V. Chagovets et al., Eur. J. Mass Spectrom. 22, 123–126 (2016)
Received: 30 June 2016 ■ Accepted: 3 August 2016 ■ Publication: 19 August 2016
123
EUROPEAN
JOURNAL
OF
MASS
SPECTROMETRY
Virtual Issue: Papers Presented at the 12th EFTMS Workshop, Matera, Italy
Peculiarities of data interpretation upon
direct tissue analysis by Fourier transform
ion cyclotron resonance mass spectrometry
Vitaliy Chagovets,a Aleksey Kononikhin,a,b Nataliia Starodubtseva,a,b Yury Kostyukevich,b,c,d Igor Popov,b,c
Vladimir Frankevicha* and Eugene Nikolaevb,c,d
a
Department of System Biology in Reproduction, Federal State Budget Institution ‘Research Center for Obstetrics, Gynecology and
Perinatology’, 4 Oparin Street, Moscow 117997, Russian Federation. E-mail: vfrankevich@gmail.com
b
Moscow Institute of Physics and Technology, 141700 Dolgoprudnyi, Moscow Region, Russian Federation
c
Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Leninskii pr., 38, bld. 2 Moscow, 119334, Russian Federation
d
Skolkovo Institute of Science and Technology, 100 Novaya Street, Skolkovo 143025 Russian Federation
The importance of high-resolution mass spectrometry for the correct data interpretation of a direct tissue analysis is demonstrated with
an example of its clinical application for an endometriosis study. Multivariate analysis of the data discovers lipid species differentially
expressed in different tissues under investigation. High-resolution mass spectrometry allows unambiguous separation of peaks with
close masses that correspond to proton and sodium adducts of phosphatidylcholines and to phosphatidylcholines differing in double
bond number.
Keywords: direct tissue analysis, ambient mass spectrometry, Fourier transform ion cyclotron resonance, endometriosis, mass spectrometry
clinical application
Introduction
Investigation of tissue composition is an important stage
in biomarker discovery and high-throughput methods are
required to obtain the molecular signatures.1 Ambient ionization mass spectrometry is a fast growing branch that allows
molecular information to be obtained from tissue samples
with minimal sample pretreatment and a set of methods
has been proposed.2–7 One of the drawbacks of the ambient
analysis is the complexity of the obtained spectra and the
lack of additional degrees of separation. This problem can be
addressed by ion mobility methods. Another way to overcome
biological sample complexity and not to lose important information is to use high-resolution with Fourier transform ion
cyclotron resonance (FT-ICR) or Orbitrap mass spectrometers.
ISSN: 1469-0667
doi: 10.1255/ejms.1425
The present paper demonstrates profits from the combination of a direct tissue spray ion source with the FT-ICR mass
spectrometer with the example of an endometriosis study.
Experimental
Methanol (HPLC grade) and formic acid were purchased from
Sigma-Aldich.
Tissue samples were collected from 30 patients with ovarian
cysts under laparoscopic surgery in the Operative Gynecology
department at the V.I. Kulakov Research Center for Obstetrics,
Gynecology and Perinatology. All the patients included in
© IM Publications LLP 2016
All rights reserved
124
Peculiarities of Data Interpretation upon Direct Tissue Analysis by FT-ICR-MS
Figure 1. Positive-ion direct tissue mass spectra of (a) ovarian cyst endometriosis and (b) the eutopic endometrium. The numbers over
the peaks correspond to items in Table 1. Annotated peaks are those giving the highest impact to tissue classification. – lipid species,
– oxidized lipid species.
the study provided written informed consent. All the procedures and study methods were approved by the Commission
of Biomedical Ethics at V.I. Kulakov Research Center for
Obstetrics, Gynecology and Perinatology.
The experimental setup is described elsewhere.7 In brief, a
slice of tissue was placed on the tip of medical needle in the
front of a LTQ FT Ultra instrument (Thermo Scientific, Bremen,
Germany), which includes a FT-ICR analyzer. Methanol with
0.1% formic acid was supplied to the tissue by a fused silica
capillary. This solution provides the extraction of compounds
from the tissue and is then sprayed on the high voltage application.
Acquired mass spectrometric data were analyzed by a
partial least squares discriminant analysis (PLS-DA) method
realized with the ropls package.8
The lipid nomenclature used throughout the paper is in
accordance with LIPID MAPS9 terminology and the shorthand
notation is summarized in Liebisch et al.10
Results and discussion
All the mass spectra of the tissue samples were acquired
in positive ion mode. The most abundant peaks were in the
range between m/z 700 and m/z 900. Figure 1 shows the mass
spectra characteristics for ovarian cyst endometriosis and
a eutopic endometrium of the same patient. PLS-DA multivariate analysis of the mass spectrometric data reveals that
molecular information obtained from the direct tissue mass
spectra is sufficient for tissues differentiation. The created
model for the ovarian vs endometrium describes 80% of the
data using the latent variables (R2) and 66% are predicted
by the model according to the cross validation, the values
showing an accuracy which can be expected to predict new
data (Q2). Variable importance in the projection (VIP) values
are obtained from PLS-DA models and used to determine
compounds with the highest impact to the latent variables
(Table 1). According to the accurate mass, most of the selected
species are phosphatidylcholines (PC). PCs are registered
in mass spectrometry as protonated molecules and adducts
with alkali metal ions such as sodium and potassium. Even
trace amounts of alkali metal ions can cause comparable
intensities of sodiated and protonated molecules. An interference between peaks of sodiated and protonated PCs is
observed for PCs with two aliphatic chains with a general
formulae PC m:n and PC (m + 2):(n + 3), where m is the total
carbon number of fatty acyls and n is the double-bond number.
Such an overlapping of peaks hampers the quantitative and
V. Chagovets et al., Eur. J. Mass Spectrom. 22, 123–126 (2016)
125
Table 1. List of masses with the highest influence on tissue differentiation according to PLS-DA analysis.
#
Accurate mass
Theoretical mass
Mass accuracy
(ppm)
[Lipid + Na]+
Elemental
composition
1
798.5441
798.5619
22
Ox PC 34:1
C 42H82NO9P
2
832.5843
832.5827
2
PC 38:4
C 46H84NO8P
3
796.5257
796.5463
26
Ox PC 34:2
C 42H80NO9P
4
782.5702
782.5670
4
PC 34:1
C 42H82NO8P
5
725.5600
725.5568
4
SM 34:1
C 39H79N2O6P
6
822.5428
822.5619
23
Ox PC 36:3
C 44H82NO9P
7
824.5575
824.5776
24
Ox PC 36:2
C 44H84NO9P
8
806.5679
806.5670
1
PC 36:3
C 44H82NO8P
9
780.5540
780.5514
3
PC 34:2
C 42H80NO8P
10
820.5287
820.5463
21
Ox PC 36:4
C 44H80NO9P
11
808.5850
808.5827
3
PC 36:2
C 44H84NO8P
12
834.5971
834.5983
1
PC 38:3
C 46H86NO8P
13
848.5570
848.5776
24
Ox PC 38:4
C 46H84NO9P
14
772.5276
772.5463
24
Ox PC 32:0
C 40H80NO9P
ppm, parts per million.
qualitative estimation of the lipid composition. The fastest and
most reliable method of interference peaks deconvolution is to
resolve the peaks at the instrumentation level. The difference
between sodiated and protonated molecules, e.g. for [PC
36:2 + Na]+ and [PC 38:5 + H]+, is 808.5851 – 808.5827 = 0.0024,
which requires a resolution of over 3.4 × 105 to separate them.
FT-ICR manifests two such cases, shown in Figure 2(a) and
(b). Resolution of the presented peaks is up to 8 × 105, which
allows the unambiguous identification of the fourth and 11th
items in Table 1 as sodium adducts of PC 34:1 and PC 36:2.
Another issue for lipid identification is the interference of
the isotopic peaks of compounds with a difference of one
double bond. In this case, a resolution of about 105 is sufficient. [PC 38:3 + Na]+ has m/z 834.5983, the third isotope of
[PC 38:4 + Na]+ has a mass 834.5891 and their difference is
834.5983 – 834.5891 = 0.0092. Resolution of these peaks is
shown in Figure 2(c), which gives the 12th item in Table 1 as
[PC 38:3 + Na]+.
Of note are the groups of peaks marked with the empty
circles which also differentiate endometrioid tissues (Figure
1). Their patterns are similar to those of closed circles: 3,1
is similar to 9,4; 6,7 to 8,11; and 13,12. The mass difference
between these groups is 16 Da. One can speculate that the
open-circle peaks correspond to oxidized products of the
respective lipids. Such a possibility has been observed previously with a different ambient ionization method.11 However,
this version needs further elaboration because the mass
accuracy for these peaks, on the assumption of oxygen attachment to the lipids, is low (Table 1).
Conclusion
An FT-ICR mass analyzer is essential for direct tissue analysis
in order to identify lipid constituents correctly and to avoid some
problems connected with features in the mass spectrometric
Figure 2. Regions of a high resolution mass spectrum comprising peaks with near-lying m/z.
126
Peculiarities of Data Interpretation upon Direct Tissue Analysis by FT-ICR-MS
investigation of lipids. The interference of protonated and
sodiated lipid species and of lipids with different double-bond
numbers are among these features.
Acknowledgments
This work was supported by Russian Science Foundation Grant
No. 16-14-00029, and N.S. acknowledges MERF Grant No.
MK-8484.2016.7 for partial support for the sample collection.
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