A Sulfoxide-Based Isobaric Labelling Reagent for Accurate Quantitative Mass Spectrometry

Article abstract of DOI:10.1002/anie.201708867

Modern proteomics requires reagents for exact quantification of peptides in complex mixtures. Peptide labelling is most typically achieved with isobaric tags that consist of a balancer and a reporter part that separate in the gas phase. An ingenious distribution of stable isotopes provides multiple reagents with identical molecular weight but a different mass of the reporter groups, allowing relative quantification of multiple samples in one measurement. Here we report a new isobaric labelling reagent, where the balancer and the reporter are linked by a sulfoxide group, which, based on the sulfoxide pyrolysis, leads to easy and asymmetric cleavage at low fragmentation energy. The fragmentation of our new design is significantly improved, yielding more intense complementary ion signals, allowing complementary ion cluster analysis as well.

Full text of DOI:10.1002/anie.201708867

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Accepted Article
Title: A sulfoxide-based isobaric labelling reagent for accurate
quantitative mass spectrometry
Authors: Michael Stadlmeier, Jana Bogena, Miriam Wallner, and
Thomas Carell
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201708867
Angew. Chem. 10.1002/ange.201708867
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A sulfoxide-based isobaric labelling reagent for accurate
quantitative mass spectrometry
Michael Stadlmeier[a],+, Jana Bogena[a],+, Miriam Wallner[a],+, Martin Wühr[b] and Thomas Carell[a],*
Abstract: Modern proteomics requires reagents for exact
quantification of peptides in complex mixtures. Peptide labelling is
most typically achieved with isobaric tags that consist of a balancer
and a reporter part that separate in the gas phase. An ingenious
distribution of stable isotopes provides multiple reagents with identical
molecular weight but a different mass of the reporter groups, allowing
relative quantification of multiple samples in one measurement.
Current generation reagents require a high fragmentation energy for
cleavage, leading to incomplete fragmentation and hence loss of
signal intensity. Here we report a new isobaric labelling reagent,
where the balancer and the reporter are linked by a sulfoxide group,
which, based on the sulfoxide pyrolysis, leads to easy and asymmetric
cleavage at low fragmentation energy. The fragmentation of our new
design is significantly improved, yielding more intense complementary
ion signals, allowing complementary ion cluster analysis as well.
After the development of new genome sequencing methods that allow
human genomics studies in just a few hours,[1] today, we are
witnessing the emergence of novel mass spectrometry methods that
enable the investigation of the complete proteomes of cells and
tissues.[2-4] The proteome is defined as the collection of all proteins
present in a sample and hence proteomes differ dramatically from cell
type to cell type and in different tissues.[5] Proteomics data therefore
provide fingerprint-type information about cellular situations and
potentially existing disease states.[6] To gain deep insight into the
proteome of biological systems, it is necessary to obtain quantitative
information about the levels of the individual proteins in the different
samples. Nowadays, this is performed with mass spectrometry. Since
exact quantification of intact proteins is difficult, the proteomes need
to be digested (trypsinated) to give the corresponding peptides. For
quantification of these peptides, methods such as metabolic
labelling,[7-8] label-free quantification[9] or isobaric labelling[10-11] are
performed. Since isobaric labelling is able to reveal even small
differences in peptide abundances and because many samples can
be compared in one measurement, it is one of the most commonly
used quantification methods.[12] A typical proteomics experiment
illustrating the procedure is shown in Fig. 1A.
[a]
M. Sc. M. Stadlmeier, M. Sc. J. Bogena, M. Sc. M. Wallner, Prof. Dr.
T. Carell
Center for Integrated Protein Science at the Department of
Chemistry, Ludwig-Maximilians-Universität München,
Butenandtstr. 5-13, 81377 Munich, Germany
E-Mail: Thomas.Carell@lmu.de
Figure 1. A) Isobaric labelling experiment for quantitative proteomics. The
samples are individually labelled with isotopologues of the reagents 1 or 2 and
combined for LC-MS² studies. Fragmentation in the gas phase yields reporter
and complementary ions for relative quantification. The reporter ion ratio is
distorted by co-isolated peptides (purple). The complementary ion clusters can
be analysed without such a distortion. B) Currently used TMT (1) reagent.
Fragmentation introduces a negative charge on the balancer, reducing the
overall charge state on the complementary ions. C) SulfOxide Tag (SOT, 2)
reported in this study with a charge-neutral fragmentation that retains all
charges on the complementary ions to facilitate fragmentation.
[b]
[+]
Prof. Dr. Martin Wühr
Department of Molecular Biology & the Lewis-Sigler Institute for
Integrative Genomics, Princeton University, Princeton, NJ 08544,
USA
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document
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The proteomes of two samples are isolated, digested and the peptides
are reacted with an isobaric labelling reagent such as the Tandem
Mass Tag (TMT) reagent 1 (Fig. 1B) or the new labelling reagent 2
(Fig. 1C) reported in this study. The prepared derivatised peptide
mixtures are next combined and the mixture is analysed by HPLC-
MS[13] or even CE-MS.[14-15] During separation, the same peptides
derived from the two samples (blue and red in Fig. 1) will feature the
same retention time due to the isobaric character of the labels. They
will consequently enter the mass spectrometer at the same time,
leading to one indistinguishable m/z-value in the full MS-scan. This
allows performing mass spectrometry-based identification by
selecting a single precursor m/z for fragmentation (MS²). Cleavage of
the isobaric labels provides two now different reporter ions (, Fig 1A),
which allows relative quantification. The sensitivity of the methods
depends on the cleavage efficiency.
To examine the fragmentation properties of SOT 2, we digested a
HEK lysate, containing all translated proteins into the corresponding
peptides following a standard protocol (SI). The obtained complex
peptide mixture (P) was divided into two portions. While one portion
was reacted with reagent 2179 (P-2179) the other was combined with
2180 to give P-2180 (pH = 8.5, 150 mM triethylammonium bicarbonate
buffer, 1.5 mg 2179 or 2180, 60 min). We subsequently quenched
unreacted reagent 2 with hydroxylamine. Next, the labelled mixtures
{P-2179+P-2180} were combined in a 1:1 ratio, and the complex mixture
was desalted and concentrated according to reported procedures.[18]
In addition to a reporter ion, a balancer-peptide conjugate, the
complementary ion, is also generated from each peptide. Because the
attached balancer retains the distinct isotope pattern from the isobaric
labelling reagent, the complementary ions allow quantification as
well.[16] This even has the advantage that co-eluting peptides (purple),
which give reporter ions indistinguishable from the reporter signals of
interest, cannot disturb the signal. This problem, known as ratio
distortion, often hinders accurate quantification based on reporter ion
analysis.[17] Again, sensitivity is determined by cleavage efficiency and
this is a drawback of the contemporary reagents.[16]
Here we report the development of a new SulfOxide Tag reagent 2
(SOT, Scheme 1 A) that fragments more easily, which improves
quantitation. Importantly, the reagent features two basic tert-amino
groups, which are protonated in the gas phase. This avoids formation
of neutral species during reagent cleavage and it increases the charge
density, which facilitates fragmentation. The SOT reagent 2 design
allows the introduction of up to eight heavy stable isotopes into the
structure generating reporters with m/z = 1, while keeping isobaricity.
This design was chosen to enable mass spectrometric quantification
of nine different samples in parallel with one single measurement. The
proposed structures of this higher 9-plex reagents are shown in the SI
(Supporting Fig. 3).
The synthesis of reagent 2 and of two isobaric isotope derivatives (2179
and 2180), which feature different reporter ion molecular weights
(179 Da and 180 Da), is shown in Scheme 1B. To our knowledge, the
masses of the generated reporter ions do not coincide with
immonium- or other frequently observed fragment ions. The synthesis
(Supporting Information) starts with the methylester of the
homocystine dimer 3, which is first converted with dimethylamino
propionic acid into the bis-amide 4. Reduction of the disulfide and
alkylation of the thiol with benzyl bromoacetate furnishes the key
intermediate 5. Cleavage of the benzylester to 6 and reaction of 6 with
1,1-dimethylethylenediamine gives the bisamide 7. Saponification of
the methylester in 7 to 8, oxidation of the sulfide to the sulfoxide 9 and
conversion of the acid provides reagent 2 as the active ester. To
access the isotopologues, we replaced the dimethylamino propionic
acid by the similar compound 10 in which one methyl group carries a
13C atom (SI) in a second synthesis. In a third synthesis, we used the
13C-labelled benzyl bromoacetate 11 (SI). This gave the
corresponding reagents 2179 and 2180 (Scheme 1A) in similar yields.
The synthesis takes roughly one week, and the overall yield is ca.
12%. Storing the reagent in pure form or even in a stock solution
(DMSO) is possible at -20 °C for several weeks.
Scheme 1. A) Depiction of the new reagent 2 and of the isotopologues 2179 and
2180. B) a) 3-(Dimethylamino)propionic acid hydrochloride, NEt3, HOBt, 60 °C,
2 h, 85%; b) Over two steps i) TCEP*HCl, NaHCO3, H2O/DMF (4:1), r.t., 10 min;
ii) Benzyl bromoacetate, r.t., 2 h, 95%; c) 10% Formic acid in MeOH, 100 wt%
Pd black, 40 °C, 2 h, 81%; d) N,N’-dimethylethane-1,2-diamine, DIPEA, PyBOP,
DMF, 40 °C, 1 h, 73%; e) LiOH, MeOH/H2O (2.5:1), r.t., 1 h, quant.; f) pH = 2,
mCPBA, H2O, r.t., 20 min, 75%; g) NHS-TFA, pyridine, DMF, r.t., 2 h, 35%.
179 Da and 180 Da are the molecular weights of the generated reporter ions.
For comparison, we performed the same experiment with the
commercially available isotopically labelled TMT reagents 1 (duplex)
according to manufacturer’s recommendations to obtain the mixture
{P-TMT126 + P-TMT127}. The peptide mixtures {P-2179 + P-2180} and
{P-TMT126 + P-TMT127} were next measured by nanoHPLC-MS2 and
the data were analysed using the MaxQuant software and a software
package developed in-house (Supplementary Figure 1&2).[19] The
obtained data are depicted in Fig. 2 and Fig. 3. As an example, Fig. 2A
shows the cleavage of the SOT reagent 2 after reaction with the
peptide DLPEHAVLK2+ (bearing two labels) in direct comparison to
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the corresponding TMT labelled peptide in the complex mixture. We
measured at a normalized fragmentation energy of 28% HCD (higher-
energy collisional dissociation), which is ideally suited to fragment
peptides for their identification. The SOT reagent 2 clearly generates
higher intensity reporter ions and in addition, we observe more
fragmentation, indicating that the reagent supports peptide
fragmentation. Fig. 2B shows an analysis of all peptides identified in
the SOT- and TMT-labelled sample and here, we see that in most
MS²-spectra, the relative reporter ion intensity for the SOT-sample is
as high as 70-100%, which is unprecedented. This enables easy
relative quantification by determining reporter ion ratios with available
software packages. In comparison, TMT-labelled peptides produce
reporter ions of lower relative intensity at this HCD-energy. Fig. 2C
shows the charge states of the intact labelled peptides before
fragmentation (precursors). In agreement with our design, we see that
the SOT reagent 2 generates labelled peptides with much higher
charge states. Importantly, more than 65% of the labelled peptides
have charge states equal to or above +3. This high charge density
facilitates the subsequent fragmentation, which results in the
formation of more complementary ions.
we saw that 58% of these fragments still contained the intact reagent
2, while 42% have lost the reporter group yielding complementary ion
clusters (Fig. 3A).
Figure 3. A) Ratio of all identified fragment ions containing either the intact label
(reporter + balancer, green) or which show loss of the reporter ions, leading to
the formation of complementary ion clusters (cleaved label, orange). *Because
one intact label produces two cleaved labels in case of SOT (2) only, the amount
of cleaved label containing fragments was divided by two to allow comparison
with the TMT data. B) Statistical analysis of the quantity of labelled fragment
ions per peptide spectral match (PSM). SOT-labelled peptides show a drastic
increase in the number of complementary ions per spectrum compared to TMT-
labelled peptides, resulting in 6–7 complementary ion clusters on average. C)
Statistical analysis of the relative MS²-scan intensities of the labelled fragment
ions per PSM. The median intensity of the complementary ions is elevated for
SOT-labelled peptides when compared to the use of TMT. D) Example MS²-
spectrum of the labelled peptide EILIPVFK4+, depicting the reporter ions (red),
seven complementary ion clusters (orange) and some fragment ions used for
identification (grey).
Figure 2. A) Comparison of fragmentation efficiency between TMT (1) and SOT
(2) on a peptide. At a normalized collision energy of 28% HCD, the SOT-labelled
peptide fragments more readily and yields high reporter ion signals. Tag =
reacted reagent. B) Statistical analysis of the reporter ion relative intensities
observed in the MS²-experiment. For SOT (2), the reporter ion exhibits an
excellent visibility, facilitating reporter ion quantification. In comparison, TMT (1)
produces reporter ions of lower intensity. C) Charge state distribution of
precursor-peptides labelled with the TMT (1) or SOT (2) duplex. By using the
SOT-reagent, higher charge states become more abundant, which should lead
to more efficient fragmentation due to higher charge density.
This balanced ratio allows the parallel identification of the (intact)
peptides with standard database search algorithms and quantification
via the abundantly formed complementary ion clusters. In case of
TMT, the amount of fragmentation is significantly lower and only 15%
of all labelled fragment ions are cleaved. This holds true even when
considering that the TMT-duplex complementary ions are
indistinguishable, because they lose CO (containing the isotope
marker) during fragmentation. Fig. 3B shows that the SOT reagent 2
creates on average 13 peptide-balancer fragments from every
precursor for later quantification, which corresponds to 6–7
complementary ion clusters. For SOT-labelled peptides, there are not
only more complementary ions compared to TMT-labelled peptides,
While the reporter ion intensity allows measuring the relative ratios
between peptides present in P-2180 versus P-2179, it is desirable to
quantify with the complementary ion clusters generated by the
balancer-peptide conjugates.[16] Because these balancer-conjugates
fragment further in the mass spectrometer, a large number of
complementary ion clusters are formed, which can all be used for
quantification. This provides higher data density and it reduces ratio
distortion, because the complementary ions are sequence specific
and are therefore distinguishable, unlike the reporter ions which are
the same for all peptides. In our experiment, we observe that the SOT
reagent 2 has ideal properties for such a complementary ion cluster
analysis. When we studied the label-containing peptide fragment ions,
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but their median relative intensity in the respective MS²-spectra is also
higher (Fig. 3C). An example spectrum is displayed in Fig. 3D. The
signals for the reporter ions (red) and of seven complementary ion
clusters (orange) are clearly visible with high relative intensities. For
identification of the peptide, multiple high intensity ions are available
(grey). Although the number of acquired MS2-scans was comparable
for the SOT- and TMT-samples (42169 for SOT, 46062 for TMT), the
peptide identification rate was lower for SOT-labelled peptides (20%
for SOT, 35% for TMT).
be particularly attractive for a targeted multiplexed approach.[20] The
most important properties of the SOT reagent 2 are that reporter ions
and complementary ion clusters are formed in parallel and that the
introduction of multiple tert-amino groups generates the expected
higher charge states which results in better peptide fragmentation. A
current drawback is that the SOT reagent leads to identification of
fewer peptides. The lower number is certainly caused by the currently
available software, which is not yet optimized for the SOT reagent. In
addition, the reagent increases the charge state, which could be
another limiting factor. Software optimization and synthesis of a
reagent that lacks the tertiary amine, can solve these problems.
Research in this direction is ongoing.[21]
To investigate whether our new reagent reduces the ratio distortion
effect, we performed an experiment in which a 1:1 labelled mixture of
HEK-lysate served as a background. Into this background, a labelled
bovine serum albumin (BSA) digest was added in a 4:1 ratio in a low
quantity (Fig. 4A). This ensures that only a small amount of BSA
peptides gets selected for isolation and fragmentation in a large
background of 1:1 labelled human peptides. This should give a large
ratio distortion. As observed previously in similar datasets with strong
distortion,[16] the normalized median reporter ion ratio for the BSA
peptides is 1.15 in case of the SOT-sample and 1.11 in case of the
TMT-sample, showing the massive distortion of the ratio towards 1:1
(Fig. 4B). We next studied the same SOT-dataset regarding the
complementary ion clusters of the BSA peptides.
Acknowledgements
We thank the Deutsche Forschungsgemeinschaft for financial support
via SPP1784, CA275 and GRK2062. We thank the
Bundesministerium für Bildung und Forschung (BMBF) for additional
support by the Excellence Cluster CiPSM. Further support from the
European Union through the Marie Curie International Training and
Mobility Network “Clickgene” (grant No. 642023) is acknowledged
M.S. thanks the Fonds der Chemischen Industrie for a predoctoral
fellowship.
Keywords: Peptide derivatization reagent, sulfoxide pyrolysis,
isobaric labelling, quantitative mass spectrometry,
complementary ion clusters, proteomics
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Figure 4. A) Experiment to show quantification of a protein with strong ratio
distortion. HEK-lysate was labelled and mixed in a 1 to 1 ratio to serve as a
background. Bovine serum albumin (BSA) was labelled and a 4 to 1 ratio was
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advantage of SOT complementary cluster analysis (SOTc).
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that allows the efficient parallel formation of reporter ions and
complementary ion clusters for peptide quantification. The reagent
helps to reduce quantification errors caused by ratio distortion.
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clusters that results from fragmentations of the tag and the peptide
backbone. These ion-clusters enable multiple quantification events
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interference than when using the intact peptide complimentary ion
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[21] For a sample of the new reagent 2 and the in house software package
used to analyze the data please visit our homepage at
www.carellgroup.de and contact us
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