Microbial Forensics Using MADLITOF MS Spectral Fingerprints

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Microbial Forensics Using MADLI-TOF MS Spectral
Fingerprints

• Koren Holland Deckman
• Gettysburg College
• Barbara C. Levin and Chad P. Nelson National
  Institute of Standards and Technology

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MAIN OBJECTIVE
TO DETERMINE THE EFFECTS OF VARIOUS EXPERIMENTAL
CONDITIONS ON THE IDENTIFICATION OF BACTERIA BY
MALDI-TOF MASS SPECTROMETRY
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SPECIFIC OBJECTIVES

• To investigate the use of matrix-assisted laser
  desorption/ionization time-of-flight (MALDI-TOF)
  mass spectrometry (MS) as a rapid means of
  identification of various bacterial genera,
  species and strains and the effects of a variety
  of environmental conditions.
• To determine the conditions under which MALDI-TOF
  MS would produce unique cellular spectral
  fingerprints from the proteomes of a variety of
  microbial organisms.  

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SPECIFIC OBJECTIVES

• To determine if the the elimination of protein
  digests and 2D-gel electrophoresis steps would
  prevent the unique cellular spectral
  fingerprints from whole cells.
• To examine the effects of cell growth stages and
  cell growth conditions on these cellular spectral
  fingerprints.

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SPECIFIC OBJECTIVES

• To test if the proteomes from the same whole
  cells grown in liquid media or as colonies grown
  on solid agar would generate the same or
  different spectral fingerprints.
• To determine the reproducibility of spectra from
  the same cells collected from different colonies
  on different days.
• To examine the effect of freezing on the cellular
  spectral fingerprints.

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METHODS AND MATERIALS
Bacteria Examined Gram Positive Bacillus
subtilis, var. globigii, ATCC 9372 Bacillus
subtilis ATCC 6051A Bacillus subtilis ATCC
82 Bacillus cereus ATCC 2 Bacillus cereus
ATCC 11778 Bacillus thuringiensis ATCC 10792
Gram Negative Escherichia coli ATCC 15597
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Bacterial growth conditions continued
Bacterial Growth Conditions Liquid LB broth
(10 g Bacto-tryptone, 5 g yeast extract, and 5
g NaCl in 1 L)
Solid Colonies of bacteria were grown on
Tryptic Soy agar prepared following label
directions.
Freezing Samples Three 1 mL samples of B.
subtilis (globigii) and E. coli Two samples
20oC for 15 days or 15 weeks and for MALDI-TOF
MS analysis at the designated given time One
sample analyzed by MALDI-TOF MS immediately
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MALDI-TOF MS
Matrix-assisted laser desorption/ionization
(MALDI) time-of-flight (TOF) mass spectrometry
(MS). Advantages 1. Accurately measures the
mass of very small amounts of material (e.g., 1
ug of peptide, protein and DNA, femtomole
amounts 10-15 mole) 2. Rapid results in 15
minutes.
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MALDI-TOF MS
Sample chamber
Flight tube
Sample plate
path of ions
m2 m1
detector
Laser

• Sample is placed on sample plate
• Irradiated with laser.
• Ions accelerate through the flight tube to the
  detector
• Applied Biosystems Voyager-DE STR
  Bio-spectrometry Workstation

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MALDI-TOF MS PROCEDURE

• Cell washed 2 ammonium chloride and resuspended
  in ammonium chloride.
• Mixed with ferulic acid matrix solution.
• Two ?L placed on sample plate and air dried.
• Nitrogen laser (337 nm) operated in a linear,
  delayed extraction and positive ion mode.
• The low mass gate m/z 1000, delay time300 ns,
  acceleration voltage 25 kV, grid voltage 90.
• Internal calibration angiotensin I (m/z 1297)
  and myoglobin (m/z 16,952).
• Each spectrum obtained by averaging 100 laser
  shots.

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RESULTS Figure 1
The MALDI-TOF MS of gram negative E. coli (Fig.
1A) and gram positive B. subtilis (strain
globigii) (Fig. 1B) are unique and easily
distinguishable from each other.
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RESULTS Figures 2 - 3

• The proteomic spectra of the different growth
  stages (log, stationary, decay) of each species
  of bacteria are visibly different.
• Key diagnostic peaks (e.g., Fig. 2 peaks c and
  d Fig. 3 peaks e and f) were found in all
  growth stages.
• These characteristic peaks may be able to be used
  as identifying biomarkers regardless of the
  growth stage

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RESULTS Figure 4

• The MALDI-TOF MS spectra generated by the fresh
  and frozen E. coli indicate that freezing has
  little or no effect on the spectral fingerprints.
  The MS spectra of samples that were stored at
  20oC for 15 days (Fig. 4B) and 15 weeks (Fig.
  4C) are almost identical to samples analyzed
  fresh (Fig. 4A).

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RESULTS Figures 5-6

• B. subtilis (Fig. 5) and E. coli (Fig. 6)
  collected on different days at ? the same optical
  density have very similar proteomic spectra. Each
  culture came from a single colony and was grown
  in LB broth at 37oC shaking at 150 RPM.

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RESULTS Figure 7
Comparison of B. subtilis globigii (ATCC 9372)
grown in liquid cultures or grown overnight on
agar plates. The spectra are very similar.
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RESULTS Figure 8
B. subtilis (globigii) ATCC 9372 mass spectra (A)
compared to B. cereus ATCC 2. The spectra are
distinctly different.
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RESULTS Figure 9
B. subtilis (globigii) ATCC 9372 compared to B.
subtilis ATCC 6051A . Although there are some
similarities, there are enough differences to be
distinctive.
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RESULTS Figure 10
B. subtilis (globigii) ATCC 9372 compared to B.
subtilis ATCC 6051A and B. subtilis ATCC 82.
Although there are some similarities, there are
enough differences to be distinctive.
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RESULTS Figure 11
Bacillus cereus ATCC 11778 compared to Bacillus
cereus ATCC 2. Although there are some
similarities, there are enough differences to be
distinctive.
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RESULTS Figure 12
Bacillus subtilis globigii ATCC 9372 (colony)
compared to Bacillus thuringiensis ATCC 10792
(colony) and Bacillus cereus ATCC 2 (liquid).
Although there are some similarities, there are
enough differences to be distinctive.
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RESULTS Figure 13
Samples stored in ammonium chloride showed
significant decay. E. coli (Fig. 13A) and B.
subtilis (Fig. 13B)
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CONCLUSIONS
The MALDI-TOF MS of whole bacterial cells were
examined under different experimental conditions.
Although the stage of growth (logarithmic,
stationary, or decay) did change the overall
spectral fingerprint, characteristic identifying
mass peaks were still present in all stages.
Freezing cells at 20oC for as long as 15 weeks
did not affect the spectra. Freshly grown
gram-positive bacteria, Bacillus subtilis (strain
globigii), gram-negative bacteria, Escherichia
coli, as well as other bacilli species, and
strains generate unique and reproducible
proteomic MS spectra. Similar spectral
fingerprints were obtained from colonies grown on
solid or in liquid media. MALDI-TOF MS can
analyze femtomole (10-15 M) concentrations of
peptides in very short periods of time (10 min)
and is the ideal instrument to analyze
environmental samples containing very low
concentrations of specific organisms and/or
biomolecules.
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ACKNOWLEDGMENT
Gettysburg College for partially funding the
sabbatical research year of Dr. Koren Holland
Deckman at NIST
E-MAIL ADDRESSES

• kholland_at_gettysburg.edu
• barbara.levin_at_nist.gov