Increasing the Multiplexing of Protein
Quantitation from 6- to 10-Plex with
Reporter Ion Isotopologues
Rosa Viner,1 Ryan Bomgarden, 2 Michael Blank,1 John Rogers2
1
Thermo Fisher Scientific, San Jose, CA, USA; 2Thermo Fisher Scientific, Rockford, IL, USA
Overview
Purpose: To develop Tandem Mass Tag™ (TMT™) 10-plex reagents by combining
current Thermo Scientific™ TMTsixplex™ reagents with four TMT6 reagent isotope
variants on multiple high-resolution mass spectrometry platforms.
Methods: HeLa cell lysates and bovine serum albumin (BSA) digests were labeled
with Thermo Scientific™ TMT10plex™ reagents. Aliquots from six, eight or all ten
channels were mixed in different ratios and analyzed on three different Thermo
Scientific™ Orbitrap™-based instruments.
Results: We have extended the multiplexing capabilities of TMT reagents from 6 to 10
without increasing the size or structure of the tag by utilizing the 6 mDa mass
difference between 13C and 15N isotopes of the mass tag reporter ions. Mass
spectrometry analysis on three different Orbitrap-based instruments using FT
MS2/MS3 shows no negative effect on the number of protein identifications or quality
of quantification in a complex digest by increasing sample multiplexing from 6 to 10.
Results
Comparison of unlabeled to TMT6-,
We have extended the previously repo
for multiplex peptide quantitation witho
the tag (Figure 1). The TMT10plex rea
between 13C and 15N isotopes which a
order to accurately quantify all ten cha
resolved. We determined the minimum
MSn resolution to be 35K at m/z 200 (
resolution, an accurate ratio calculatio
a mass tolerance window of up to 10 p
FIGURE 1. Thermo Scientific TMT10
mass for TMT6plex tags (blue), TMT
TMT10-12
Introduction
Amine-reactive isobaric tags (TMT or iTRAQ®) enable concurrent identification and
multiplexed quantification of proteins in different samples using tandem mass
spectrometry. Increasing the number of isobaric tag-labeled samples that can be
compared in a single experiment is highly desirable. However, it has been reported
that increasing multiplexing by using larger tagging molecules significantly decreases
the number of identified and quantified proteins and peptides.1 Here, we demonstrate
that 10-plex multiplexing can be achieved with high resolution mass spectrometry
utilizing the 6 mDa mass difference between 15N and 13C stable isotopes by combining
current TMTsixplex reagents with four TMT6 isotope variants without loss of protein
identifications and quality of quantification in a complex digest.
Methods
TMT10-127N
127.1247610
TMT10-128N
128.1281158
TMT10-129N
129.1314706
TMT10-130N
130.1348254
Sample Preparation
HeLa cell lysates (Thermo Scientific, Rockford, IL) and bovine serum albumin (BSA,
Sigma, St.Louis, MO) were reduced, alkylated and digested with LysC and/or trypsin.
Samples were then labeled with TMT10plex reagents (Figure 1) according to
manufacturer’s instructions. Aliquots from six, eight or all ten channels were mixed in
equimolar ratios. To assess the impact of peptide co-isolation on quantification, HeLa
digest labeled with Thermo Scientific™ TMT8plex™ reagents was spiked with BSA
digest labeled with TMT10plex reagents mixed at fixed ratios (16:8:4:2:1:1:2:4:8:16).
TMT10-131
131.1381802
FIGURE 2. Optimal resolution settin
LC-MS
A Thermo Scientific™ EASY-nLC 1000™ HPLC system and Thermo Scientific EASYSpray™ source with Thermo Scientific™ Acclaim™ PepMap™100 2 cm x 75 μm trap
column and Thermo Scientific™ EASY-Spray™ PepMap™ RSLC C18 25 or 50 cm x
75 μm ID column were used to separate peptides with a 5-25% acetonitrile gradient in
0.1% formic acid over 60 min for single proteins or 210 min at a flow rate of
300 nL/min for HeLa digest. Samples (500 ng injections) were analyzed on Thermo
Scientific™ Q Exactive, Orbitrap Elite™ and Orbitrap Fusion™ Tribrid™ mass
spectrometers using FT HCD MS2 or synchronous precursor selection (SPS) MS3
fragmentations (Orbitrap Fusion MS).
Data Analysis
Thermo Scientific™ Proteome Discoverer™ software version 1.4 was used to search
MS/MS spectra against the Swiss-Prot® human+BSA database using SEQUEST HT®
search engine. Static modifications included carbamidomethylation (C) and TMTsixplex
(N-terminal, K). Dynamic modifications included methionine oxidation and deamidation
(N,Q). Resulting peptide hits were filtered for maximum 1% FDR using the Percolator
algorithm.2 The TMT10plex quantification method within Proteome Discoverer software
was used to calculate the reporter ratios with mass tolerance ±10 ppm without
applying the isotopic correction factors. Only peptide spectra containing all reporter
ions were designated as “quantifiable spectra”. A protein ratio was expressed as a
median value of the ratios for all quantifiable spectra of the peptides pertaining to that
protein. For spiked in BSA experiments with data originating from the MS2 HCD
method, a precursor co-isolation filter of 25% was applied. This eliminated peptides
where contributions from co-eluting, nearly isobaric peptide species could interfere
significantly with the reporter ion signals coming from the peptide of interest. For data
sets obtained with the MS3-based method which contained both MS2 and MS3
spectra, CID(MS2) spectra were used for peptide identification and HCD(MS3) that
contained reporter ions were used for quantification3.
2 Increasing the Multiplexing of Protein Quantitation from 6- to 10-Plex with Reporter Ion Isotopologues
To benchmark the performance of new
Lys-C, trypsin HeLa cell digest and an
(equimolar 6-, 8- or 10-plex) on three
methods used for Orbitrap Elite, Q Ex
in Table 1. Compared to unlabeled He
samples consistently identified at leas
identified proteins/peptides with 8plex
labeled samples, depending on the in
samples consistently quantified more
compared to unlabeled samples.
ng capabilities of TMT reagents from 6 to 10
the tag by utilizing the 6 mDa mass
of the mass tag reporter ions. Mass
rbitrap-based instruments using FT
e number of protein identifications or quality
creasing sample multiplexing from 6 to 10.
AQ®) enable concurrent identification and
ferent samples using tandem mass
obaric tag-labeled samples that can be
desirable. However, it has been reported
r tagging molecules significantly decreases
oteins and peptides.1 Here, we demonstrate
with high resolution mass spectrometry
en 15N and 13C stable isotopes by combining
T6 isotope variants without loss of protein
in a complex digest.
kford, IL) and bovine serum albumin (BSA,
ated and digested with LysC and/or trypsin.
ex reagents (Figure 1) according to
six, eight or all ten channels were mixed in
peptide co-isolation on quantification, HeLa
MT8plex™ reagents was spiked with BSA
mixed at fixed ratios (16:8:4:2:1:1:2:4:8:16).
HPLC system and Thermo Scientific EASYcclaim™ PepMap™100 2 cm x 75 μm trap
ray™ PepMap™ RSLC C18 25 or 50 cm x
eptides with a 5-25% acetonitrile gradient in
oteins or 210 min at a flow rate of
0 ng injections) were analyzed on Thermo
nd Orbitrap Fusion™ Tribrid™ mass
chronous precursor selection (SPS) MS3
™ software version 1.4 was used to search
uman+BSA database using SEQUEST HT®
ed carbamidomethylation (C) and TMTsixplex
luded methionine oxidation and deamidation
for maximum 1% FDR using the Percolator
method within Proteome Discoverer software
with mass tolerance ±10 ppm without
nly peptide spectra containing all reporter
ctra”. A protein ratio was expressed as a
ble spectra of the peptides pertaining to that
th data originating from the MS2 HCD
5% was applied. This eliminated peptides
y isobaric peptide species could interfere
oming from the peptide of interest. For data
which contained both MS2 and MS3
peptide identification and HCD(MS3) that
ntification3.
Comparison of unlabeled to TMT6-, 8- or 10-plex labeled HeLa digests.
We have extended the previously reported4,5 TMT8plex set of reagents to a 10plex set
for multiplex peptide quantitation without increasing the size or altering the structure of
the tag (Figure 1). The TMT10plex reagents utilize the 6 mDa mass difference
between 13C and 15N isotopes which are measured by high-resolution LC-MSn. In
order to accurately quantify all ten channels, all reporter ions need to be baseline
resolved. We determined the minimum resolution to separate all ten channels for an
MSn resolution to be 35K at m/z 200 (~50K at m/z 120) as shown in Figure 2. At this
resolution, an accurate ratio calculation for the all reporter ions can be achieved using
a mass tolerance window of up to 10 ppm without applying isotope correction factors.
FIGURE 1. Thermo Scientific TMT10plex Reagents structures and reporter ion
mass for TMT6plex tags (blue), TMT8plex (green) and TMT10plex (orange).
TMT10-126
126.1277261
TMT10-127N
TMT10-127C
127.1247610
127.1310809
TMT10-128N
TMT10-128C
TMT10-129N
TMT10-129C
TMT10-130N
TMT10-130C
128.1281158
128.1344357
129.1314706
129.1377905
130.1348254
130.1411453
TMT10-131
131.1381802
Parameter
Q Exactive MS
FT MS1
Resolution settings
(FWHM at m/z 200)
140,000
Target value
3e6
Injection time
120
FT MS2
HCD
Resolution settings
(FWHM at m/z 200)
35,000
Target value
1e5
Injection time
250
Isolation width (Da)
2
Top N
10
FIGURE 3. Identification and quantifica
three Orbitrap instruments. Number of
protein groups (B) are shown at 1% FD
quantifiable proteins and peptides is a
of two replicate runs for each sample.
A.
ETD cleavage site
40000
HCD cleavage site
35000
30000
FIGURE 2. Optimal resolution settings to resolve isotope variants.
Q Exactive MS
Transient 128 ms
RP 35K@m/z 200
Peptides
erum albumin (BSA) digests were labeled
agents. Aliquots from six, eight or all ten
d analyzed on three different Thermo
TABLE 1. Instrument settings used fo
25000
20000
15000
10000
5000
0
Total
Quantified
Q Exactive MS
OT Elite MS
Transient 96 ms
RP 30K@m/z 400
B.
Tot
Orb
4500
4000
OT Fusion MS
Transient 128 ms
RP 60K@m/z 200
3500
3000
To benchmark the performance of new higher multiplexing reagents, we prepared a
Lys-C, trypsin HeLa cell digest and analyzed unlabeled and TMT-labeled samples
(equimolar 6-, 8- or 10-plex) on three different Orbitrap-based instruments. Acquisition
methods used for Orbitrap Elite, Q Exactive and Orbitrap Fusion MS are summarized
in Table 1. Compared to unlabeled HeLa digest, HCD MS2 of TMT6plex-labeled
samples consistently identified at least 80% as many protein groups, with no loss in
identified proteins/peptides with 8plex and 10plex samples compared to TMT6plexlabeled samples, depending on the instrument (Figure 3). All three multiplexed
samples consistently quantified more than 98% of identified peptides and proteins
compared to unlabeled samples.
Protein groups
™ (TMT™) 10-plex reagents by combining
reagents with four TMT6 reagent isotope
spectrometry platforms.
Results
2500
2000
1500
1000
500
0
Total
Quantified
Q Exactive MS
Thermo Scientific Poster Note • PN ASMS13_W617_RViner_e 06/13S 3
Tota
OrO
Quantitative precision and accuracy.
TABLE 1. Instrument settings used for TMT experiments.
or 10-plex labeled HeLa digests.
ed4,5 TMT8plex set of reagents to a 10plex set
increasing the size or altering the structure of
nts utilize the 6 mDa mass difference
measured by high-resolution LC-MSn. In
els, all reporter ions need to be baseline
esolution to separate all ten channels for an
0K at m/z 120) as shown in Figure 2. At this
or the all reporter ions can be achieved using
m without applying isotope correction factors.
ex Reagents structures and reporter ion
plex (green) and TMT10plex (orange).
126.1277261
TMT10-127C
127.1310809
Parameter
Q Exactive MS
Orbitrap Elite MS
Orbitrap Fusion MS
140,000
144,000
120,000
Target value
3e6
1e6
2e5
Injection time
120
120
50
FT MS2
HCD
HCD
HCD
35,000
36,000
60,000
Target value
1e5
5e4
1e5
Injection time
FT MS1
Resolution settings
(FWHM at m/z 200)
Two main factors contribute to decrease
tags: 1) interference in reporter ion mas
of precursor ions. Interference from bac
addressed by scanning with high resolu
been recently addressed by using a mu
Orbitrap Velos or Orbitrap Elite instrume
isolation width and fragmentation at the
FIGURE 4. Synchronous Precursor S
quantification.
Precursor
Resolution settings
(FWHM at m/z 200)
250
250
120
Isolation width (Da)
2
2
2
Top N
10
15
3 sec
TMT10-128C
TMT10-129C
129.1377905
TMT10-130C
130.1411453
FIGURE 3. Identification and quantification results for TMT experiments on
three Orbitrap instruments. Number of total peptide identifications (A) and
protein groups (B) are shown at 1% FDR for 500ng HeLa digest. The number of
quantifiable proteins and peptides is also shown. Results represent an average
of two replicate runs for each sample.
A.
ETD cleavage site
40000
HCD cleavage site
35000
25000
Unlabeled
20000
TMT6
15000
TMT8
10000
TMT10
FIGURE 5. Accuracy and precision o
HeLa matrix using MS2 vs MS3 meth
and observed (B) ratios normalized t
represent an average of two replicate
A.
20
5000
0
Total
Quantified
Q Exactive MS
OT Elite MS
Transient 96 ms
RP 30K@m/z 400
B.
Total
Quantified
OT Elite
Orbitrap
EliteMS
MS
Total
Quantified
OT Fusion
MSMS
Orbitrap
Fusion
4500
4000
OT Fusion MS
Transient 128 ms
RP 60K@m/z 200
3500
Protein groups
2500
Unlabeled
2000
TMT6
TMT8
1500
TMT10
1000
500
0
Total
Quantified
Q Exactive MS
Total
Quantified
OT Elite
MSMS
Orbitrap
Elite
Total
Quantified
OT Fusion
MS
Orbitrap
Fusion
MS
TMT 8 HeL
16
14
12
10
8
6
4
2
B.
20
126
127N
127C
128N
BSA alone
18
BSA+ HeLa SPS M
16
BSA+ HeLa MS2 (
14
12
10
8
6
4
2
0
4 Increasing the Multiplexing of Protein Quantitation from 6- to 10-Plex with Reporter Ion Isotopologues
TMT 10 BS
18
0
3000
igher multiplexing reagents, we prepared a
yzed unlabeled and TMT-labeled samples
ferent Orbitrap-based instruments. Acquisition
ive and Orbitrap Fusion MS are summarized
digest, HCD MS2 of TMT6plex-labeled
0% as many protein groups, with no loss in
nd 10plex samples compared to TMT6plexument (Figure 3). All three multiplexed
an 98% of identified peptides and proteins
BSA (16:8:4:2:1:1:2:4:8:16
Expected BSA peptide reporter Ion ratios
Q Exactive MS
Transient 128 ms
RP 35K@m/z 200
Peptides
30000
s to resolve isotope variants.
FTMS3 HCD of
selected notches
Observed BSA peptide reporter ion ratios
128.1344357
126
127N
127C
128N
Orbitrap Fusion MS
144,000
120,000
1e6
2e5
120
50
HCD
HCD
Two main factors contribute to decreased quantitative accuracy when using isobaric
tags: 1) interference in reporter ion mass regions and 2) interference from co-isolation
of precursor ions. Interference from background ions in the reporter region can be
addressed by scanning with high resolution (Figure 2). Co-isolation interference has
been recently addressed by using a multi-notch (SPS )MS3 experiment on an LTQ
Orbitrap Velos or Orbitrap Elite instrument4,6 and by combining a narrow precursor
isolation width and fragmentation at the apex of the LC.7
FIGURE 4. Synchronous Precursor Selection MS3 TMT workflow for accurate
quantification.
36,000
60,000
5e4
1e5
250
120
2
2
15
3 sec
ults for TMT experiments on
ptide identifications (A) and
0ng HeLa digest. The number of
wn. Results represent an average
ntified
MS
350
3500
300
3000
2500
MS2
2000
MS3
1500
200
150
100
50
500
0
250
Total
Quantified
0
To assess the impact of peptide co-isolation on q
samples were equally mixed (1:1:1:1:1:1:1:1) an
TMT10plex reagents mixed at different ratios (16
fmol BSA + 500 ng HeLa digest) was then analyz
narrow precursor isolation (1.2 amu) by HCD MS
fragmentation/quantification method (Figures 4 &
method significantly improved quantitative accur
as shown in Figure 5B. The percent of proteins q
approach was over 95% with less than 25% loss
improved scan rate of the Orbitrap Fusion MS (F
outperform previously reported single notch MS3
of quantified proteins and quantitative accuracy.3
Reporter ions
Unlabeled
TMT6
TMT8
TMT10
Total
Higher-resolution analysis, at least 35K@ m
TMT10plex reagent reporter ion measurem

A.
Precursor co-isolation effect in complex sa
extent by relying on the reporter ion intens

The Orbitrap Fusion MS demonstrated sign
of peptide identifications and quantifiable p
compared to the previous series of instrum

A novel FT MS3 Synchronous Precursor S
Orbitrap Fusion MS showed only ~25% los
excellent 95% quantitation rate with signific
accuracy for BSA digest peptides spiked in
BSA (16:8:4:2:1:1:2:4:8:16) + HeLa (10:10:10:0:10:10:10:0:10:10)
20
OT Fusion
MSMS
Orbitrap
Fusion
TMT8
TMT10
Total
Quantified
OT Fusion
MS
Orbitrap
Fusion
MS
TMT 10 BSA, 500 fmol:129:64:32:16:8:8:16:32:64:129
18
TMT 8 HeLa, 500 ng
16
14
12
10
B.
References
8
6
4
2
0
TMT6
Mass spectrometry analysis on three differ
shows no negative effect on number of pro
quantification in complex digest by increas

Quantified
Unlabeled

FIGURE 5. Accuracy and precision of TMT10 BSA quantitation spiked into TMT8
HeLa matrix using MS2 vs MS3 methods on Orbitrap Fusion MS. Expected (A)
and observed (B) ratios normalized to 126 reporter ion intensity. Results
represent an average of two replicate runs for each sample.
Expected BSA peptide reporter Ion ratios
S
MS
400
4000
Conclusion
20
Observed BSA peptide reporter ion ratios
ntified
FTMS3 HCD of
selected notches
B
HeLa
4500
1000
MS2 IT CID
Multi-notch isolation
5 fragments selected
Precursor
A.
Peptides
itrap Elite MS
FIGURE 6. Identification and quantification of
into TMT8-labeled HeLa using MS2 vs MS3 me
Number of HeLa proteins (A) and BSA peptid
1% FDR. Results represent an average of two
Protein groups
Quantitative precision and accuracy.
periments.
126
127N
127C
128N
128C
129N
129C
130N
130C
131
BSA alone
18
BSA+ HeLa SPS MS3
16
BSA+ HeLa MS2 ( 1.2 amu isolation)
14
12
10
8
6
iTRAQ is a trademark of Applera Corporation. Tandem Mass Ta
plc. SEQUEST is a registered trademark of the University of Wa
Science, Ltd. Swiss-Prot is a registered trademark of Institute S
Switzerland. All other trademarks are the property of Thermo Fi
4
2
0
1. Pichler, P.; Kocher, T.; Holzmann, J.; Maza
Mechtler, K. Anal. Chem. 2010, 82: 6549−6
2. Kall, L. Canterbury, J, Weston, J., Noble, W
2007, 4: 923-925.
3. Viner, R.; Scigelova, M.; Zeller, M.; Opperm
V. Thermo Fisher Scientific Application Not
4. McAlister, G., Huttlin, E.L.; Haas, W.; Ting,
Kuhn, K.; Pike, I.; Grothe, R.A,; Blethrow, J
7469-7478.
5. Werner, T., Becher, I.; Sweetman,G.; Doce
Chem. 2012. 84: 7188-7194.
6. Ting, L.; Rad, R.; Gygi, S. P.; Haas, W., Na
7. Savitski, M. M.; Sweetman, G.; Askenazi, M
Bantscheff, M. Anal. Chem. 2011, 83: 8959
126
127N
127C
128N
128C
129N
129C
130N
130C
131
This information is not intended to encourage use of these prod
intellectual property rights of others.
Thermo Scientific Poster Note • PN ASMS13_W617_RViner_e 06/13S 5
A.
B.
HeLa
4500
4000
350
3500
300
3000
2500
MS2
2000
MS3
1500
MS2
200
MS3
150
50
500
0
250
100
1000
MS2 IT CID
Multi-notch isolation
5 fragments selected
BSA
400
Peptides
Selection MS3 TMT workflow for accurate
FIGURE 6. Identification and quantification of TMT10-labeled BSA digest spiked
into TMT8-labeled HeLa using MS2 vs MS3 methods on the Orbitrap Fusion MS.
Number of HeLa proteins (A) and BSA peptide (B) identifications are shown at
1% FDR. Results represent an average of two replicate runs for each sample.
Protein groups
.
ed quantitative accuracy when using isobaric
ss regions and 2) interference from co-isolation
ckground ions in the reporter region can be
ution (Figure 2). Co-isolation interference has
ulti-notch (SPS )MS3 experiment on an LTQ
ent4,6 and by combining a narrow precursor
apex of the LC.7
Total
Quantified
0
Sequence
coverage
Total
Quantified
To assess the impact of peptide co-isolation on quantification, TMT8plex-labeled HeLa
samples were equally mixed (1:1:1:1:1:1:1:1) and spiked with BSA labeled with
TMT10plex reagents mixed at different ratios (16:8:4:2:1:1:2:4:8:16). The sample (500
fmol BSA + 500 ng HeLa digest) was then analyzed on an Orbitrap Fusion MS using
narrow precursor isolation (1.2 amu) by HCD MS2 or a novel SPS MS3
fragmentation/quantification method (Figures 4 & 5).4 The SPS( multi-notch) MS3
method significantly improved quantitative accuracy for the spiked BSA peptide digest
as shown in Figure 5B. The percent of proteins quantified employing the MS3
approach was over 95% with less than 25% loss in identified proteins due to the
improved scan rate of the Orbitrap Fusion MS (Figure 6). These results significantly
outperform previously reported single notch MS3 acquisition methods for both number
of quantified proteins and quantitative accuracy.3,6
Reporter ions
Conclusion
of TMT10 BSA quantitation spiked into TMT8
hods on Orbitrap Fusion MS. Expected (A)
to 126 reporter ion intensity. Results
e runs for each sample.
) + HeLa (10:10:10:0:10:10:10:0:10:10)

Mass spectrometry analysis on three different Orbitrap-based instruments
shows no negative effect on number of protein identifications or quality of
quantification in complex digest by increasing TMT multiplexing from 6 to 10.

Higher-resolution analysis, at least 35K@ m/z 200, is required for the accurate
TMT10plex reagent reporter ion measurements.

Precursor co-isolation effect in complex samples can be addressed to a large
extent by relying on the reporter ion intensities extracted from MS3 spectra.

The Orbitrap Fusion MS demonstrated significant improvements in the number
of peptide identifications and quantifiable proteins using TMT reagents
compared to the previous series of instruments.

A novel FT MS3 Synchronous Precursor Selection method implemented on the
Orbitrap Fusion MS showed only ~25% loss of identified proteins and an
excellent 95% quantitation rate with significantly improved quantification
accuracy for BSA digest peptides spiked into a complex proteome matrix.
A, 500 fmol:129:64:32:16:8:8:16:32:64:129
La, 500 ng
References
128C
129N
129C
130N
130C
131
MS3
( 1.2 amu isolation)
1. Pichler, P.; Kocher, T.; Holzmann, J.; Mazanek, M.; Taus, T.; Ammerer, G.;
Mechtler, K. Anal. Chem. 2010, 82: 6549−6558.
2. Kall, L. Canterbury, J, Weston, J., Noble, W.S., MacCoss, M. Nature Meth.
2007, 4: 923-925.
3. Viner, R.; Scigelova, M.; Zeller, M.; Oppermann, M.; Moehring, T.; Zabrouskov,
V. Thermo Fisher Scientific Application Note #566, 2012
4. McAlister, G., Huttlin, E.L.; Haas, W.; Ting, L.; Jedrychowski, M.P.; Rogers, J.C.;
Kuhn, K.; Pike, I.; Grothe, R.A,; Blethrow, J.D.; Gygi, S.P. Anal Chem. 2012. 84:
7469-7478.
5. Werner, T., Becher, I.; Sweetman,G.; Doce,C.; Savitski, M.; Bantscheff, M. Anal
Chem. 2012. 84: 7188-7194.
6. Ting, L.; Rad, R.; Gygi, S. P.; Haas, W., Nature Methods 2011, 8 , 937-940.
7. Savitski, M. M.; Sweetman, G.; Askenazi, M.; Marto, J. A.; Lang, M.; Zinn, N.;
Bantscheff, M. Anal. Chem. 2011, 83: 8959−8967.
iTRAQ is a trademark of Applera Corporation. Tandem Mass Tag, TMT are trademarks of Proteome Sciences
plc. SEQUEST is a registered trademark of the University of Washington. Mascot is a trademark of Matrix
Science, Ltd. Swiss-Prot is a registered trademark of Institute Suisse de Bioinformatique (Sib) Foundation
Switzerland. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
128C
129N
129C
130N
130C
131
This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
6 Increasing the Multiplexing of Protein Quantitation from 6- to 10-Plex with Reporter Ion Isotopologues
www.thermoscientific.com
©2013 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. iTRAQ is
a trademark of Applera Corporation. Tandem Mass Tag, TMT are trademarks of Proteome Sciences plc. SEQUEST is a registered
trademark of the University of Washington. Mascot is a trademark of Matrix Science, Ltd. Swiss-Prot is a registered trademark of
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