Anal. Chem. 2003, 75, 1628-1637
Detection of Cyclic Lipopeptide Biomarkers from Bacillus Species Using Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Angelo J. Madonna and Kent J. Voorhees*
Department of Chemistry, Colorado School of Mines, Golden, Colorado 80401 Nelli I. Taranenko, Victor V. Laiko, and Vladimir M. Doroshenko
MassTech Inc., Subsidiary of SESI, Burtonsville, Maryland 20866
A novel approach to microbial detection using atmospheric pressure matrix-assisted laser desorption/ionization with an ion trap mass spectrometer to analyze whole cell bacteria is introduced. This new approach was tested with lyophilized spores and cultures of Bacillus globigii (BG) grown on agar media for 4 days or longer. At each stage of growth, it was found that biomarkers, identified as cyclic lipopeptides known as fengycin and surfactin, could be detected by pulsed ultraviolet laser irradiation of intact BG cells (∼5 mg) cocrystallized with r-cyano-4hydroxycinnamic acid. Furthermore, definitive amino acid sequence information was obtained by performing tandem mass spectrometry on the precursor ions of the cyclic lipopeptides. The investigation was broadened to include the examination of aerosolized BG spores collected from the atmosphere and directly deposited onto double-sided tape. Subsequent analysis of the recovered spores resulted in the production of mass peaks consistent with fengycin. Other Bacillus species were analyzed for comparison and showed mass spectral peaks also identified as originating from various cyclic lipopeptides. Further studies were conducted using a pulsed infrared laser as the excitation source to analyze BG cells (∼5 mg) suspended in a matrix of 0.03 M ammonium citrate and glycerol resulting in the production of ions characteristic of fengycin and surfactin. The discovery of matrix-assisted laser desorption/ionization (MALDI) by Karas and Hillenkamp has had an enormous influence on the growing popularity of biological mass spectrometry (MS).1 This analytical procedure is finding extensive use by scientists intent on gaining a fundamental understanding of the processes driving living systems. One particularly useful aspect of MALDI is the ability to analyze whole microorganisms and biological tissues at the molecular scale without need of physical or chemical preprocessing steps.2-7 This capability has caught the * Corresponding author. E-mail:
[email protected]. Fax: 303 273-3629. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301.
1628 Analytical Chemistry, Vol. 75, No. 7, April 1, 2003
attention of researchers concerned with developing rapid methods for detecting infectious microorganisms. MALDI-MS has been shown to be very effective in discriminating between whole-cell microorganisms based on the production of mass spectra with unique distributions of taxonomically relevant protein/peptide signals.8-10 The resulting mass spectra are generated within minutes using simple, nonrigorous methods that are applicable to a broad range of microorganisms. In this capacity, MALDI-MS could play a significant role in safeguarding public health by pinpointing sources of infectious diseases before they have the chance of becoming widely dispersed. Moreover, MALDI-MS generally provides very high sensitivity and high sample throughputs with short duty cycles and requires negligible quantities of inexpensive consumables. Recently, a newly developed MALDI source that operates under atmospheric pressure has been described.11 This MALDI source is compatible with commercially available mass spectrometers that are fitted with standard electrospray ionization sources. When compared with conventional vacuum MALDI, atmospheric pressure (AP) MALDI has been shown to yield less analyte fragmentation while providing higher resolving power for individual components in complex mixtures.12,13 An added benefit of this new ionization source is the ability to perform both sample handling and MALDI analysis under normal atmospheric condi(2) Holland, R. D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Persons, C. C.; Voorhees, K. J.; Lay, J. O., Jr. Rapid Commun. Mass Spectrom. 1996, 10, 1227-1232. (3) Claydon, M. A.; Davey, S. N.; Edwards-Jones, V.; Gordon, D. B. Nat. Biotechnol. 1996, 14, 1584-1586. (4) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751-60. (5) Stoeckli, M.; Chaurand, P.; Hallanhan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493-496. (6) Amiri-Eliasi, B.; Fenselau, C. Anal. Chem. 2001, 73, 5228-5231. (7) Bundy, J. L.; Fenselau, C. Anal. Chem. 2001, 73, 751-757. (8) van Barr, B. L. M. FEMS Mircobiol. Rev. 2000, 24, 193-219. (9) Dalluge, J. J. Fresenius J. Anal. Chem. 2000, 866, 701-711. (10) Lay, J. O. J. Trends Anal. Chem. 2000, 19, 507-516. (11) Laiko, V. V.; Baldwin, M. A.; Burlingame, A. L. Anal. Chem. 2000, 72, 652657. (12) Laiko, V. V.; Moyer, S. C.; Cotter, R. J. Anal. Chem. 2000, 72, 5239-5243. (13) Doroshenko, V. M.; Laiko, V. V.; Taranenko, N. I.; Berkout, V. D.; Lee, H. S. Int. J. Mass Spectrom., in press. 10.1021/ac020506v CCC: $25.00
© 2003 American Chemical Society Published on Web 02/27/2003
tions. This latter characteristic is highly suitable for interfacing mass spectrometers to sample collection/concentration devices that operate at atmospheric pressure. The prospect for such an arrangement was the incentive for the current study to evaluate whether AP-MALDI could generate vital taxonomic information from whole-cell bacteria as effectively as presently observed using conventional vacuum MALDI. For this purpose, a quadrupole ion trap mass spectrometer equipped with an AP-MALDI source was used to investigate Bacillus globigii at different stages of growth, as well as from aerosolized samples collected using a high-capacity air sampler. Several other species of bacteria from the Bacillus genus were also analyzed for comparison. The developed methodologies and results from this work are presented below. EXPERIMENTAL SECTION Bacillus Species and Culture Conditions. Each organism utilized in this study was acquired as a lyophilized stock culture. Bacillus globigii var. Niger (BG) was obtained from Dugway Proving Grounds (Dugway, UT), Bacillus cereus (ATCC 14579), Bacillus (ATCC 61), Bacillus licheniformis (ATTC 14580), Bacillus megaterium (ATCC 14581), and Bacillus subtilis (ATCC 6051) were purchased from the American Type Culture Collecton (Rockville, MD), and Bacillus sphaericus was received from the Armed Forces Institute of Pathology (Washington, DC). All cultivation steps were performed in a sterilized biosafety level II cabinet using standard microbiology procedures. The lyophilized cultures were initially rehydrated in trypticase soy broth for 24 h prior to being streaked onto trypticase soy agar plates and subsequently incubated at 37 °C. Analyte Preparation. For analysis using a nitrogen UV laser, bacterial colonies (∼5 mg) were harvested from agar plates with an inoculation loop, suspended in 1 mL of water/acetonitrile (50: 50 v/v), and then briefly vortexed to give homogeneous suspensions. The prepared suspensions were spotted onto the MALDI target plate in 1-µL aliquots and evaporated to dryness at room temperature. The bacterial residues were then overlaid with 1 µL of matrix solution prepared from 8 mg of R-cyano-4-hydroxycinnamic acid (R-CHCA) dissolved in 1 mL of water/85% formic acid/ acetonitrile (70:13:17 v/v/v) and then analyzed by AP-MALDI following crystal formation. With this method, a detection limit of ∼1.0 × 107 spores/mL (∼10 000 spores/µL) was achieved. The prepared suspensions were enumerated with a Petroff-Hausser counting chamber (Hausser Scientific Partnership, Horsham, PA). When an erbium:yttrium aluminum garnet (Er:YAG) infrared laser (IR) was employed, colonies were (∼5 mg) suspended in diluted ammonium citrate (0.03 M) and then admixed with glycerol/water (50:50 v/v). Microliter aliquots of this mixture were then applied to the MALDI sample plate and analyzed immediately by AP-IR-MALDI. All solutions were prepared from doubledeionized water and HPLC-grade solvents sold by Sigma-Aldrich Corp. (St. Louis, MO). Air Sampling. Airborne BG spores were collected (sampling rate 1000 L of air/min) using a four-stage virtual impactor (SCP Dynamics, Inc, Minnesota, MN) designed to fractionate and collect airborne particles of respirable size (1-10 µm) with 50% overall sampling efficiency. Five-milligram quantities of pure lyophilized BG spores (or mixed with dry yeast or gelatin mix) were ground into fine powders and disseminated above the air sampler by placing the powder samples between gloved hands and vigorously clapping. The spores were collected for analysis by placing a
MALDI sample plate covered with double-sided tape directly in line with the outlet of the concentrated sample. Following collection (
