: July 11, 2014 7500
dx.doi.org/10.1021/ac501254b | Anal. Chem. 2014, 86, 7500−7507
Analytical Chemistry
Article
laser ablation electrospray ionization (LAESI),54 and rapid evaporative ionization-mass spectrometry (REIMS),55 bacterial analysis has been examined in a preliminary fashion. In several of these cases, microorganism colonies were examined directly. Paper spray mass spectrometry (PS-MS) has been successful in qualitative and quantitative mass spectrometric analysis of complex mixtures27−29 but has not been previously applied to bacterial discrimination. This is undertaken in the present study. Principal component analysis (PCA) is commonly used for exploratory investigation of the complex information contained in full mass spectral data sets, allowing simultaneous consideration of all spectral variables and their intercorrelation.56 Nearly all of the chemical information content of the original m/z variables is reorganized and compacted into a set of orthogonal principal components (PCs).57 The projections of the data objects (i.e., the samples) onto the PCs are called scores, while the importance of each original spectral variable in defining a certain PC is given by its loading coefficient. Both scores and loading values can be represented in twodimensional scatter plots. The relationship between the score and loading plots is evident from the codirectionality of objects and variables in related score and loading plots.56 This paper presents the application of PS-MS in the rapid discrimination of bacteria (1−2 min). Due to the large amount of information acquired in full scan mass spectra, multivariate statistical methods are necessary to manage the MS data and extract chemical features from them. In this study, a preliminary PCA was used to explore the differences among bacteria based on PS mass spectra. Then taking advantage of the fact that most of the variance in the data set is available in a few PCs, the generation of which acts as a data reduction technique, linear discriminant analysis (LDA) was applied as a supervised discriminant classification method to quantify the ability of PSMS chemical signatures in discriminating among bacteria.58 This approach has been previously reported for other types of samples to tackle classification problems by using PCs scores as variables in LDA.59,60
analysis. All bacterial culturing activities were performed in a biological safety cabinet and biohazardous materials were autoclaved. All experiments were performed under Institutional Review Board guidelines IBC protocol #070-004-10 “Novel tissue, Biological fluid and Bacteria Evaluation by Mass Spectrometry” as amended. One strain of 16 clinically relevant bacteria were analyzed; see Table S1 of the Supporting Information. Several of these species were selected for their high level of similarity by either phenotypic or genotypic methods.35,61 Multiple bacterial strains were not included and are not anticipated to effect species level discrimination, based on genetic homology between strains.62 Instrumentation. Mass spectrometric analysis was performed using a linear ion trap mass spectrometer (LTQ, Thermo Fisher Scientific, San Jose, CA). Spectra were collected with automatic gain control (AGC) activated and a maximum ion trap injection time of 50 ms and three microscans. Full scan negative (−) and positive (+) ion spectra were obtained over the range of m/z 200−2000 in profile mode. The temperature of the MS capillary was set at 275 °C. The tube lens voltage was set at −100 and 0 V in the negative and positive ion modes, respectively. The capillary voltage was maintained at −50 and 0 V in the negative ion mode and positive ion mode, respectively. The voltage used for paper spray ionization was −3.5 kV in the negative ion mode and 2.8 kV in the positive ion mode. Tandem mass spectrometry (MS/MS) was carried out using collision-induced dissociation (CID) for structural elucidation. An isolation window of 1.0−1.8 (m/z units) and normalized collision energy of 15%−25% (manufacturer’s unit) were chosen. The identities of highly abundant ions have been confirmed by high resolution mass measurements acquired using an Orbitrap mass spectrometer (Exactive, Thermo Fisher Scientific, San Jose, CA). The Orbitrap instrumental conditions in the negative ion mode were as follows: maximum injection time of 50 ms; two microscans; m/z range of 200−2000; activated AGC; −25 V capillary voltage; and −165 V tube lens voltage. Paper Spray-MS Methods. A triangular piece of filter paper, 10 mm (height) by 8 mm (base), was held with the apex in line with the inlet of the mass spectrometer at a distance of 5 ± 1 mm from it.63 High voltage was supplied via the mass spectrometer using copper clips attached to the paper triangles. Approximately 100 μg (≤5 bacterial colonies) of material was selected from the culture plates using sterile inoculation loops and smeared onto the middle of the surface of the paper triangle. Fifteen microliters of solvent was then applied by pipet, with the selections of solvent and volume being guided by evaluations of spray duration and mass spectral quality (Supporting Information, Figure S1). PS-MS spectra were recorded for 1−2 min/sample and are displayed without background subtraction. With the aim of improving classification, a one-step PS-MS experiment was developed in which positive and negative mode information was collected from the same samples. This was accomplished by switching the polarity of the instrument during data acquisition during a single solvent addition (pure methanol). Signal in this bipolar method was obtained on average for 60 s, negative ion spectra were collected for the first 30 s, and positive ion data were collected for the remaining 30 s. Multivariate Statistics. PCA and LDA A list of m/z values and corresponding ion abundances from averaged mass spectra were imported into Matlab (MathWorks, Inc., Natick, MA),
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EXPERIMENTAL SECTION Chemicals and Materials. Methanol (HPLC grade) was purchased from Mallinckrodt Baker Inc. (Phillipsburg, NJ). Sodium dodecyl sulfate (SDS), 3-[(3-cholamido-propyl)dimethylammonio]-1-propanesulfonate (CHAPS), and octylβ-D-glucopyranoside were purchased from Sigma-Aldrich (St. Louis, MO). The culturing medium, trypticase soy agar supplemented with 5% sheep blood (TSAB), was purchased from Remel (Lenexa, KS). Sterile inoculation loops were purchased from Copan Diagnostics, Inc. (Murrieta, CA). Whatman grade 1 cellulose filter paper was purchased from Whatman International Ltd. (Maidstone, England). Copper clips were purchased from McMasters-Carr (Chicago, IL). Microorganism Culturing. The bacterial isolates, supplied by bioMérieux, Inc. (Hazelwood, MO), were cultured from frozen samples stored at −80 °C on TSAB in cryotubes. Bacteria were aliquoted from frozen storage by sterile wooden splints, streaked on TSAB plates, and incubated at 37 ± 1 °C for approximately 24 h. Bacteria were subcultured from an isolated colony, removing it using a sterile loop, and incubated for an additional 24 h in a VWR forced air incubator (Chicago, IL). Mass spectral analysis was performed on samples taken from the subcultured plates after 24 h, any plates appearing to have two bacterial colony morphologies were excluded from 7501
dx.doi.org/10.1021/ac501254b | Anal. Chem. 2014, 86, 7500−7507
Analytical Chemistry
Article
(30°) resulted in greater currents (>1.5 μA) suggesting that the electric field strength exceeded the threshold required for stable electrospray and resulted in a gaseous discharge. It was determined empirically that such discharges adversely affected spectral quality leading to increased noise and spectral complexity.67 The less acute angle of 45° yielded spray currents consistent with typical electrospray plume formation (0.1−0.5 μA) and resulted in higher quality mass spectra. Therefore, it was decided that a paper tip angle of 45° was most suitable under the conditions tested. The addition of compounds which reduce the surface tension (e.g., surfactants) of electrosprayed solvents is known to enhance ionization efficiency.68 Multiple surfactants were explored including CHAPS, SDS, and octyl-β-D-glucopyranoside. The addition of CHAPS, a zwitterionic surfactant, to the spray solvent (methanol) resulted in stable mass spectral signals of greater intensity compared to other surfactant additives. CHAPS was detectable in both the positive and in the negative mode as the protonated molecule and the chloride adduct, m/z 615.5 and 649.5, respectively. A deleterious effect upon positive mode PS-MS was noted with CHAPS, likely the consequence of charge competition, while negative mode PS performance was improved significantly with
