Application Note # ET-23
Accurate Molecular Formula Determination
and Identification of Molecules with
> 1100 m/z with UHR-TOF
Introduction
Methods
In the course of identification and structural characterization
of compounds, unambiguous sum formula calculation is an
essential step. However, with increasing molecular mass
the numbers of possible sum formulae increase exponentially. With molecules as small as 500 m/z, it is impossible to
get just one sum formula suggestion from an accurate mass
measurement. Even if a mass accuracy of 0.1 ppm is assumed, and application of constraints to exclude chemically
"impossible" molecules are applied, at 1000 m/z at 0.1 ppm
mass accuracy, more than 2200 sum formulae would still be
calculated (>10.000 for mass accuracy 1 ppm). [1]
HPLC: Ultimate 3000 Rapid Separation LC (Dionex).
Column: Zorbax XDB-C8 2.1x50 mm, 1.8 μm (Agilent).
Column Temp: 30 °C.
Mobile Phase: A = H2O, B = MeOH (each 0.1% HCOOH).
Gradient: Linear gradient 85 –99.5% in 3 min, hold 5 min.
Flow rate: 0.2 mL/min.
Injection volume: 1 μL.
Sample: bacterial metabolite, provided by CVUA Stuttgart.
MS: micrOTOF-Q II (ESI-Qq-TOF MS and maXis 4G
(UHR-TOF MS; Bruker). Ionization: ESI(+). Scan range: m/z
50-1300. Scan mode: Full scan, MSMS 1171.
To effectively overcome this issue, the combination of
accurate mass information with isotopic pattern derived
both from full scan and MS/MS spectra, can be used to
significantly extend the m/z range for reliable sum formula
suggestion to values higher than 1000. In this study, the
unique capabilities of the maXis™ 4G UHR-TOF was applied
to a bacterial metabolite sample to accurately identify an
unknown compound with a large m/z value.
A solution containing a bacterial metabolite that gave an MS
signal at about m/z 1171 was analyzed using the conditions
above. An additional run in MS/MS mode was performed,
acquiring full scan MS/MS spectra for a precursor mass of
m/z 1171. Sum formula suggestions were calculated using
DataAnalysis™ 4.0 SR2 (Bruker) applying different settings.
ƒƒ F
Full scan and MSMS spectrum
Intens. 1.
+MS, 3.31-3.64min #(194-214)
5
x10
Intens. 1.
x104
+MS2(1171.0000), 54-66eV, 3.51-3.83min #(205-224)
499.2999
+K - 1.0077
2.5
+Na - 1.0071
1.25
+NH4 - 1.0069
a)
2.0
357.2379
1170.7112
b)
1.00
556.3567
741.4618
1.5
0.75
1175.6665
1.0
0.5
1175.6648
698.4184
0.50
940.5803
0.25
[M+H]+
1153.6838
883.5223
1191.6397
1125.6856
172.1320
0.0
0.00
1155 1160 1165 1170 1175 1180 1185 1190
m/z
200
400
600
800
1000
m/z
Fig. 1: Full scan (a) and MSMS spectrum (b) of the
analyzed metabolite. The adduct pattern observed
in the full scan spectrum allows for a clear assignment of the [M+H]+ signal.
Results
Full scan mass spectra on the micrOTOF-Q II gave an exact
mass value of m/z 1170.7112 for the bacterial metabolite
with a mass accuracy of 5 ppm. Additional MS signals of
lower intensity allowed for the characterization of the observed ion as ammonium (presence of [M+H]+, sodium, and
potassium adducts (Fig. 1a and 1b).
Sum Formula Calculations
b)
c)
a)
Fig. 2: Sum formula calculations for m/z 1170.7112 using a calculation window of 5 ppm and chemical constraints: The number of sum
formula suggestions depends on the predefined element range.
Allowing only C, H, N, O as elements, 30 sum formula
suggestions were calculated using the 5 ppm mass accuracy window (Fig. 2a). If additional elements were allowed,
the number of theoretical suggestions increased rapidly to
several hundred, even if structural constraint rules were
applied. (Fig. 2b, c). The data evaluation software allowed
the rating of results according to the matching of experimental and theoretical isotope patterns. Taking isotope
pattern information into account the number of meaningful
suggestions was reduced to about 12 (Fig 3).
The additional data analysis tool SF3D (Smart Formula
3D™, included in DataAnalysis 4.0 SR2) allowed for the
verification of sum formula suggestions by combining full
scan MS and MS/MS data. This software also takes the
fragments‘ accurate mass and isotope pattern information
into account. Only six sum formulae remained, which could
explain a specified minimum number of fragment signals, of
which only one could be found in online available databases
(Fig. 5).
Analysis using an UHR-TOF-MS maXis 4G system showed
the benefit of enhanced mass accuracy, since this approach
only left a minimum of sum formula (Fig. 4). Thus the bacterial metabolite could putatively be identified as cereulide.
SmartFormula3D
Fig. 4: Principle of SmartFormula3D
and result for maXis data: only two
sum formula candidates remained.
Isotope Pattern Match
Intens.
1.
x105
+MS, 3.31-3.64min #(194-214)
1170.7112
1.5
1171.7142
experimental
1.0
1172.7166
1173.7186
0.5
0.0
x105 1.
2.0
C 57 H 100 N 7 O 18 ,1170.71
1170.7119
1.5
simulated
patterns
1171.7152
1.0
1172.7186
1173.7220
0.5
0.0
x105 1.
1170.7079
2.0
1.5
C 52 H 100 N 9 O 20 ,1170.71
1171.7112
1.0
1172.7145
1173.7179
0.5
0.0
x105 1.
C 68 H 100 N O 15 ,1170.71
1170.7087
2.0
1171.7121
1.5
1.0
1172.7154
0.5
1173.7188
0.0
x105 1.
C 81 H 92 N 3 O 4 ,1170.71
1170.7082
2.0
1171.7116
1.5
1.0
1172.7149
0.5
0.0
1170
1173.7183
1174.7216
1171
Fig. 3: Result ranking for isotope pattern match.
1172
1173
1174
1175 m/z
Online Search Results
result no. 1
Utilizing the combined hardware and software capability of the
maXis 4G and SmartFormula 3D platforms combining accurate
mass measurement, isotope pattern, full scan MS and MS/MS
information and an online search tool allows for sum formula
generation and identification of compounds even at >1000 m/z
values.
Keywords
Instrumentation & Software
Small molecule identification
maXis 4G UHR-TOF
Sum formula generation
micrOTOFQ II
SmartFormula3D
CompoundCrawler
For research use only. Not for use in diagnostic procedures.
References
1. Kind, T., Fiehn, O., Metabolomic database annotations via query of elemental compositions: Mass
accuracy is insufficient even at less than 1 ppm. BMC
Bioinformatics 2006, 7:234
Acknowledgment
Many thanks to Roland Perz (CVUA Stuttgart,
Germany) for providing this sample!
Authors
Wiebke Lohmann; Petra Decker; Aiko Barsch
Bruker Daltonik GmbH, Bremen, Germany
Bruker Daltonik GmbH
Bruker Daltonics Inc.
Bremen · Germany
Phone +49 (0)421-2205-0
Fax +49 (0)421-2205-103
[email protected]
Billerica, MA · USA
Phone +1 (978) 663-3660
Fax +1 (978) 667-5993
[email protected]
www.bruker.com/maXis
Fremont, CA · USA
Phone +1 (510) 683-4300
Fax +1 (510) 490-6586
[email protected]
Bruker Daltonics is continually improving its products and reserves the right
Conclusions
to change specifications without notice. © Bruker Daltonics 04-2011, ET-23 #275620
Fig. 5: Online search result for
the sum formula suggestions
from SmartFormula 3D: only one
compound and structure can be
assigned