Letter
pubs.acs.org/ac
Mass Defect-Based Pseudo-Isobaric Dimethyl Labeling for Proteome
Quantification
Yuan Zhou,†,‡ Yichu Shan,† Qi Wu,†,‡ Shen Zhang,†,‡ Lihua Zhang,*,† and Yukui Zhang†
†
National Chromatographic Research and Analysis Center, Key Lab of Separation Sciences for Analytical Chemistry, Dalian Institute
of Chemical Physics, Chinese Academy of Sciences, Dalian, China
‡
University of Chinese Academy of Sciences, Beijing, China
S Supporting Information
*
ABSTRACT: Discovering differentially expressed proteins in various biological samples requires proteome quantification
methods with accuracy, precision, and wide dynamic range. This study describes a mass defect-based pseudo-isobaric dimethyl
labeling (pIDL) method based on the subtle mass defect differences between 12C/13C and 1H/2H. Lys-C protein digests were
labeled with CD2O/13CD2O and reduced with NaCNBD3/NaCNBH3 as heavy and light isotopologues, respectively. The
fragment ion pairs with mass differences of 5.84 mDa were resolved by high-resolution tandem mass spectrometry (MS/MS) and
used for quantification. The pIDL method described here resulted in highly accurate and precise quantification results with
approximately 100-fold dynamic range. Furthermore, the pIDL method was extended to 4-plex proteome quantification and
applied to the quantitative analysis of proteomes from Hca-P and Hca-F, two mouse hepatocarcinoma ascites syngeneic cell lines
with low and high lymph node metastasis rates.
M
interference is common for MS/MS level-based quantification
methods.9,10
To solve these problems, several innovative methods have
been recently developed. Isobaric peptide termini labeling
(IPTL)11−13 is an elegant solution in which the amino groups
of the N-termini and C-termini of Lys-C protein digests were
crosswise labeled with heavy/light isotope reagents according
to the slightly different chemical properties of α- and ε-NH2.
Fragment ion pairs specific to the labeled peptides were used
for peptide/protein quantification. Although IPTL improved
the quantification accuracy compared to other reported MS/
MS level methods, the side reactions that are inherent in the
multistep labeling can adversely affect quantification accuracy
and dynamic range.12
NeuCode SILAC14 is another strategy based on subtle mass
differences resulting from the mass defects of 12C/13C,
14
N/15N, and 1H/2H. The small differences present when
ethods of stable-isotope incorporation with mass
spectrometry (MS)-based proteome quantification
have advanced rapidly in the past decade. Peptide samples
can be differentially tagged with heavy or light isotopes by
metabolic labeling1,2 or chemical labeling.3,4 The mass
differences can be distinguished at either the MS or tandem
mass spectrometry (MS/MS) level. Dimethyl labeling,3 a
chemical labeling method, is widely used for proteome
quantification at the MS level. Several advantages of this
method include quick reaction, high labeling efficiency, low
cost, and applicability to different types of samples including
tissues, cells, and body fluids.5 Isobaric tags for relative and
absolute quantitation (iTRAQ),4 an MS/MS-based method,
allows the proteome quantification of up to eight samples
simultaneously and provides more precise quantification results
than the MS level quantification method.6,7 Although these
strategies have been widely used for proteome quantification,
their accuracy and dynamic range are limited by the signal-tonoise ratio and the increased MS spectral complexity leads to
fewer quantified proteins for MS level-based quantification
approaches.8 In addition, ratio distortion caused by precursor
© 2013 American Chemical Society
Received: September 6, 2013
Accepted: November 1, 2013
Published: November 1, 2013
10658
dx.doi.org/10.1021/ac402834w | Anal. Chem. 2013, 85, 10658−10663
Analytical Chemistry
Letter
fractions were pooled as equal-interval fractions, such as
fraction 1 mixed with fraction 21 and fraction 20 mixed with
fraction 40.17 All of the fractions were then lyophilized in a
SpeedVac, and the samples were stored at −20 °C until use.
LC−MS/MS Analysis. All of the samples were resuspended
in water containing 0.1% formic acid (FA). The samples were
analyzed using a low-pH RPLC−ESI-MS/MS system consisting
of a quaternary surveyor MS pump (Thermo, CA) and a LTQOrbitrap Velos mass spectrometer (Thermo, CA) with a
nanospray source. Buffer C (98% H2O + 2% ACN + 0.1% FA)
and Buffer D (2% H2O + 98% ACN + 0.1% FA) were used for
gradient separation. Peptides were separated on a homemade
C18 column (150 mm × 75 μm i.d., Agela, Tianjin, China).
Gradient elution was performed using a gradient of 5% to 35%
acetonitrile in 0.1% FA with run times of 49 min for the BSA
samples, 94 min for the Hca-P samples, and 50 min for the
Hca-P and Hca-F mixture at a flow rate of 300 nL/min.
Peptides were detected using a LTQ-Orbitrap Velos mass
spectrometer in a data-dependent acquisition mode. For each
cycle, survey scan MS was acquired from m/z 350 to 1 800 at a
resolution of 60 000 at m/z = 400. For optimization of MS/MS
resolution, the three most intense ions were selected for MS/
MS scan by HCD at resolutions of 15k, 30k, and 60k,
respectively. For the quantification of the peptides, the six most
intense ions were selected for MS/MS scan at a resolution of
30k in centroid mode. The dynamic exclusion function was set
to a repeat count of 1, and an exclusion duration of 40 s was
used. The normalized collision energy was set to 40%. The
temperature of the ion transfer capillary was set to 200 °C. The
spray voltage was set to 2.0 kV. One microscan was used for
each MS and MS/MS scan.
Data Analysis. The acquired raw files were analyzed by
MaxQuant18 (version 1.2.2.5). The Andromeda19 program,
which is embedded in MaxQuant, was used to search the peak
lists against BSA.fasta or the target-decoy IPI-mouse database
(version 3.68, 56 729 entries). Common contaminants were
added to this database. Cysteine carbamidomethylation was
searched as a fixed modification, whereas N-terminal acetylation
and methionine oxidation were searched as variable modifications. Heavy- and light-labeled samples were searched
independently using dimethyl (+34.063 12 Da) and dimethyl
(+34.063 12) N-termini and K as variable modifications. For
the four-plex sample, dimethyl (+30.038 01)/dimethyl
(+34.063 12 Da) or dimethyl (+30.043 85)/dimethyl
(+34.068 96 Da) of N-termini and K were set as light/heavy
labels for quantification in two independent search. The two
results were then combined. Peptide identification was based
on a search with an initial mass deviation of up to 6 ppm for the
precursor ions and an allowed fragment mass deviation of 20
ppm. Enzyme specificity was set to trypsin for BSA samples or
Lys-C for the complex samples. Two mis-cleavages were
allowed and a minimum of six amino acids per identified
peptide was required. A FDR of 0.01 for proteins and peptides
was required.
All of the raw files were converted to *.mgf files using
pXtract (part of the pFind program20). The intensity values of
the peptide a, b, and y fragment ion pairs were extracted from
the mgf files using java scripts built in-house to calculate the H/
L ratio of the peptide. The ratio of each PSM was obtained by
calculating the ratio of the total intensities of the heavy and
light paired fragment ions.8 Peptide and protein ratios were
calculated as the median of all spectra matching the same
peptide and the median of all the quantified unique peptides
lysine (K) is composed of these different isotopes enable
proteome quantification with good accuracy and dynamic
range. However, it suffers from long cycle time to acquire the
MS1 spectra since the resolution of MS1 was as high as 480k.
We hypothesized that the incorporation of 12C/13C and
1
H/2H into Lys-C protein digests by dimethyl labeling would
generate a new fragment ion-based quantification method,
which was termed as pseudo-isobaric dimethyl labeling (pIDL).
Compared to other reported fragment ion-based quantification
methods, such as IPTL12 and QITL,15 by our proposed
method, the labeling of the amino groups of N termini and K
were performed simultaneously, beneficial to improve quantification accuracy and precision and decrease the side reaction.
■
METHODS
Sample Preparation. Approximately 2 × 106 Hca-F or
Hca-P cells were inoculated subcutaneously and grown in the
abdominal cavity of inbred Chinese 615 mice for 7 days.16 The
cells in ascites were collected. The cells from both cell lines
were then washed three times with cold 1× PBS buffer, further
homogenized in 2 mL of lysis buffer (1% (v/v) protease
inhibitor cocktail in 8 M urea,) using a Tissue Tearor from
Biospec Products (Bartlesville, OK) at approximately 10 000
rpm for 1 min, sonicated at 100 W for 100 s, and centrifuged at
25 000g for 40 min at 4 °C. The supernatant was collected and
proteins were precipitated by the addition of cold acetone.
After centrifugation, the pellets were lyophilized in a SpeedVac
(Thermo Fisher Scientific, San Jose, CA) and subsequently
resuspended in 8 M urea. Protein concentration was
determined by the Bradford assay (Beyotime, Nantong,
China), and the samples were stored at −20 °C until use.
Disulfide bonds were reduced by incubating the protein with
DTT at 56 °C for 1 h, followed by alkylation of the proteins by
IAA in the dark at room temperature for 40 min. The solution
was then diluted to 1 M urea with 50 mM phosphate buffer
(pH 8.0). Finally, trypsin was added to the BSA samples at a
ratio of 1:50 (enzyme/protein, w/w) and incubated at 37 °C
for 24 h. For the Hca-F or Hca-P protein samples, Lys-C was
added at a weight ratio of 1:50 (enzyme/protein) and
incubated at 37 °C for 24 h.
Dimethyl Labeling. Both BSA and Lys-C Hca-P digests
were labeled with 13CD2O and NaCNBH3 (light labeling, L)
and with CD 2O and NaCNBD3 (heavy labeling, H),
respectively, as previously described.5 Next, the two differently
labeled BSA digests were mixed at ratios of H/L=1:100, 1:50,
1:10, 1:5, 1:1, 5:1, 10:1, 50:1 and 100:1; Hca-P digests were
mixed at ratios of H/L = 1:1, 5:1, 10:1, and 40:1. All the mixed
samples were then desalted by C18-trap column and
lyophilized in a SpeedVac. All the samples were then stored
at −20 °C until use.
Lys-C Hca-P protein digests were labeled by 13CH2O,
NaCNBH3 (30L) and 13CD2O, NaCNBH3 (34L), whereas
Hca-F protein digests were labeled by CH2O, NaCNBD3
(30H), and CD2O, NaCNBD3 (34H). After the samples were
quenched with a 1% NH3 solution, they were mixed at a ratio of
1:1:1:1 (w/w). The mixture of labeled peptides was separated
by high-pH RPLC using an Agilent 1290 Infinity LC system
(Santa Clara, CA). Buffer A (100% H2O + 25 mM NH4FA, pH
= 9.5) and Buffer B (10% H2O + 90% ACN + 25 mM NH4FA)
were used for gradient separation. The gradient elution was
performed using 2% B (0−2 min), 2−5% B (2−3 min), 5−35%
B (3−78 min), 35−80% B (78−78.1 min), and 80% B (78.1−
82 min), with fractions collected every 2 min. The resulting
10659
dx.doi.org/10.1021/ac402834w | Anal. Chem. 2013, 85, 10658−10663
Analytical Chemistry
Letter
Figure 1. The pIDL method and the effect of MS/MS resolution on quantification. (a) Mass increments for one amino group labeled with different
types of commercially available formaldehyde and sodium cyanoborohydride. (b) Schematic representation of the mass defect-based pseudo-isobaric
dimethyl labeling method. Lys-C protein digests were labeled with 13CD2O and NaCNBH3, in addition to CD2O and NaCNBD3, as light and heavy
isotopologues, respectively. The mass difference was 11.68 mDa, which is indistinguishable at the MS level. However, high-resolution MS/MS is
capable of resolving fragment ions with a mass difference of 5.84 mDa, which allows proteome quantification. (c) MS/MS spectrum of
TYFPHFDVSHGSAQVK. (d) Resolved fragment ions with the MS/MS resolution at 15k, 30k, and 60k (from top to bottom).
from a protein.7,21 Proteins quantified at least twice and with at
least a 2-fold change were considered to be differentially
expressed proteins.
lymph node metastasis rates16 as model samples to investigate
the performance of the pIDL method.
Effects of MS/MS Resolution on Proteome Quantification. Collision-induced dissociation (CID) in the ion trap is
widely used for peptide identification. However, it suffers from
low resolution and 1/3 cutoff. However, in the pIDL method,
only a 5.84 mDa mass difference between light and heavy
isotopologues, which can be resolved by high MS/MS
resolution, was used for quantification. Therefore, we selected
higher-energy collisional dissociation (HCD) as the collision
mode and Orbitrap as the mass analyzer. The resolving power
of Orbitrap-type mass analyzer decreases with increasing m/z of
the measured ion.22 Since the resolution equals the ratio
between M and ΔM for larger ions with larger m/z, the ΔM is
larger than 5.84 mDa, so they cannot be resolved. Thus, only
fragment ions in the low mass range can be resolved by a
current LTQ-Orbitrap Velos mass spectrometer. Higher
resolution enables more fragment ion pairs to be resolved,
but longer cycle time is needed to collect the spectra.
Therefore, we investigated the effect of MS/MS resolution
(15k to 60k at m/z = 400) on proteome quantification. When
peptide quantification was performed, to ensure the fragment
ions were correctly selected, the mass tolerance of fragment
ions was set as 2 mDa. Taking TYFPHFDVSHGSAQVK from
hemoglobin as an example (Figure 1c,d), we plotted the
distinguished fragment ion pairs at the resolution described
■
RESULTS AND DISCUSSION
IDL Method for Proteome Quantification. Currently,
four types of formaldehyde (CH2O, 13CH2O, CD2O, and
13
CD2O) and two types of sodium cyanoborohydride
(NaCNBD3 and NaCNBH3) are commercially available
(Figure 1a). The labeling reaction introduced two carbon
atoms and four hydrogen atoms to the peptides. If 13CD2O +
NaCNBH3 was applied, the mass increment is 34.063 12, which
is the mass of (13CD2)2. If CD2O + NaCNBD3 was applied, the
mass increment is 34.068 96, which is the mass of (CD3)2-H2.
Other combination of different labeling reagents can also be
used to generate pseudo-isobaric labeling.
We used Lys-C to digest proteins because its cleavage site is
lysine (K). N- and C-terminal amino groups of peptides can be
pseudo-isobaric dimethyl labeled simultaneously. Therefore, all
of the a-, b-, and y-type fragment ion counterparts would have
mass shifts of 5.84 mDa (Figure 1b). The fragment ions with
such subtle mass differences can be resolved by high-resolution
MS/MS, which were used for proteome quantification. In this
study, we selected two mouse hepatocarcinoma ascites
syngeneic cell lines with low (Hca-P) and high (Hca-F)
10660
dx.doi.org/10.1021/ac402834w | Anal. Chem. 2013, 85, 10658−10663
Analytical Chemistry
Letter
Figure 2. Quantification results using the pIDL method. (a) Box plots showing the PSMs, peptides, and proteins measured at a mixing ratio of 1:1.
(b) Box plots showing the ratios measured (box and whiskers) and the expected ratios (red dashed line) at mixing ratios of 5, 10, and 40. (c)
Comparison of the expected ratios versus the measured ratios. Differentially labeled BSA digests were mixed at ratios of 100:1, 50:1, 10:1, 5:1, 1:1,
1:5, 1:10, 50:1, and 100:1. The average of three measured ratios (red cross) and SD values (error bar) are displayed. (d) The two quantification
results obtained by four-plex pIDL method demonstrated a strong correlation for analyzing the differentially expressed proteins of Hca-F and the
Hca-P cell lines (R2 = 0.887).
above. Only one fragment ion pair was used for quantification
at the resolution of 15k, whereas 5 and 10 fragment ion pairs
were used for quantification at resolutions of 30k and 60k,
respectively. At the 30k resolution, 99.5% of all spectra can be
used for quantification, and less cycle time was required than at
the 60k resolution (Supporting Information S-Table 1).
Therefore, the 30k MS/MS resolution was utilized in the
following study.
Accuracy and Dynamic Range. We next investigated the
accuracy and precision of the pIDL method with heavily and
lightly labeled Hca-P protein digests at mixing ratios of 1, 5, 10,
and 40. The 1:1 ratio yielded 5374 peptide-spectrum matches
(PSMs) using the pIDL method, of which 99.1% were
quantified in three replicate runs. Additionally, over 99.9% of
all the ratios ranged from 0.5 to 2 and the 25 and 75 percentiles
were 0.959 and 1.100, respectively (Figure 2a). All identified
proteins can be quantified with ratios ranging from 0.700 to
1.44, and the RSD values were less than 10% for 87.2% of the
proteins that were quantified at least twice in the three replicate
runs. For heavily and lightly labeled Hca-P protein digests at
mixing ratios of 5, 10, and 40, the measured ratios were 5.48,
11.15, and 45.65, respectively, with relative errors of 9.6%,
11.5%, and 14.1%, respectively (Figure 2b). Thus, no significant
ratio compression was observed, which demonstrates the
quantification accuracy of pIDL in a wide dynamic range.
We further evaluated the dynamic range of pIDL method
according to refs 12, 15, and 23 by analyzing the mixtures of
tryptic BSA digests with H/L ratios of 100:1, 50:1, 10:1, 5:1,
1:1, 1:5, 1:10, 1:50, and 1:100 (w/w). According to the pIDL
method shown in Figure 1b, only peptides with K as C terminal
were used for BSA quantification. The quantification results
were listed in S-Table 2 in the Supporting Information. We
plotted the average values of measured ratios acquired from the
three replicate runs against their expected ratios. Good linearity
(y = 0.988x − 0.110,) across a 100-fold dynamic range with R2
= 0.999 (Figure 2c). All of the mixtures were precisely
quantified with RSD values lower than 9%.
Complete labeling of all peptides is imperative in accurate
protein quantification. Both identified labeled and unlabeled
peptides were used to calculate the labeling efficiency with
variable modification of the N-terminal amine or lysine residue
set as dimethyl (+34.068 96 Da or +34.063 12 Da, respectively)
during the database search. Using the heavily and lightly labeled
Hca-P protein digest ratio of 1:1 as an example, the values
obtained were ≥99.9% for lysine and ≥99.4% for N-terminal
amines (Supporting Information S-Table 3).
10661
dx.doi.org/10.1021/ac402834w | Anal. Chem. 2013, 85, 10658−10663
Analytical Chemistry
Letter
to provide a novel proteome quantification technique. Because
of the high labeling efficiency via a one-pot reaction, specific
fragment ion pairs for quantification, and enhanced purity of
fragment ions by high-resolution MS/MS, such a strategy
showed advantages of wide dynamic range, high accuracy, and
good precision. Furthermore, pIDL was also successfully used
as a 4-plex to improve the reproducibility of proteome
quantification. All these results demonstrated that pIDL
might become a promising technique to achieve the large
scale proteome quantification.
Because fragment ions have higher specificity for peptides
than reporter ions using the pIDL method, the ratio
compression caused by coelution was reduced. Besides,
fragment ions were purified since contaminating ions and
fragment ions can be resolved at the MS/MS resolution of 30k
(m/z=400). To ensure the fragment ions were correctly
selected, the mass tolerance of fragment ions was set as 2
mDa. Using ETYGDMADCCEK from BSA with H/L=1 as an
example (Supporting Information S-Figure 1), many contaminating ions around the lightly and heavily labeled a1 ions were
resolved, thus the a1 ion pair used for quantification was
purified. The 2 mDa mass tolerance of a1 ion ensured the
correct a1 ions were correctly selected for further analysis.
Several fragment ion-based methods were developed, such as
IPTL,12 QITL15 and IVTAL,24 by which only the PSM with at
least 4 pairs of fragment ions were used for peptide
quantification to obtain accurate quantification results. In our
method, we compared the obtained H/L ratios obtained from 1
to 10 fragment ion pairs, as shown in Supporting Information
Figure 2. The median values of different pairs ranged from 1.01
to 1.05, which showed no significant difference among the
results by t test. Additionally, the SD values obtained from the
assay ranged from 0.0618 to 0.225 (Supporting Information SFigure 2). Therefore, by our proposed method, even the
fragment-ion-pair number as low as 1 is capable of providing
accurate quantification results.
Four-Plex pIDL Method and Its Application. The pIDL
method is based on the subtle mass defects between 12C/13C
and 1H/2H and results in three isotopic peaks by calculating the
incorporation of these six reagents according to Figure 1a. Each
peak is capable of quantifying two samples. Therefore,
quantification data of up to six samples (six-plex) can be
acquired simultaneously using the pIDL method.
We analyzed the proteomes from the Hca-P and Hca-F cell
lines using the four-plex pIDL method. Hca-P protein digests
were labeled by 13CH2O, NaCNBH3 (30L) and 13CD2O,
NaCNBH3 (34L), whereas Hca-F protein digests were labeled
by CH2O, NaCNBD3 (30H), and CD2O, NaCNBD3 (34H). In
this method, two quantification results can be achieved in just
one LC−MS experiment. In total 2403 proteins were quantified
by both light (30H/30L) and heavy (34H/34L) isotopologues.
The ratio of 30H/30L and the ratio of 34H/34L showed
excellent correlation (R 2 = 0.887, Figure 2d), which
demonstrates the high reproducibility of protein quantification
obtained using the 4-plex pIDL method. For the four-plex pIDL
method, the proteins quantified at least twice were further
analyzed. In total, 2517 unique proteins were reliably
quantified, of which 80 proteins were up-regulated in Hca-F
and 114 proteins were down-regulated in Hca-F (Supporting
Information S-Table 4). The average RSD value of all
quantified proteins was 11.76%, demonstrating good quantification precision. However, in the four-plex pIDL method,
about 55% of all the peptides can be fragmented by both lightly
and heavily labeled isotopologues due to the limited scan speed
of the mass spectrometer. Meanwhile, about 75% of all the
quantified proteins can be quantified by both +30 and +34
isotope clusters. We believe that more peptides labeled with
both light and heavy reagents can be fragmented with the
development of the mass spectrometer.
■
ASSOCIATED CONTENT
S Supporting Information
*
(S-Figure 1) Spectrum of a1 ion of ETYGDMADCCEK (H/L
= 1) from BSA; (S-Figure 2) effect of fragment ion pair
numbers of PSMs on quantification results; (S-Table 1) effect
of resolving power on protein quantification; (S-Table 2)
quantification results of heavily and lightly labeled BSA digests
with different ratios; (S-Table 3) dimethyl labeling efficiency at
N-terminal α-amine group and ε-amine group of lysine in the
peptides; and (S-Table 4) differentially expressed proteins in
Hca-P and Hca-F cell lines. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Phone and fax: +86-41184379720.
Author Contributions
Yuan Zhou and Yichu Shan contributed equally to this work.
Y.Z., L.Z., and Y.Z. designed the experiments, analyzed the
data, and wrote the paper. Y.S. developed software for data
analysis. Q.W. and S.Z. participated in isotope labeling of
protein samples and data analysis.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The work was supported by the China State Key Basic
Research Program (Grant 2012CB910604), National Natural
Science Foundation (Grants 20935004 and 21005079), and
The Creative Research Group Project by NSFC (Grant No.
21021004). We also thank Prof. Shujuan Shao from Dalian
Medical University for providing Hca-P and Hca-F samples.
■
REFERENCES
(1) Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 6591−6596.
(2) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.;
Steen, H.; Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1, 376−
386.
(3) Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H. Anal. Chem.
2003, 75, 6843−6852.
(4) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker,
K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.;
Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.;
Jacobson, A.; Pappin, D. J. Mol. Cell. Proteomics 2004, 3, 1154−1169.
(5) Boersema, P. J.; Raijmakers, R.; Lemeer, S.; Mohammed, S.;
Heck, A. J. Nat. Protoc. 2009, 4, 484−494.
(6) Choe, L.; D’Ascenzo, M.; Relkin, N. R.; Pappin, D.; Ross, P.;
Williamson, B.; Guertin, S.; Pribil, P.; Lee, K. H. Proteomics 2007, 7,
3651−3660.
■
CONCLUSIONS
In summary, on the basis of the subtle mass defect differences
between 12C/13C and 1H/2H, the pIDL method was developed
10662
dx.doi.org/10.1021/ac402834w | Anal. Chem. 2013, 85, 10658−10663
Analytical Chemistry
Letter
(7) Mertins, P.; Udeshi, N. D.; Clauser, K. R.; Mani, D. R.; Patel, J.;
Ong, S. E.; Jaffe, J. D.; Carr, S. A. Mol. Cell. Proteomics 2012, 11, M111
014423.
(8) Zhang, G.; Neubert, T. A. Mol. Cell. Proteomics 2006, 5, 401−411.
(9) Ting, L.; Rad, R.; Gygi, S. P.; Haas, W. Nat. Methods 2011, 8,
937−940.
(10) Wenger, C. D.; Lee, M. V.; Hebert, A. S.; McAlister, G. C.;
Phanstiel, D. H.; Westphall, M. S.; Coon, J. J. Nat. Methods 2011, 8,
933−935.
(11) Koehler, C. J.; Strozynski, M.; Kozielski, F.; Treumann, A.;
Thiede, B. J. Proteome Res. 2009, 8, 4333−4341.
(12) Koehler, C. J.; Arntzen, M. O.; Strozynski, M.; Treumann, A.;
Thiede, B. Anal. Chem. 2011, 83, 4775−4781.
(13) Koehler, C. J.; Arntzen, M. O.; de Souza, G. A.; Thiede, B. Anal.
Chem. 2013, 85, 2478−2485.
(14) Hebert, A. S.; Merrill, A. E.; Bailey, D. J.; Still, A. J.; Westphall,
M. S.; Strieter, E. R.; Pagliarini, D. J.; Coon, J. J. Nat. Methods 2013,
10, 332−334.
(15) Yang, S. J.; Nie, A. Y.; Zhang, L.; Yan, G. Q.; Yao, J.; Xie, L. Q.;
Lu, H. J.; Yang, P. Y. J. Proteomics 2012, 75, 5797−5806.
(16) Song, B.; Tang, J. W.; Wang, B.; Cui, X. N.; Hou, L.; Sun, L.;
Mao, L. M.; Zhou, C. H.; Du, Y.; Wang, L. H.; Wang, H. X.; Zheng, R.
S.; Sun, L. World J. Gastroenterol. 2005, 11, 1463−1472.
(17) Song, C.; Ye, M.; Han, G.; Jiang, X.; Wang, F.; Yu, Z.; Chen, R.;
Zou, H. Anal. Chem. 2010, 82, 53−56.
(18) Cox, J.; Mann, M. Nat. Biotechnol. 2008, 26, 1367−1372.
(19) Cox, J.; Neuhauser, N.; Michalski, A.; Scheltema, R. A.; Olsen, J.
V.; Mann, M. J. Proteome Res. 2011, 10, 1794−1805.
(20) Wang, L. H.; Li, D. Q.; Fu, Y.; Wang, H. P.; Zhang, J. F.; Yuan,
Z. F.; Sun, R. X.; Zeng, R.; He, S. M.; Gao, W. Rapid Commun. Mass
Spectrom. 2007, 21, 2985−2991.
(21) Bantscheff, M.; Lemeer, S.; Savitski, M. M.; Kuster, B. Anal.
Bioanal. Chem. 2012, 404, 939−965.
(22) Scigelova, M.; Makarov, A. Proteomics 2006, 6 (Suppl2), 16−21.
(23) Sohn, C. H.; Lee, J. E.; Sweredoski, M. J.; Graham, R. L.; Smith,
G. T.; Hess, S.; Czerwieniec, G.; Loo, J. A.; Deshaies, R. J.;
Beauchamp, J. L. J. Am. Chem. Soc. 2012, 134, 2672−2680.
(24) Nie, A. Y.; Zhang, L.; Yan, G. Q.; Yao, J.; Zhang, Y.; Lu, H. J.;
Yang, P. Y.; He, F. C. Anal. Chem. 2011, 83, 6026−6033.
10663
dx.doi.org/10.1021/ac402834w | Anal. Chem. 2013, 85, 10658−10663