Enhanced sample multiplexing for nitrotyrosine-modified proteins using combined precursor isotopic labeling and isobaric tagging

Article abstract of DOI:10.1021/ac202000v

Current strategies for identification and quantification of 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) generally rely on biotin/avidin enrichment. Quantitative approaches have been demonstrated which employ isotopic labeling or isobaric tagging in order to quantify differences in the relative abundances of 3NT-modified proteins in two or potentially eight samples, respectively. Here, we present a novel strategy which uses combined precursor isotopic labeling and isobaric tagging (cPILOT) to increase the multiplexing capability of quantifying 3NT-modified proteins to 12 or 16 samples using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively. This strategy employs "light" and "heavy" labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides. Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled 3AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT ⁰, TMT⁶, and iTRAQ⁸ reagents and discuss limitations of the strategy.

Full text of DOI:10.1021/ac202000v

Article
pubs.acs.org/ac
Enhanced Sample Multiplexing for Nitrotyrosine-Modified Proteins
Using Combined Precursor Isotopic Labeling and Isobaric Tagging
Rena A. S. Robinson* and Adam R. Evans
̃
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
ABSTRACT: Current strategies for identification and quanti-
fication of 3-nitrotyrosine (3NT) post-translationally modified
proteins (PTM) generally rely on biotin/avidin enrichment.
Quantitative approaches have been demonstrated which
employ isotopic labeling or isobaric tagging in order to
quantify differences in the relative abundances of 3NT-
modified proteins in two or potentially eight samples,
respectively. Here, we present a novel strategy which uses
combined precursor isotopic labeling and isobaric tagging
(
cPILOT) to increase the multiplexing capability of quantify-
ing 3NT-modified proteins to 12 or 16 samples using
commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively.
This strategy employs “light” and “heavy” labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides.
Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled
3
AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in
0
6
8
vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT , TMT , and iTRAQ reagents and discuss
limitations of the strategy.
1
1,15,29−34
yrosine nitration is a common post-translational mod-
peptides.
These approaches rely on reduction of the
T
ification (PTM) to proteins that occurs in vivo especially
NO group to an NH group in Tyr to form 3-aminotyrosine
2
2
under conditions of oxidative and nitrosative stress. This
(3AT). A biotin tag is incorporated to 3AT, and using avidin
affinity chromatography, biotinylated peptides can be isolated.
Quantitative methods for determining differences in the relative
1
−6
modification is associated with a number of pathologies
7
8−11
including neurodegenerative disorders such as Alzheimer’s
1
2,13
7,14
and Parkinson’s diseases
and cardiovascular disease.
abundances of 3NT-modified proteins have been reported
Reaction of NO• with superoxide anion (O2 ) forms
•−
which rely on amine-reactive chemistry.
35−37
Isotopic labeling
−
peroxynitrite (ONOO ), which incorporates a NO group to
methods which allow precursor ion quantification have been
2
1
5
2
37
the side chains of Tyr (or Trp, Phe, Cys, and Met) residues
leading to formation of 3-nitrotyrosine (3NT). Consequences
of 3NT modification to proteins are that it can alter protein
structure, lead to aggregation, increase susceptibility to
degradation, and cause reduced enzymatic activity.
tionally, nitration of proteins may disrupt cell signaling events
that rely on phosphorylation of tyrosine residues.
Proteomic methods to identify nitrated proteins have been
recently reviewed.
demonstrated with H-phenylisothiocyanate and 1-(6-methyl-
36 35
[D0/D3]-nicotinoyloxy)succinimide. In a recent report, the
isobaric tag for relative and absolute quantitation (iTRAQ)
reagent was used to tag 3AT peptides (after the blocking of N-
termini and lysine residues with an acetyl group). In this
approach, no enrichment of 3NT-peptides is carried out;
however, upon doing precursor ion scanning for specific
iTRAQ reporter ions (e.g., m/z 114 and 117) 3NT-modified
1
5,16
Addi-
1
5,17,18
5
,14,18−20
35
The most widely used method to
peptides can be easily identified. Although it was only
detect 3NT-modified proteins is gel electrophoresis combined
with Western blotting detection using an anti-3NT monoclonal
or polyclonal antibody or immunoprecipitation of 3NT-
partially demonstrated, this approach can quantify a total of
eight samples using the iTRAQ 8-plex reagent kit.
Herein, we sought to increase the multiplexing capability of
quantitative proteomics to assess differences in the relative
abundances of 3NT-modified proteins. Through combined
precursor isotopic labeling and isobaric tagging (cPILOT), we
developed a novel strategy that uses commercially available
tandem mass tags (TMT) and iTRAQ reagents in order to
increase the number of samples that can be compared in a
19−21
containing proteins.
Two-dimensional Western blotting
relies however on mass spectrometry (MS) identification of
protein spots and often provides little or no information on the
specific site of 3NT modification. Matrix-assisted laser
2
2,23
desorption/ionization (MALDI)-MS,
mass spectrometry (MS/MS),
electrospray tandem
and liquid chromatog-
raphy coupled with MS/MS have also been employed to
2
2,24,25
26−28
identify nitrated residues in tryptic peptides.
Received: August 2, 2011
Accepted: April 17, 2012
Published: April 17, 2012
Recently, a number of groups have employed enrichment
strategies to isolate, identify, and quantify 3NT-modified
©
2012 American Chemical Society
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Article
single analysis to 12 and 16, respectively. Additionally, the
presence of reporter ions in the mass spectra can be used to
identify nitrated peptides and help to eliminate false positive
identifications that can arise from shotgun proteomics experi-
enzyme:protein and incubated at 37 °C for 24 h. Samples were
flash frozen with liquid nitrogen and stored at −80 °C until
further use.
Synthesis of N-Acetoxy-H -succinimide and N-Acetox-
3
3
8
2
ments. cPILOT builds upon currently used quantitative
y- H -succinimide Reagents. The procedure for synthesis of
N-acetoxy-H -succinimide and N-acetoxy- H -succinimide is
3
3
5,39
2
proteomics strategies.
Proof-of-principle cPILOT experi-
3
3
3
9,40
ments are presented using simple mixtures of in vitro nitrated
bovine serum albumin (BSA) peptides and peptides from
mouse splenic proteins analyzed with nanoflow liquid
chromatography tandem mass spectrometry (LC-MS/MS)
using a LTQ-Orbitrap Velos MS.
described elsewhere.
Briefly, 1.9478 g of N-hydroxysuccini-
mide (NHS, Sigma Aldrich) was added to 4.8 mL of >99%
acetic anhydride (Sigma Aldrich) or 1.9451 g of NHS was
2
added to 4.4 mL of 99% H -acetic anhydride (Sigma Aldrich).
6
Reactions occurred at room temperature under nitrogen with
constant stirring for 15 h. White crystals were collected, washed
thoroughly with hexane, and allowed to dry under vacuum.
METHODS
■
Product yield was 1.9909 and 1.5085 g for N-acetoxy-H -
In Vitro Nitration of BSA. One milligram of BSA (Sigma
Aldrich; St. Louis, MO) was dissolved in 500 μL of phosphat−e
buffer saline (PBS), and a 50 μL aliquot of ∼40 mM ONOO
3
2
succinimide and N-acetoxy- H -succinimide, respectively. NMR
3
analysis confirmed formation of the appropriate succinimide
ester (>95% purity).
(
Millipore; Billerica, MA) was added to the solution. The
3
NT Reduction and Isobaric Tagging of Peptides. 3NT
sample was incubated at room temperature for 30 min under
constant stirring (hereafter referred to as 3NT-BSA). The
reaction was quenched by flash freezing in liquid nitrogen. A
control BSA sample was treated in the same manner with the
was reduced to 3AT by the addition of 0.25 M sodium
dithionite in a 150 mol excess to peptide mixtures for a 30
minute incubation at room temperature. For experiments using
0
−
the TMT reagent, 50 μg of the light and heavy labeled 3AT-
exception of addition of ONOO to the buffer solution.
BSA peptides was pooled into a single mixture. To this sample
Samples were stored at −80 °C until further use.
0
(
100 μg) one vial of TMT dissolved in anhydrous dimethyl
Spleen Homogenization. Mouse spleen tissue was
homogenized in an ice-cold phosphate buffer saline (PBS)
solution containing 8 M urea with 100 passes of a Wheaton
homogenizer. Homogenate solution was collected, sonicated,
and centrifuged at 13 000 rpm for 10 min (4 °C). Supernatants
were collected and protein concentrations determined using the
BCA assay according to the manufacturer’s instructions (Pierce
Thermo; Rockford, IL). Samples were stored at −80 °C until
further use.
sulfoxide was added directly to the sample according to the
manufacturer’s instructions. Reactions incubated at room
temperature under constant stirring for 1 h. Excess hydroxyl-
amine hydrochloride (Sigma Aldrich) was used to quench the
reaction. For experiments using the TMT 6-plex reagents
6
(
hereafter referred to as TMT ), light and heavy labeled 3AT-
BSA peptide samples were split into six mixtures each. Each
6
sample was then reacted with the appropriate TMT reagent
Immunochemical Measurement of 3NT-BSA Level.
Control and 3NT-BSA samples (5 μL) were incubated at room
temperature for 20 min with 5 μL of SDS (12% v/v) and 10 μL
of Laemmli buffer [0.125 M Tryzma base, pH 6.8, SDS (4% v/
v), and glycerol (20% v/v)]. Samples (250 ng) were blotted
onto a nitrocellulose membrane and blocked with BSA (3% w/
v) in a PBS solution containing 0.04% (v/v) Tween-20 and
according to the manufacturer’s instructions. Next, portions of
6
the light and heavy labeled TMT -derivatized samples were
mixed into a new sample tube to generate the desired mass
ratios for reporter ions (discussed below). Overall, the total
mass of light and heavy labeled peptides was equal after
pooling. For experiments using the iTRAQ 8-plex reagents
8
(hereafter referred to as iTRAQ ), light and heavy labeled
0
.10 M NaCl (hereafter referred to as Wash blot). A 1:2000
dilution of rabbit polyclonal anti-3NT primary antibody
Sigma-Aldrich) was added, and the blot was incubated for 3
peptide samples (100 μg) were each split into two equal
8
aliquots. The appropriate iTRAQ reagent was dissolved in 70
(
μL of ethanol and directly added to the light or heavy labeled
peptide samples. Reactions incubated at room temperature
under constant stirring for 1 h. Light and heavy labeled peptide
samples were then pooled into a single mixture to generate the
desired iTRAQ8 reagent ratios (discussed below). Finally,
samples were cleaned using Waters Oasis HLB C18 cartridges
and dried using a speedy-vac.
h on a rocker. The blot was rinsed for 5 min in Wash blot three
times and subsequently incubated with a 1:8000 dilution of
antirabbit IgG alkaline phosphatase secondary antibody (Sigma-
Aldrich) in Wash blot for 1 h at room temperature on a rocker.
The membrane was washed as described above and developed
using a solution of 0.2 mM nitrotetrazolium blue chloride and
0
.4 mM 5-bromo-4-chloro-3-indolyl phosphate dipotassium in
LC-MS/MS. Samples were separated on a nanoflow Eskigent
2D LC system using reversed-phase chromatography. Samples
were reconstituted in a water solution with 0.1% formic acid (1
an alkaline phosphatase buffer (0.1 M Tris, 0.1 M NaCl, 5 mM
MgCl2; pH 9.5). The dried blot was scanned on a Canon
scanner and saved as a .TIF image.
Trypsin Digestion. Desired amounts of samples were
subject to trypsin digestion as follows. DTT (0.25 M; Thermo
Fisher; Pittsburgh, PA) was added in a 1:40 protein:reagent
mole excess and incubated at 37 °C for 2 h. Iodoacetamide
−1
μg·μL ) and separated on a nanoflow Eksigent 2D LC system.
Buffer A was composed of 97:3 H O:acetonitrile (ACN) with
2
0.1% formic acid. Buffer B was composed of ACN with 0.1%
formic acid. Samples were injected onto a trapping column
(100 μm i.d. × 2 cm) packed with 200 Å C18 material
(Michrom Bioresources Inc., Auburn, CA) using an autosam-
pler. After a short wash, samples were eluted to a pulled-tip
analytical column (75 μm i.d. × 13.2 cm) packed with 100 Å
C18 material (Michrom Bioresources Inc.). Gradient elution
was performed as follows (% buffer A:% buffer B): 90:10 for 10
min, ramp to 70:30 over 90 min, ramp to 40:60 over 30 min,
ramp to 20:80 over 5 min, hold for 10 min, followed by column
(
1
0.25 M; Acros Organics; Morris Plains, NJ) was added in a
:80 protein:reagent mole excess at 0 °C for 2 h in the dark
followed by addition of 1:40 protein:reagent mole excess L-
cysteine at room temperature for 30 min. Tris buffer (0.2 M
tris, 10 mM CaCl , pH 8.0) was added to the mixture until the
final volume was four times the original volume. TPCK-treated
trypsin (Sigma Aldrich) was added to each sample at 2% w:w
2
4
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Scheme 1. Schematic Representation of the Chemical Derivatization Steps Performed in the cPILOT Strategy
2
equilibration. Eluted peptides were ionized using the nanospray
source (∼1.7 kV) of an LTQ-Orbitrap Velos MS. The MS was
operated using data-dependent acquisition with the following
parameters: full FT parent scans at 60 000 resolution over the
m/z range 300−1600, top seven most intense ions were
selected for CID (isolation width 3 m/z, 35% normalized
collision energy) and HCD (10 ms activation time, isolation
width 3 m/z, 45% normalized collision energy). CID and HCD
MS/MS spectra were recorded in the LTQ and Orbitrap (7500
resolution), respectively.
light (-H ) or heavy (- H ) label, respectively. The second step
3
3
involves the use of sodium dithionite to reduce 3NT to 3AT.
The third major step uses commercially available TMT
reagents (Scheme 1) or the iTRAQ reagent to react with
3AT according to the manufacturer’s instructions. Finally, the
derivatized samples are mixed in a 1:1 light:heavy ratio and
analyzed by LC-MS/MS. We reduce sample loss and potential
loss of low-abundance 3NT-modified peptides by performing a
single cleanup step which occurs after isobaric tagging.
Validation of in Vitro Nitration and Isotopic
Acetylation of BSA. Immunochemistry and LC-MS/MS
experiments were initially performed in order to confirm
nitration and acetylation of BSA. BSA 3NT modification sites
Database Searching and Data Analysis. Raw data files
were searched against the BSA sequence (Swiss Prot Entry
P02769) or mouse International Protein Index Database with
SEQUEST in Proteome Discoverer 1.2 (Thermo). Database
searching parameters include precursor mass tolerance of 15
ppm, fragment mass tolerance of 1.0 Da, static modification of
carbamidomethylation to cysteine, dynamic modifications of
light and heavy acetyl groups at the N-terminus and lysine
residues, methionine oxidation, and special modification for
TMT- or iTRAQ-derivatized 3AT residues. Masses for these
have been well characterized using various enrichment
34,35,41−44
methods.
In total, BSA has 21 Tyr residues (Swiss
0
6
modifications were 239.163 (TMT ), 244.174 (TMT ), and
3
8
19.216 Da (iTRAQ ). False discovery rates of p < 0.05 and
0.01 were set using decoy database searching such that only
medium and high confidence peptides, respectively, were used
for analyses. Intensity and area information for light and heavy
labeled peptides were provided in the software output (.msf
file). Manual calculation of relative reporter ion peak intensity
(
or peak area) ratios was performed due to limitations in the
software output for this cPILOT strategy.
RESULTS AND DISCUSSION
■
Combined Precursor Isotopic Labeling and Isobaric
Tagging (cPILOT). Herein we present a novel approach,
cPILOT, which combines isotopic labeling with isobaric tagging
strategies such as TMT and iTRAQ in order to extend the
number of 3NT-modified samples that can be multiplexed in a
single experiment to 12 and 16, respectively. The strategy
consists of three primary steps as shown in Scheme 1:
acetylation of primary amine groups, reduction of 3NT groups
to 3AT, and isobaric tagging (using TMT or iTRAQ) of 3AT
groups. Nitrated tryptic peptides are initially reacted with N-
Figure 1. Validation of in vitro nitration. (a) Immunochemical blot of
−
BSA incubated with buffer (control) and ONOO after probing with
primary anti-3NT antibody. (b) Example MS/MS spectrum obtained
for a doubly charged peak at t = 117.4 min with m/z 829.3837 that
r
acetoxy-H -succinimide ester or isotopically labeled N-acetox-
2+
3
has been assigned as [(Acetyl)DAFLGSFLY(NO )EYSR + 2H] .
2
2
y- H -succinimide ester in order to acetylate primary amine
Corresponding CID generated b- and y-type fragment ions are labeled
3
groups (i.e., N-termini or lysine residues) with an isotopically
in the spectrum.
4
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0
Figure 2. Demonstration of chemical derivatization using TMT reagents. (a) Integrated parent mass spectrum at t = 54.00−54.45 min across the
r
m/z range 912−920 in which a doubly charged isotopic pair is observed at m/z 913.4019 and 916.4206 for the light and heavy labeled peptide,
respectively. (b) Overlay of the MS/MS spectra obtained after CID of the light and heavy labeled peaks shown in a. It should be noted that each
peptide peak was isolated individually. MS/MS spectra from the light and heavy labeled peptides have been superimposed in this spectrum. b- and y-
type fragment ions (as labeled in the plot) have led to the peptide assignment of [(Acetyl)EY(NH-TMT )EATLEEccAK(Acetyl) + 2H] . The
lowercase letter “c” in the peptide sequence corresponds to carbamidomethylated cysteine. (c) Overlay of the MS/MS spectra obtained after HCD
of the light and heavy labeled peaks shown in a. (d) Zoomed-in low m/z region of the HCD spectra shown in c for the light (lower left) and heavy
0
2+
(
upper right) labeled peptide peaks.
0
Prot Entry P02769) that could potentially be nitrated. Figure 1a
shows an immunoblot with a dark band corresponding to the
3AT-BSA Isotopic Labeling and TMT . Having validated
the presence of nitration and acetylation, 3NT-BSA samples
were reduced to 3AT-BSA, split into two equal aliquots, and
isotopically labeled with either a light or a heavy acetyl group.
Acetylated 3AT-BSA samples were then derivatized with the
3
NT-BSA sample. Under similar reaction conditions, without
−
the presence of ONOO , BSA control samples showed no
immunoreactivity, verifying generation of in vitro nitrated BSA.
Validation of the first step of cPILOT was tested by tryptically
digesting and acetylating (light label) 3NT-BSA followed by
LC-MS/MS analysis on an LTQ-Orbitrap Velos MS. Nitrated
peptides should be shifted in mass according to the number of
added acetyl (42.0106 Da) and nitro (44.9851) groups. Figure
0
TMT reagent that generates reporter ion m/z 126 and pooled
in a 1:1 ratio light:heavy mixture. Figure 2a shows an example
pair of doubly charged peaks at m/z 913.4019 and 916.4206
that have a total mass shift of 6 Da. This mass shift indicates the
presence of two acetylations to the peptide. Upon independent
CID MS/MS of the light and heavy precursor peaks (Figure
2b) this pair has been identified as the peptide [(Acetyl)EY-
1b shows an example of the MS/MS spectrum obtained upon
fragmentation of a peak with a precursor m/z 829.3837. On the
basis of the b- and y-type fragment ions detected in the CID
MS/MS analysis, the peptide has been assigned as [(Acetyl)-
0
2+
(NH-TMT )EATLEEccAK(Acetyl) + 2H] . This tryptic
peptide has been modified with both N-terminal and Lys acetyl
groups, carbamidomethyl groups to Cys, and addition of the
2
+
DAFLGSFLY(NO )EYSR + 2H] . Acetylation of the N-
2
0
terminus was observed in this spectrum as well as the y and y5
TMT reagent to Tyr376. Identification of this peptide is
4
singly charged fragment ions locating the position of nitration
to Tyr355. In this spectrum only Tyr355 was observed as 3NT;
however, we also observe nitration of Tyr357 (data not shown).
Overall, in these experiments we observed nitration of 15 Tyr
residues in which MS/MS spectra were manually validated. The
location of the 3NT-modification sites are as follows: Tyr54,
Tyr108, Tyr161, Tyr163, Tyr171, Tyr173, Tyr179, Tyr180,
Tyr355, Tyr357, Tyr364, Tyr376, Tyr424, Tyr475, and Tyr520.
further supported by the HCD MS/MS spectra obtained for
the light and heavy precursor ions (Figure 2c). Confirmation of
the TMT derivatization can be determined by the presence of
the reporter ion m/z 126.1258 in the HCD spectra for both the
light and the heavy labeled precursor ions (Figure 2d).
It should be noted that fragmentation of acetylated tryptic
peptides generates an immonium ion at m/z 143.118, which
45
upon loss of NH produces a fragment peak at m/z 126.092.
3
4
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6
Figure 3. Multiplexing of 12 independent samples using TMT reagents. (a) Integrated parent mass spectrum at t = 32.65−33.99 min of the doubly
r
charged isotopic pair observed at m/z 488.2824 and 489.7917 for the light and heavy labeled peptide, respectively, that has been assigned as
(Acetyl)AW(NH-TMT )SVAR + 2H] . (b) Zoomed-in low m/z region of the HCD MS/MS spectra obtained upon isolation and fragmentation of
6
2+
[
the light labeled peak shown in a. (c) Zoomed-in low m/z region of the HCD MS/MS spectra obtained upon isolation and fragmentation of the
heavy labeled peak shown in a.
This immonium ion −NH fragment can be detected in the
single experiment that involves identification of nitrated
peptides. Figure 3a shows a precursor ion MS spectrum
obtained for a pair of peaks in 3AT-BSA whereby light and
3
HCD MS/MS spectrum of the light precursor peak; however,
0
at R = 7500 it is resolved from the TMT reporter ion m/z
6
126.1258 in the Orbitrap MS. For lower resolution MS it will
heavy acetylated peptides were derivatized with the TMT
be difficult to discern the contribution of the relative peak
intensity that is derived from acetylated immonium ions −NH3
and TMT reporter ions. This is not the case for the heavy
reagents (reporter ions m/z 126−131). The particular peptide
pair shown at m/z 488.2824 and 489.7917 has been assigned to
6
2+
[(Acetyl)AW(NH-TMT )SVAR + 2H] . The corresponding
mass shift of 3 Da confirms the presence of only one acetyl
group which has been detected on the N-terminus. Addition-
ally, this peptide contains a Trp residue that was nitrated in
precursor ions, in which the immonium ion −NH is shifted by
3
3
Da. For example, in the HCD MS/MS spectrum shown in the
upper right of Figure 2d the immonium ion −NH occurs at m/
z 129.1083. For TMT reagents, in which reporter ions are
3
6
6
vitro as validated by the mass shift corresponding to the TMT
generated at m/z 126−131, lower resolution MS will not be
able to distinguish immonium ion vs reporter ion signal
generated at m/z 126 and 129. Therefore, these reporters
would have to be avoided in low-resolution instruments
especially for analysis of more complex mixtures in which the
relative abundances across samples would not be known a
priori. Another approach is to use iTRAQ reagents in which
reporter ions occur at lower m/z values (i.e., 113−121); this
approach is presented below.
reagent in CID and HCD MS/MS spectra (data not shown).
In order to assess the ability of this approach to measure
quantitative differences across multiple samples, we prepared a
3NT-BSA sample and split it into 12 equal aliquots whereby 6
aliquots were acetylated with the light label and an additional 6
aliquots were acetylated with the heavy label. Each sample was
6
then derivatized with a particular TMT reagent, and samples
were pooled to generate the following mass ratios. For aliquots
6
containing 3AT light labeled and TMT tagged peptides, the 6
Twelve Sample Multiplexing with TMT6 Reagents.
Isobaric tagging strategies have an inherent cost which is
augmented with the desired sample multiplexing capability for
an experiment. This makes analyses expensive for projects
involving a large N, use of technical and/or biological replicates,
and experimental validation (e.g., forward and reverse tagging).
Herein, we demonstrate how cPILOT can be employed in
order to maximize the cost of isobaric tagging strategies for a
aliquots were mixed in a 1:50:10:1:2:0.5 ratio corresponding to
reporter ions m/z 126:127:128:129:130:131. The 6 aliquots
6
containing 3AT heavy labeled and TMT tagged peptides were
mixed in a 1:0.5:2:1:10:50 ratio corresponding to reporter ions
m/z 126:127:128:129:130:131. Overall, the total mass ratios of
the derivatized light labeled:heavy labeled tryptic peptides was
1:1. This is reflected in the precursor peaks measured in the
parent MS (Figure 3a). Figure 3b and 3c shows the presence of
4
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8
Figure 4. Demonstration of chemical derivatization using a single iTRAQ reagent (i.e., reporter ion m/z 113). (a) Integrated parent mass spectrum
at t = 44.93−47.83 min in which a doubly charged isotopic pair is observed at m/z 949.3803 and 952.3989 for the light and heavy labeled peptide,
r
8
2+
respectively, that has been assigned as [(Acetyl)ETY(NH-iTRAQ )GDmADccEK(Acetyl) + 2H] . Lowercase “m” and “c” correspond to
methionine oxidation and cysteine carbamidomethylation, respectively. (b and c) Zoomed-in low m/z region of the HCD MS/MS spectra obtained
for the light and heavy labeled peptide peaks shown in a, respectively.
6
each of the TMT reagents for the light and heavy labeled
precursor ions, respectively. The observed relative abundance
ratios for the light precursor peak (Figure 3b) according to
peak areas are 1.0:38:8.5:1.2:2.9:1.2, and the ratios for the
heavy precursor peak (Figure 3c) are 1.0:5.6:3.6:1.7:4.0:11. The
correlation in measured versus expected reporter ion ratios is
the ion signal for each of the reporter ions and hence
inaccuracies in the measured relative abundances. This is
consistent with the compressed and overestimated reporter ion
6
ratios that were detected in the [(Acetyl)AW(NH-TMT )-
SVAR + 2H]2+ and other peptides detected in this data set.
Similar issues with underestimation of reporter ion abundance
2
2
46,47
linear for both the light (R = 0.9991) and the heavy (R =
.7956) precursor ions. However, the measured reporter ion
ratios have been previously discussed.
Solutions to reduce
0
quantitation errors might involve the use of proton transfer
4
8,49
ratios are compressed in some cases and overestimated in
others deviating from expected reporter ion ratios from sample
preparation. While some error in quantitation can be related to
errors during sample processing, upon closer inspection of the
precursor and CID MS/MS and HCD MS/MS spectra we
attribute the inaccuracy in measured reporter ion ratios to
overlap of the isotopically labeled precursor ions that occurs
reactions to charge reduce precursor ions,
incorporation of
3
50
a MS step, or use of different isotopically labeled reagents
(e.g., dimethylation) that will increase the mass shift between
51
light and heavy labeled precursors in the parent mass scan.
This is the first demonstration of a quantitative proteomics
approach that couples precursor isotopic labeling with isobaric
tagging to enhance sample multiplexing capabilities for 3NT-
modified peptides. Roughly speaking, the relative abundances
for the cPILOT 12-plex study presented above are reflective of
the expected abundances based on sample pooling for most of
the reporter ions. The ability to distinguish immonium ions is
indicated in these spectra (Figure 3b and 3c) by the two
resolved peaks at m/z 126 and 129 in both HCD MS/MS
spectra. On the basis of experimental design, cPILOT offers
inter- and intrasample quantification. For example, cPILOT
6
during isolation in the LTQ. The [(Acetyl)AW(NH-TMT )-
SVAR + 2H]2 peptide shown in Figure 3 is doubly charged
and contains a single label; thus, precursor ions are shifted in
mass by 1.5 m/z. The isolation window used in these studies
was 3.0 m/z (±1.5 m/z). Therefore, it is highly possible that
ions from the heavy labeled precursor m/z were isolated with
the light labeled precursor m/z and vice versa. Isolation of both
precursor ions in one MS scan event would cause an overlap in
+
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Analytical Chemistry
Article
could be employed to control (light) vs diseased (heavy)
samples whereby information regarding the relative change in
abundance across multiple N could be assessed at the level of
precursor ion signal (i.e., average of all control or disease
biological replicates), and intrasample variation can be assessed
at the level of reporter ion signal.
MS/MS spectra (data not shown) this peak has been assigned
8
3+
as [(Acetyl)HPEY(NH-iTRAQ )AVSVLLR + 3H] . The issue
of overlapping precursor ion isolation for the isotopically
labeled pairs is better demonstrated in this data than could be
delineated with the data presented in Figure 3. Theoretically,
the HCD MS/MS spectrum (Figure 5) should only contain the
reporter ions at m/z 113 and 115 since it was generated upon
isolation and fragmentation of the light labeled precursor peak.
However, there is a signal observed from the heavy labeled
precursor ion peak as the reporter ions at m/z 117 and 118 are
also present in the spectrum. Detection of the heavy labeled
reporter ion peaks confirms that overlap in precursor ion
isolation is occurring in the LTQ and is consistent with the
inaccurate abundances measured in the 12-plex experiments
(Figure 3b and 3c).
Figure 5 only displays the HCD MS/MS spectrum obtained
for the light labeled precursor peak; however, accurate reporter
ion abundances for both isotopically labeled peaks are
observed. The expected reporter ion ratios for the light labeled
peak is 1:1 for m/z 113:115 and for the heavy labeled peak 1:4
for m/z 117:118. For the light labeled precursor peak, the
measured reporter ion abundances based on peak intensity are
1:1.1 for m/z 113:115. The heavy labeled precursor peak
measured reporter ion abundances is 1:4.0 for m/z 117:118.
Similar ratio values are obtained using peak areas as opposed to
peak intensities for abundance calculations (see table in Figure
5). These data provide evidence to support that cPILOT allows
differences in relative protein abundances to be measured
across multiple samples.
3AT-BSA Isotopic Labeling and iTRAQ. Figure 4 shows
the extension of cPILOT to iTRAQ multiplex reagents. The
doubly charged peptide pair at m/z 949.3803 and 952.3989
shifted in mass by 6 Da (Figure 4a) has been assigned to
8
2+
[
(Acetyl)ETY(NH-iTRAQ )GDmADccEK(Acetyl) + 2H] .
The number of observed acetyl groups agrees with labeling of
the N-terminus and a Lys residue, carbamidomethylation of
8
Cys, and tagging of Tyr108 with the iTRAQ reagent that
produces reporter ion m/z 113. Addition of the iTRAQ8
reagent to Tyr108 is also confirmed by the presence of the
reporter ion m/z 113 in the HCD MS/MS spectra of both the
light and the heavy labeled precursor ions (Figure 4b and 4c,
respectively). This peptide also contains an oxidized Met
residue in which oxidation could have occurred during the in
vitro nitration reaction. Due to the lower m/z range (i.e., 113−
1
21) of iTRAQ reporter ions there is no overlap with the
acetylated immonium ion −NH that occurs at m/z 126.
3
Multiplexing with iTRAQ Reagents. We designed
another proof-of-principle experiment to assess whether or
not overlap in reporter ion abundances occurs during precursor
ion isolation. Nonoverlapping reporter ion m/z values for
isotopically labeled peptides were purposefully selected for this
experiment. Briefly, two aliquots of light labeled 3AT-BSA were
8
derivatized with iTRAQ reagents that produce reporter ions
In order to demonstrate the potential capability of this
approach to detect nitrated peptides in complex mixtures, the
cPILOT workflow was carried out on tryptic peptides from
mouse splenic proteins. On the basis of the aforementioned
issues, only a four-plex experiment was designed which allowed
us to track the occurrence of precursor ion overlap and evaluate
the reporter ion quantitation. The peptide mixture was split
into four individual aliquots, acetylated with either light or
m/z 113 and 115 and mixed in a 1:1 ratio. Two equal aliquots
of heavy labeled 3AT-BSA were derivatized with iTRAQ8
reagents that produce reporter ions m/z 117 and 118 and
mixed in a 1:4 ratio. Light and heavy labeled samples (1:1 ratio)
were combined into a single mixture for analysis. Figure 5
shows the low m/z region of the HCD MS/MS spectra
generated upon isolation and fragmentation of the light labeled
precursor peak at m/z 548.9827. On the basis of CID and HCD
8
heavy labels, derivatized with iTRAQ reagents, and mixed in a
1
1
:1 ratio for m/z 113 and 114 and 1:1 ratio for m/z 115 and
16, respectively. Figure 6a shows a precursor mass spectrum
for a doubly charged precursor peak pair at m/z 810.972 that is
shifted by 3 Da. This pair has been assigned as the
8
2+
[
(Acetyl)APGISY(NH -iTRAQ )QRLVR + 2H] peptide
2
belonging to phosphoinositide 3-kinase regulatory subunit 5
protein (IPI00400180.1). From the HCD MS/MS spectra
obtained from the light and heavy labeled precursors it is
evident that this peptide was nitrated as indicated by the
presence of the four reporter ions. Additionally, the observed
peak intensity ratios for reporter ions are similar to the
expected values. It is still apparent from the MS/MS spectra
that there is overlap in the precursor isolation indicated by the
presence of the reporter ions at m/z 115 and 116 in the HCD
spectra (Figure 6b) taken upon isolation of the light precursor
peak and the presence of m/z 113 and 114 in the HCD spectra
taken upon isolation of the heavy precursor peak (Figure 6c).
Under ideal conditions, only reporter ions at m/z 113 and 114
should be present in HCD spectra from the light labeled
precursor (similarly only m/z 115 and 116 should be observed
for the heavy precursor). Overall, we identified 371 peptides
which corresponds to 96 mouse splenic proteins. Five proteins
were identified that each contain a single nitrated peptide:
Figure 5. Quantitative demonstration of four multiplexed samples
using iTRAQ reagents. Zoomed-in low m/z region of the HCD MS/
MS spectra obtained upon isolation and fragmentation of the light
8
labeled peak at t = 67.48 min and m/z 548.9827 that has been
r
8
3+
assigned as [(Acetyl)HPEY(NH-iTRAQ )AVSVLLR + 3H] . Ob-
served reporter ion peak intensities and peak areas are displayed in the
tables (top).
phosphoinositide 3-kinase regulatory subunit 5 protein
8
[(Acetyl)APGISY(NH -iTRAQ )QRLVR], nebulin [(Acetyl)-
2
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8
Figure 6. Application of cPILOT to mouse spleen tissue using iTRAQ reagents in a four-plex experiment. (a) Parent mass spectrum at t = 105.03
r
min of the doubly charged isotopic pair observed at m/z 810.972 and 812.481 for the light and heavy labeled peptide, respectively,
8
2+
[
(Acetyl)APGISY(NH -iTRAQ )QRLVR + 2H] of the phosphoinositide 3-kinase regulatory subunit 5 protein (IPI00400180.1) protein from
2
spleen tissue. (b) Zoomed-in low m/z region of the HCD MS/MS spectra obtained upon isolation and fragmentation of the light labeled peak
shown in a. (c) Zoomed-in low m/z region of the HCD MS/MS spectra obtained upon isolation and fragmentation of the heavy labeled peak shown
in a.
8
DY(NH -iTRAQ )DLREDAISIK(Acetyl)], Ep300 protein
Finally, for application of cPILOT to complex peptide mixtures
in which protein abundances are not known a priori it is
necessary to have efficient chemical reactions in order to reduce
complexity in data analysis and interpretation. Isotopic labeling
and commercial TMT and iTRAQ reactions have efficiencies
2
8
[
(Acetyl)KVEGDM(Ox)Y(NH -iTRAQ )ESANNR], Isoform
of Bullous pemphigoid antigen 1 [(Acetyl)-
SIDELNSAWDSLNK(Acetyl)AW(NH -iTRAQ )K(Acetyl)],
and Npas3 protein [(Acetyl)VERY(NH -iTRAQ )-
VESEADLR]. We note that a low percentage (i.e., 1.6%) of
nitrated peptides was identified with modifications on Tyr or
Trp residues from the entire data set. Additional enrichment
steps (i.e., immunoprecipitation of 3NT-modified peptides
prior to cPILOT) may be necessary in order to identify larger
numbers of nitrated peptides in complex mixtures.
Caveats to obtaining accurate quantitative information with
cPILOT relies on minimal to no overlap in the precursor ion
isolation for isotopically labeled peaks, efficient chemical
2
1
8
2
8
2
1
5,52−54
reported to be greater than 98%.
Our results are
consistent with these reported efficiencies.
CONCLUSION
■
This work reports on the first quantitative proteomics approach
that combines precursor ion isolation with isobaric tagging
strategies such as TMT and iTRAQ for analysis of 3NT-
modified proteins. cPILOT can extend sample multiplexing
6
capabilities to 12 and 16 samples with commercial TMT and
reactions, and minimal errors during sample preparation. The
N-acetoxy- H -succinimide reagent used to isotopically label
8
iTRAQ reagents, respectively, and provide identification of
2
3
nitrated peptides through detection of reporter ions in HCD
MS/MS spectra. Despite the observed limitations with the
reagents used for this cPILOT strategy, accurate quantitative
information could be obtained with appropriate precursor
tagging reagents and/or improvements to experimental design.
As 3NT modifications are low abundance, we also note that this
approach can be combined with prior enrichment procedures
for nitrated peptides such as immunoprecipitation or with
selected ion monitoring experiments to search for reporter ions.
Primary advantages with cPILOT include the ability to use
tryptic peptides in these studies can be substituted with other
reagents that will increase the mass shift between light and
heavy labeled peptides, thus eliminating compression and
overestimation of reporter ion abundance ratios. Our laboratory
is currently exploring other precursor isotopic labeling reagents
and/or experimental schemes to improve upon these issues.
Errors associated with sample loss were minimized during these
experiments by performing “one-pot” reactions and using a
single sample cleanup step prior to LC-MS/MS analysis.
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Analytical Chemistry
Article
reporter ion tags to identify nitrated peptides, some reduction
of costs associated with purchasing commercial isobaric tag kits,
and increasing the number of samples analyzed in a single
experiment. The ability to increase multiplexing of proteomics
experiments is an attractive area for many researchers that are
examining expression differences in numerous biological
samples and conditions.
(23) Sarver, A.; Scheffler, N. K.; Shetlar, M. D.; Gibson, B. W. J. Am.
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(
AUTHOR INFORMATION
Corresponding Author
Phone: 412-624-8167. Fax: 412-624-8611. E-mail: rena@pitt.
M. H.; Lacan, G.; Melega, W. P.; Camp, D. G., II; Smith, R. D.; Smith,
■
D. J.; Squier, T. C.; Bigelow, D. J. Biochemistry 2006, 45, 8009−22.
(28) Willard, B. B.; Ruse, C. I.; Keightley, J. A.; Bond, M.; Kinter, M.
*
Anal. Chem. 2003, 75, 2370−6.
edu.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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Biochem. 2003, 320, 214−22.
The authors acknowledge the University of Pittsburgh and the
Society of Analytical Chemists in Pittsburgh for funds to
support this work. We also thank Sage Bowser in the
Department of Chemistry NMR Facility for help with
validation of synthesized reagents and Daret K. St. Clair at
the University of Kentucky for mouse spleen tissue.
(32) Zhang, Q.; Qian, W. J.; Knyushko, T. V.; Clauss, T. R.; Purvine,
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