Mass spectrometry provides accurate
characterization of two genetic marker
types in Bacillus anthracis
Matthew N. Van Ert1, Steven A. Hofstadler2, Yun Jiang2, Joseph D. Busch1,
David M. Wagner1, Jared J. Drader2, David J. Ecker2, James C. Hannis2, Lynn Y. Huynh1,
James M. Schupp1, Tatum S. Simonson1, and Paul Keim1,3
BioTechniques 37:642-651 (October 2004)
Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable num-
ber tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform. Here
we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to
characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis. The results obtained with ESI-FTICR-
MS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent
products. However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR
products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and
VNTR PCR products. In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approxi-
mately 1 sample per min. This technology represents a significant advance in our ability to rapidly characterize B. anthracis isolates
using VNTR and SNP loci.
INTRODUCTION diversity (8,9). SNPs, discovered by se- a methodology that minimizes post-
quencing the entire protective antigen PCR processing and evaluates SNP and
Current interest in Bacillus anthra- gene (pagA) from diverse B. anthra- VNTR loci on a single platform.
cis, the etiological agent of the disease cis isolates (3), have also been used to Mass spectrometry is rapidly emerg-
anthrax, stems from its potential use as determine phylogenetic relationships ing as a sensitive and accurate method
a bioterrorism or biowarfare agent. The within this pathogen. Appropriately, for characterizing PCR products. While
use of B. anthracis spores in the mail- multiple locus VNTR analysis (MLVA) PCR products have been analyzed on
related bioterrorism events of 2001 em- and pagA SNP typing played an impor- several different mass spectrometry
phasized the need for rapid, high-reso- tant role in differentiating and identify- platforms including quadrupole (11–
lution molecular typing methods for ing B. anthracis strains following the 14), time-of-flight (TOF; Reference
this pathogen. Until recently, however, 2001 bioterrorism events (10). Unfor- 15), and quadrupole ion traps (16,17),
development of these methods has been tunately, current methods for generat- no other platform can simultaneously
hampered by the highly monomorphic ing data for SNP and VNTR markers provide the resolution and mass accu-
nature of the B. anthracis genome (1). require two different reagent systems, racy obtainable on the Fourier trans-
Variable number tandem repeats electrophoresis-based detection, and, in form ion cyclotron resonance (FTICR)
(VNTRs) and single nucleotide poly- the case of SNPs, extensive post-PCR platform. Electrospray ionization Fou-
morphisms (SNPs) represent sources of processing that is not only cumbersome rier transform ion cyclotron resonance
variation in the genome and are recog- but also introduces a potential source of mass spectrometry (ESI-FTICR-MS)
nized as powerful tools for examining laboratory contamination. Furthermore, has been used to detect double-strand-
genetic relationships within B. anthra- performing separate assays to collect ed PCR products at the attomolar level
cis (2–7). VNTRs have been used suc- data on SNP and VNTR loci can con- (18), genotype simple and compound
cessfully to discriminate among closely tribute to difficulties in sample and data short tandem repeat sequences (18–20),
related strains of B. anthracis and have tracking that can ultimately impact data characterize PCR products generated
facilitated detailed epidemiological quality. Rapid genotyping of B. anthra- from amplification of 16S to 23S rDNA
analyses of local patterns of anthrax cis would, therefore, be facilitated by intergenic spacer regions (ISRs) from
1Northern Arizona University, Flagstaff, AZ, 2Isis Pharmaceuticals, Inc., Carlsbad, CA, and 3TGen, Phoenix, AZ, USA
642 BioTechniques Vol. 37, No. 4 (2004)
Table 1. PCR Product Size, Position, and States for Seven pagA SNPs
Base Compositions as
SNP pagA Nucleotide Frequency in 23 Determined by ESI-FTICR-MS
Name Product Size Positiona SNP State Isolatesb [SNP State (A:G:C:T)]c
PAGA01 77 1998 T↔C 16↔7 T (17:13:14:33)
PAGA02 84 2883 A↔G 0↔23f G (25:21:14:24)
PAGA03d 65 3481 C↔T 0↔23f TT (29:12:9:15)d
PAGA04d 65 3496 TC (29:12:10:14)d
PAGA05e 69 3602 T↔C 14↔9 TT (33:10:8:18)e
PAGA06e 69 3606 C↔T 1↔22 CC (33:10:10:16)e
PAGA07 63 3672 G↔A 0↔23f A (29:9:4:21)
SNP, single nucleotide polymorphism; ESI-FTICR-MS, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.
aAs reported previously by Price et al. (3), nucleotide positions are based on the 4235-bp pXO1 sequence containing pag in its entirety (GenBank® accession no.
bDNA from one isolate (pXO1 negative) in this study did not support amplification of pagA loci.
cThe reported base compositions of the products are the only ones consistent with the measured molecular masses and the mass measurement uncertainties.
dPAGA03 and PAGA04 are separated by 15 nucleotide positions in a single PCR product. Base changes and product base compositions are reported sequen-
tially for each SNP.
ePAGA05 and PAGA06 are separated by 4 nucleotide positions in a single PCR product. Base changes and product base compositions are reported sequentially
for each SNP.
fPAGA02 mutation has only been observed in one South African Bacillus anthracis isolate. PAGA03 and PAGA07 mutations are observed only in Sverdlovsk
clinical samples (3).
Bacillus cereus strains (21), and de- described heat lysis method (6). Brief- than 100 bp (Table 1). Similarly, only
tect heterogeneity in 16S to 23S rDNA ly, a 1.0-μL inoculating loop was used VNTR products between 100–200 bp
ISRs within the genome of a single B. to transfer a portion of a B. anthracis were examined in this study (Table 2).
cereus strain (21,22). colony into 200 μL of TE [Tris-HCl,
As first demonstrated by the groups pH 8.0, 1.0 mM ethylenediamine tetra- pagA SNP PCR
of Aaserud et al. (23) and Muddiman et acetate (EDTA)]. The colony material
al. (24), accurate mass measurements was dispersed by vortex mixing and The PCR conditions for the pagA
obtained by high-performance mass was incubated at 95°C for 20 min. Fol- SNP PCR products (Table 1) were as
spectrometry can be used to unambigu- lowing heat lysis, cellular debris was follows: initial denaturation at 95°C for
ously derive base compositions from removed by centrifugal filtration using 5 min, followed by 34 cycles of 95°C
double-stranded DNA constructs using an Ultra-Free®-MC GV .22 μm cen- for 20 s, 60°C for 30 s, and 72°C for
the mathematical constraints imposed trifugal filter unit (Millipore, Billerica, 30 s. Each 20-μL reaction contained 1
by the complementary nature of the MA, USA) at 5000× g for 5 min. The μL of template DNA, 1× PCR buffer,
two strands. More recently, Jiang and filtrate was diluted 1:10 and was used 3.0 mM MgCl2, 0.1 mM dNTPs, 0.05
Hofstadler (25) demonstrated that high- as a template to support subsequent U Platinum® Taq DNA polymerase
resolution ESI-FTICR-MS measure- PCR for VNTR and SNP analyses. (Invitrogen, Carlsbad, CA, USA), and
ments could be used to unambiguously 0.2 μM of both the forward and reverse
calculate the base composition of 120 ESI-FTICR-MS PCR Product primers. Following a 2-fold dilution of
bp PCR products in a high-throughput Design products in molecular-grade water, 20
modality. Here we demonstrate the po- μL were removed for ESI-FTICR-MS
tential of ESI-FTICR-MS for rapid ge- PCR product characterization via analysis, and the remaining 20 μL were
notyping of B. anthracis strains using ESI-FTICR-MS is most effective for used for downstream single-base exten-
VNTR and SNP loci and compare this products with fragment sizes less than sion analysis.
technology to current methodologies. 200 bp because of the increased diffi-
culty in generating single-stranded spe- Single-Base Extension and Genotype
cies in the gas phase for larger products Analyses
MATERIALS AND METHODS and the inherent difficulty in obtaining
isotopic resolution for higher molecu- Single-base extension analysis was
DNA Isolation lar weight species. Thus, to simplify performed according to the ABI Prism®
analysis of VNTR and SNP products in SNaPshot™ protocol (Applied Biosys-
DNA was obtained from 24 diverse the same mass spectrometry run, SNP tems, Foster City, CA, USA). Briefly,
B. anthracis isolates using a previously PCR products were designed at less the single nucleotide primer extension
Vol. 37, No. 4 (2004) BioTechniques 643
Table 2. VNTR Loci Information and Size Data for Observed Alleles as Detected by Gel Electrophoresis and ESI-FTICR-MS
Allele Size Ranges Observed Allele Sizes and Base
as Determined by Compositions as Determined by
VNTR Locus Repeat Size Motif Gel Electrophoresis ESI-FTICR-MS
(bp) (bp) [bp (A:G:C:T)]a
BaVNTR12 2 AT 112.44–112.55 113 (39:20:19:35)
114.45–114.57 115 (40:20:19:36)
pXO2 2 AT 134.96 135 (38:22:26:49)
136.92–137.03 137 (39:22:26:50)
138.98–139.04 139 (40:22:26:51)
140.98–141.15 141 (41:22:26:52)
143.06 143 (42:22:26:53)
155.14 155 (48:22:26:59)
pXO1 3 AAT 119.33 120 (51:14:15:40)
122.43 123 (53:14:15:41)
125.42 126 (55:14:15:42)
128.48–128.57 129 (57:14:15:43)
131.52–131.54 132 (59:14:15:44)
134.53–134.64 135 (61:14:15:45)
143.61 144 (67:14:15:48)
CG3 5 TAATA 152.92–152.93 153 (61:14:23:55)
157.87–158.05 158 (64:14:23:57)
BaVNTR35 6 TGATTG 103.03 103 (28:13:19:43)
109.13–109.26 109 (29:15:19:46)
115.43–115.5 115 (30:17:19:49)
121.78 121 (31:19:19:52)
vrrB2 9 CAACAATAT 151.4–151.62 153 (54:21:51:27)
159.50–159.67 162 (59:21:53:29)
168.49–168.63 171 (64:21:55:31)
VNTR, variable number tandem repeats; ESI-FTICR-MS, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.
aThe reported base compositions of the VNTR products are the only ones consistent with the measured molecular masses and the mass measurement
method involves the use of an unlabeled DNA polymerase, and 0.2 μM of both Prism 377 Automated Fluorescent DNA
oligonucleotide primer that is designed the forward and reverse primers. The Sequencer. GeneScan® software (Ap-
to anneal directly adjacent to the nu- PCR thermal cycling parameters for plied Biosystems) was used to analyze
cleotide base of interest. An extension VNTR loci pXO1, pXO2, vrrB2, and the gel images and assign sizes to the
reaction is then performed in the pres- CG3 were identical. Reactions were alleles. Custom macros (available upon
ence of four possible dye-labeled ter- incubated for 5 min at 94°C, followed request from the corresponding author,
minators, resulting in the incorporation by 34 cycles of 94°C for 20 s, 60°C for Paul Keim) created in Genotyper® soft-
of a single fluorophore-labeled ddNTP. 20 s, and 65°C for 20 s. The final step ware (Applied Biosystems), which use
We detected the labeled primer exten- was 65°C for 5 min. PCR thermal cy- fixed bin size assignments, allowed for
sion products by electrophoresis on an cling parameters for BaVNTR12 and the automated scoring of alleles.
ABI Prism 3100 Genetic Analyzer (Ap- BaVNTR35 involved an incubation for
plied Biosystems), and genotypes were 5 min at 94°C; 35 cycles of 94°C for 30 Electrospray Ionization Fourier
manually assigned via differential base s, 53.9°C for 30 s, and 72°C for 45 s; Transform Ion Cyclotron Resonance
incorporation at the mutation site. with a final step of 72°C for 5 min. Mass Spectrometry
VNTR PCR VNTR Electrophoresis and Prior to mass spectrometric analysis,
Genotype Analysis PCR products were purified and desalt-
VNTR loci yielding products short- ed using a 96-well automated purifica-
er than 200 bp in size (Table 2) were The VNTR PCR products obtained tion protocol as previously described
amplified using PCR. Each 20-μL re- from each B. anthracis isolate were (25). Briefly, products from single 20-
action contained 1.0 μL of template pooled equally and diluted 1:6 in mo- μL aliquots of crude PCRs were bound
DNA, 1× PCR buffer, 2.0 mM MgCl2, lecular-grade water. Products were elec- to weak anion exchange ZipTips® (Mil-
0.2 mM dNTPs, 0.04 U Platinum Taq trophoretically analyzed using an ABI lipore, Bedford, MA, USA) and rinsed
644 BioTechniques Vol. 37, No. 4 (2004)
repeatedly with 40 mM NH4HCO3, a CTC HTS PAL autosampler (LEAP mode whereby ions were accumulated
followed by multiple rinses with a so- Technologies, Carrboro, NC, USA), in the hexapole ion reservoir simultane-
lution containing 20% MeOH. Elution which was triggered by the FTICR data ously with ion detection in the trapped
of the final purified/desalted PCR prod- station. Samples were injected with an ion cell. Following a 1.2-ms transfer
ucts was accomplished with a 10-μL integrated fluidics handling system, event, during which ions were trans-
aliquot of 1 M NH4OH, with each elu- which allowed for differential control ferred to the trapped ion cell, ions were
ent directly dispensed into a well of a of flow rate for fast rinsing between subjected to a 1.6-ms chirp excitation
96-well plate. Prior to analysis by ESI- sample injections (26). During analy- corresponding to 500–8000 m/z (mass/
MS, the eluent was diluted 1:1 with a sis, PCR products were introduced charge ratio). Data were acquired over
solution containing 50% MeOH and 50 into the mass spectrometer at a flow an m/z range of 500–5000 (1 M data
mM piperidine/imidizole. A small oli- rate of 1.25 μL/min. A source poten- points over a 225-kHz bandwidth).
gonucleotide (SH2; 5′-CGTGCATG- tial of -6 kV was applied to produce a
GCGG-3′) was added into the solution stable ion current. Countercurrent dry-
as an internal mass standard at a final ing gas was heated at 130°C to assist RESULTS
concentration of 50 nM. desolvation. Ions were accumulated in
A modified Apex II 70e (Bruker an external ion reservoir comprising an ESI-FTICR-MS analysis correctly
Daltonics, Billerica, MA, USA) ac- rf-only hexapole, a skimmer cone, and identified all 24 VNTR alleles (Table 2)
tively shielded FTICR-MS was utilized an auxiliary electrode for 2.3 s prior and all 11 pagA SNP alleles (Table 1),
in negative ionization mode. Desalted to transfer into the trapped ion cell for which were also independently deter-
sample aliquots were injected directly mass analysis. Spectral acquisition was mined by electrophoretic-based analyses.
from sealed 96-well microplates using performed in the continuous duty cycle Figure 1A includes an electrophoretic gel
Figure 1. Electrophoretic and ESI-FTICR-MS analyses of three alleles in the pXO2 VNTR locus. (A) Electrophoretic gel image illustrating size data for
6 VNTR loci across 24 diverse isolates of Bacillus anthracis. The gel lanes and isolate identifications are sequential from left to right, and isolates/lanes 1, 5,
10, 15, and 20 are labeled for reference purposes. Each gel lane contains all VNTR products for a given isolate. The box and zoom highlight 3 different alleles
for the pXO2 locus, which differ by 2 bp. (B) Deconvoluted monoisotopic molecular weights and corresponding base compositions for the same 3 alleles
obtained using ESI-FTICR-MS analysis. Note that alleles differ only in the AT repeat. The products are monoadenylated (+A) due to the terminal transferase
activity of the Taq DNA polymerase. Additional peaks are present in the spectra, representing the nonadenylated and mono-adenylated complementary strand
of the product. The base compositions of the VNTR products given in the figure are the only ones consistent with the measured molecular masses and the mass
measurement uncertainties. VNTR, variable number tandem repeats; ESI-FTICR-MS, electrospray ionization Fourier transform ion cyclotron resonance mass
spectrometry; MW, molecular weight.
646 BioTechniques Vol. 37, No. 4 (2004)
image with size data for the 6 VNTR loci DNA from one isolate did not support nonadenylated forward-strand prod-
examined across 24 B. anthracis isolates. amplification of any of the pagA SNP uct in Figure 2B (molecular weight =
On the gel image, adjacent VNTR loci loci or the VNTR pXO1 locus (Figure 23617.875 Da) within a 1 ppm mass
are labeled with different fluorophores, 1A, isolate #3), indicating that the iso- measurement uncertainty (±0.002 Da)
which allowed for separation among sim- late was pXO1 negative. In total, the and 20 base compositions consistent
ilar-sized products from different loci. In pagA SNPs resolved the 23 pXO1 posi- with the nonadenylated reverse-strand
total, the 24 unique VNTR alleles sizes, tive B. anthracis isolates into 5 unique product (23802.066 ± 0.002 Da). Taking
as measured by GeneScan, ranged from genotypes (data not shown). into account that the base compositions
103–171 bp in length (Table 2). The red The mass measurement accuracy of of the two strands are complementary,
box and zoom in Figure 1A indicate size the 7 Tesla FTICR instrument (Apex the list of putative base compositions
data for 3 alleles from the pXO2 VNTR 70e ESI-FTICR mass spectrometer; can be culled, leaving only possibili-
locus. Figure 1B gives an example of the Bruker Daltonics) is generally better ties in which the base composition of
corresponding data output from ESI-FTI- than 1.5 ppm when an internal mass the forward strand is complementary
CR-MS analysis of these same 3 alleles, standard is used to post-calibrate each to that of the reverse strand. The use
including size data and base pair com- spectrum. The monoisotopic molecular of Taq DNA polymerase somewhat
position. It is important to note that al- weights for each strand are derived by complicates this situation because the
though the products illustrated in Figure an “averagine-like” fitting routine (27) individual strands are adenylated and
1B are labeled with a fluorescent moiety that fits the observed isotope envelope are therefore, strictly speaking, not
to accommodate fluorescent detection on to the distribution expected for a DNA complementary. This situation is rem-
an electrophoretic platform, ESI-FTICR- molecule of the approximate measured edied by driving the mono-adenylation
MS does not require labeled products. mass. For PCR products in this size to completion, adjusting the measured
The deconvoluted monoisotopic mo- range, an accurate molecular weight molecular weights by the mass of an
lecular weights and corresponding base determination of an individual strand adenosine (313.0576 Da), and using
pair compositions (Figure 2B) accurately is insufficient for base composition de- the adjusted masses to calculate the
reflect the two base pair size differences termination. For example, there are 41 base composition.
among these three alleles and the known base compositions consistent with the While Muddiman and coworkers
repeat structure of this locus
(Table 2, ±AT). For all VNTR
alleles, the sequence composi-
tions determined by ESI-FTI-
CR-MS were consistent with
the size differences measured
by GeneScan and predicted
by the base composition of the
repeat structures of the loci
(Table 2). Collectively, the 6
VNTR loci resolved the 24
B. anthracis isolates into 23
unique genotypes (data not
Figure 2 compares data
output from the ABI Prism
SNaPshot assay (Figure 2A)
and ESI-FTICR-MS analy-
sis (Figure 2B) of 2 alleles in
the pagA01 SNP locus. Both
techniques provided unam-
biguous scoring of the two-
allele states, however, unlike
the SNaPshot assay, ESI-
FTICR-MS also provides
very precise measurements
of the molecular mass of the
different products. For all 11 Figure 2. Electrophoretic and ESI-FTICR-MS analyses of two alleles in the PAGA01 SNP locus. (A) Electro-
SNP alleles, the base compo- pherograms indicating results of single-base extension analysis of both alleles (C, T) of the Bacillus anthracis pagA01
mass spectra and derived base
sitions determined by ESI- locus. (B) DeconvolutedTESI-FTICRthe pagA01 locus. corresponding monoisotopic molecular weights and in the figure
compositions of C and alleles of The base compositions of the SNP products given
FTICR-MS analysis were are the only ones consistent with the measured molecular masses and the mass measurement uncertainties. ESI-FTICR-
consistent with SNaPshot MS, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry; SNP, single nucleotide poly-
single-base extension results. morphism.
648 BioTechniques Vol. 37, No. 4 (2004)
(28) have shown several methods by product, ESI-FTICR-MS could accu-
which the nontemplated adenylation rately and reliably detect these sequence
can be eliminated using alternative en- variants. Thus, ESI-FTICR-MS has the
zymes such as Pfu and kodakaraensis potential to characterize both VNTR and
(KOD), we chose to use products gen- SNP loci more accurately than tradition-
erated under identical conditions (in- al electrophoretic-based methods.
cluding fluorescently labeled primers) In addition to potential increased
to allow a direct comparison of the two accuracy, ESI-FTICR-MS provides
detection approaches. Consequently, several other advantages over tradi-
the base compositions of the deconvo- tional VNTR and SNP analysis plat-
luted spectra shown in Figure 2B are forms. (i) ESI-FTICR-MS does not
annotated with “+A” to be consistent require product labeling, so there is no
with the measured molecular weights. need for expensive fluorescently la-
This precision allowed the unambigu- beled amplification primers for VNTR
ous determination of the single change analysis. (ii) Other than a simple de-
in base pair composition. ESI-FTICR- salting step, there is no post-PCR pro-
MS also characterized the products on cessing of the reaction, which is re-
both the forward and reverse strands, quired by single-base extension assays
which provided two measurements for for SNP analysis. (iii) Analysis is rap-
each SNP allele (Figure 2B). id, and complete analysis of 96 sam-
ples requires approximately 96 min.
(iv) Although ESI-FTICR-MS is cur-
DISCUSSION rently only applicable to products less
than 200 bp in size, multiple SNP and
Our results indicate that ESI-FTICR- VNTR loci can be analyzed simultane-
MS is just as accurate as traditional, ously, which promises to significantly
electrophoretic-based platforms in iden- increase the throughput of these analy-
tifying alleles at VNTR and SNP loci in ses. Preliminary studies in our labora-
B. anthracis. Furthermore, ESI-FTICR- tory suggest that similar analyses can
MS provides complete base composi- be performed on a low-cost bench-top
tion of the VNTR and SNP products. ESI-TOF mass spectrometer; while
This additional information will allow not offering the same mass resolu-
researchers to overcome several prob- tion as the FTICR platform, the mass
lems with the current methodologies. accuracy of the ESI-TOF instrument
Although not illustrated in this study, we (approximately 10 ppm) is sufficient
have observed that, in rare cases, some to provide unambiguous base compo-
VNTR loci may have multiple repeat sitions for products less than 200 bp.
motifs. If one of the repeat motifs is a Broad deployment of this genotyping
multiple of the other motif (e.g., 2 and 4 protocol on an ESI-TOF-based sys-
bp), they can produce alleles with iden- tem is an attractive alternative to ESI-
tical sizes but different sequence com- FTICR because of the significantly
positions. These differences are indistin- smaller footprint and cost of the TOF
guishable using electrophoretic analyses instruments. Further validation studies
and would be scored as the same allele. are presently under way, which will be
Through accurate base composition reported in detail elsewhere.
analysis, ESI-FTICR-MS could render In conclusion, we have demonstrat-
these products distinguishable from one ed that ESI-FTICR-MS is a potentially
another, thereby increasing genetic reso- powerful method for high-throughput
lution. While analyzing large, diverse genotyping of B. anthracis. The use
strain collections of B. anthracis and ge- of ESI-FTICR-MS to simultaneously
netic near-neighbors, we have observed examine multiple genetic marker types
that products may contain additional represents an attractive alternative to
unexpected SNPs in close proximity traditional methods in terms of cost-ef-
to the target SNP. In these situations, fectiveness, efficacy, throughput, and
single-base extension assays will not information content. This technology
detect these additional sequence vari- will significantly advance the ability
ants, which may interfere with the assay to accurately and rapidly identify and
and lead to errors. However, because it characterize isolates of B. anthracis
interrogates the entire sequence of the and other pathogens.
650 BioTechniques Vol. 37, No. 4 (2004)
ACKNOWLEDGEMENTS L.W. Mayer, and T. Popovic. 2002. Molecular ESI-FTICR mass spectrometry. Anal. Chem.
subtyping of Bacillus anthracis and the 2001 70:1203-1207.
bioterrorism-associated anthrax outbreak. 22.Muddiman, D.C., D.S. Wunschel, C. Liu, L.
We would like to thank Jeff Henrik- Emerg. Infect. Dis. 8:1111-1116. Pasa-Tolic, K.F. Fox, A. Fox, G.A. Anderson,
son, Chris Allender, and Ryan Easter- 11.Krahmer, M.T., Y.A. Johnson, J.J. Walters, and R.D. Smith. 1996. Characterization of
day for their technical assistance. This K.F. Fox, A. Fox, and M. Nagpal. 1999. Elec- PCR products from Bacilli using electrospray
work was supported by funding from trospray quadrupole mass spectrometry analy- ionization FTICR mass spectrometry. Anal.
the U.S. Department of Energy, Chemi- sis of model oligonucleotides and polymerase Chem. 68:3705-3712.
chain reaction products: determination of base 23.Aaserud, D.J., N.L. Kelleher, D.P. Little, and
cal, and Biological Nonproliferation substitutions, nucleotide additions/deletions, F.W. McLafferty. 1996. Accurate base com-
Program and by the Federal Bureau of and chemical modifications. Anal. Chem. position of double-strand DNA by mass spec-
Investigation. 71:2893-2900. trometry. J. Am. Soc. Mass Spectrom. 7:1266-
12.Johnson, Y.A., M. Nagpal, M.T. Krahmer, 1269.
K.F. Fox, and A. Fox. 2000. Precise molecular 24.Muddiman, D.C., G.A. Anderson, S.A. Hof-
weight determination of PCR products of the stadler, and R.D. Smith. 1997. Length and
rRNA intergenic spacer region using electro- base composition of PCR-amplified nucleic
STATEMENT spray quadrupole mass spectrometry for dif- acids using mass measurements from elec-
ferentiation of B. subtilis and B. atrophaeus, trospray ionization mass spectrometry. Anal.
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Vol. 37, No. 4 (2004) BioTechniques 651