Catch-and-Release Reagents for Broadscale Quantitative Proteomics
Analyses
Carlos A. Gartner, Joshua E. Elias, Corey E. Bakalarski, and Steven P. Gygi*
Department of Cell Biology, Harvard Medical School, 240 Longwood Ave, Boston, Massachusetts 02115
Received November 16, 2006
The relative quantification of protein expression levels in different cell samples through the utilization
of stable isotope dilution has become a standard method in the field of proteomics. We describe here
the development of a new reductively cleavable reagent which facilitates the relative quantification of
thousands of proteins from only tens of micrograms of starting protein. The ligand features a novel
disulfide moiety that links biotin and a thiol-reactive entity. The disulfide is stable to reductive conditions
employed during sample labeling but is readily cleaved under mild conditions using tris-(2-carboxyethyl)
phosphine (TCEP). This unique chemical property allows for the facile use of immobilized avidin in a
manner equivalent to the use of conventional reversible-binding affinity resins. Target peptides are
bound to avidin resin, washed rigorously, then cleaved directly from the resin, resulting in simplified
sample handling procedures and reduced nonspecific interactions. Here we demonstrate the stability
of the linker under two different reducing conditions and show how this “catch-and-release (CAR)”
reagent can be used to quantitatively compare protein abundances from two distinct cellular lysates.
Starting with only 40 µg protein from each sample, 1840 individual proteins were identified in a single
experiment. Using in-house software for automated peak integration, 1620 of these proteins were
quantified for differential expression.
Keywords: peptide • proteome • isotope • labeling • quantification • cleavable • cysteine • mass spectrometry (MS)
• chromatography (HPLC) • automated • parallel • avidin • biotin • purification
Introduction
Quantification of protein abundance changes has had a
major impact on our understanding of their roles in health and
disease. Changes in the expression of a protein resulting from
a particular stimulus may indicate a role for that protein, either
causal or effectual, in biological responses to that stimulus. A
wide variety of factors with the potential to affect protein
expression have been studied including the administration of
xenobiotics,1 cell cycle progression states,2 and cancer,3,4 to
name a few. Typically, the broadest proteome coverage has
been sought to maximize identification of gene products that
may play specific roles in the biological condition under
consideration. Toward this goal, the development of new
reagents and strategies that maximize procedural simplicity and
expand quantification results are greatly valued by the investigator.
The relative quantification of protein expression levels in
different cell samples through the utilization of stable isotope
dilution has become a standard method in the field of
proteomics.5-7 We describe here a new reductively cleavable
linker with chemical properties that make it ideal for use with
isotope labeling protein quantification techniques. It exploits
biotin as an affinity label for cysteine-containing peptide
* To whom correspondence should be addressed.
[email protected]; tel, (617) 432-3155; FAX, (617) 432-1144.
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Journal of Proteome Research 2007, 6, 1482-1491
Published on Web 02/21/2007
E-mail,
fractionation and takes full advantage of conventional avidin’s
high affinity while maximizing specificity. The “catch-andrelease (CAR)” reagent contains a novel hindered disulfide
moiety incorporated into a linker that is completely stable to
reductants employed during alkylation of sample cysteines but
is readily cleaved with phosphine reductants. Although reducible disulfide-containing linkers have been employed in conjunction with biotin for target purification in the past,8,9 their
labile nature, particularly with respect to the potential for sulfur
scrambling with free cysteines, render these reagents far too
reactive for use in quantitative proteomics applications. However, the development of a moiety that is inert to disulfide
scrambling and is selectively cleavable ensures its utility as a
reagent for quantitative applications and allows for the use of
disposable immobilized avidin instead of its less desirable
monomeric counterpart. Captured peptides can be thoroughly
washed and gently eluted with concomitant removal of cleaved
biotin-containing byproducts in a single step resulting in
enriched target peptides derivitized with small mass tags that
can be enriched with heavy isotopes. These tags were shown
to provide linear and reproducible relative quantification, over
a range of 2 orders of magnitude, of cysteine-containing
peptides from a test protein differentially labeled with lightor heavy-labeled CAR reagents. Importantly, the small tag
addition did not have a negative impact upon the quality of
any MS/MS spectrum examined. Finally, as a proof-of-principle
10.1021/pr060605f CCC: $37.00
 2007 American Chemical Society
CAR for Quantitative Proteomics Analyses
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Figure 1. Catch-and-release (CAR) reagents and strategy for their utilization in protein profiling. (a) Structure of the reagent showing
its principle structural features. The unique disulfide moiety imparts upon the linker a marked stability to reduction by DTT, but maintains
sensitivity to TCEP-induced cleavage. The jagged line adjacent to the disulfide group denotes a mixture of isopropyl stereoisomers.
The asterisks represent carbon atoms that carry isotopic density (13C) in the reagent’s heavy version. (b) General procedure for CAR
analysis. Samples are fractionated and trypsinized after labeling protein cysteine residues. Capture of biotinylated peptides on
conventional immobilized avidin and removal of impurities are followed by elution of target analytes directly from the resin by reduction
with TCEP. The masses added to the peptides are very small (145 and 150 amu in light and heavy reagents, respectively).
experiment, we applied the method to compare the steadystate protein expression differences of two widely studied and
disparate cancer cell lines (HeLa and HEK-293 cells). Forty
micrograms of starting material from each cell type were used
to identify more than 2000 proteins, over 85% of which were
quantified. A number of these ratios were confirmed by
Western blot analysis.
Results
Stability of the Hindered Disulfide Linker to Reductive
Conditions. The CAR reagent was designed with a unique
disulfide bridge adjacent to a polyethoxyether linker that
connects biotin and a cysteine-reactive entity (Figure 1a). It is
sterically hindered by a carbon framework making it inert to
cross-reactivity with other cysteine thiols or to reduction by
dithiothreitol (DTT) under alkylating conditions. Tagged cysteine-containing peptides can be bound to conventional immobilized avidin, washed thoroughly, and readily eluted using
tris-(2-carboxyethyl) phosphine (TCEP) as the reductant (Figure
1b).
For the described reagent to be effective, the disulfide must
be stable under conditions employed during reduction of
sample disulfides as premature reduction would result in poor
target recovery and irreproducible quantitative measurements.
To demonstrate the stability of the linker under these condi-
tions, the iodoacetyl portion of the molecule was first quenched
with D-penicillamine (D-Pen), a sterically hindered cysteine
analog that does not react with the reagent’s disulfide moiety,
even under extreme conditions (data not shown). This was
done to avoid complications inherent to observation of multiple
potentially reactive centers (the disulfide and the iodoacetyl
functionalities) and their interactions with reductants. In each
of these experiments, 5 pmol of the light CAR reagent/D-Pen
conjugate were diluted in buffer alone (control) or incubated
with varying amounts of DTT or TCEP under multiple conditions varying reaction temperature, duration, and reductant
(Figure 2a). After stopping the reactions with formic acid, 5
pmol of heavy CAR reagent/D-Pen conjugate (containing five
13
C atoms) were added as an internal standard. An amount of
sample corresponding to approximately 300 fmol of internal
standard was subjected to capillary LC/MS analysis on a linear
ion trap mass spectrometer. Each analysis was extracted as
three separate chromatograms: One corresponded to the mass
of the singly charged uncleaved heavy D-Pen conjugate (MH+
) 832.4 amu) as the internal standard, and the second
corresponded to the singly charged uncleaved light D-Pen
conjugate being tested for stability to reduction (MH+ ) 827.4
amu). The third chromatogram corresponded to the mass of
the singly charged cleaved biotin-containing product derived
from the light D-Pen conjugate (MH+ ) 535.3 amu).
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Figure 2. Measurement of CAR reagent stability to reductive conditions. (a) Conditions were tested for cleavage of a CAR reagent/Dpenicillamine conjugate. (b) Control incubation (no reductant) yielded equal amounts of target (light conjugate, red trace) and internal
standard (heavy conjugate, black trace). (Inset) Mass spectrum of parent ions pertaining to both light and heavy conjugates. (c) Incubation
with 10 mM DTT at room temperature shows no degradation of analyte relative to internal standard after 1 h. (d) Incubation at 50 °C
with 10 mM TCEP cleaved over 97% of the conjugate in 1 h. The cleaved biotin-containing product eluted as a single peak just before
20 min.
Relative to control (Figure 2b), DTT in protein alkylation
buffer containing 6 M urea and 0.05% SDS was completely
ineffective in reducing the disulfide moiety at room temperature, even at 10 mM reductant concentration (Figure 2c). In
this case, the light D-Pen conjugate abundance was indistinguishable from that of the heavy internal standard and no trace
of cleaved product was observed. In contrast, 10 mM TCEP in
buffer containing 20% methanol reduced >97% of the light
D-Pen conjugate starting material within 60 min at 50 °C (Figure
2d). The appearance of cleaved product occurred concurrently
with reduction in this case. In each example shown, the inset
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depicts the mass spectrum derived from the CAR/D-Pen
conjugates eluting just after 20 min in the chromatograms.
Results of all reductive cleavage studies are summarized to
include other conditions tested (Figure 3).
Validation of CAR Reagent Use for Relative Quantification
of a Test Protein: Linearity Studies with R-Lactalbumin.
Bovine R-lactalbumin was used as a test protein to demonstrate
the utility of CAR reagents for relative quantification of a protein
by analysis of its cysteine-containing peptides. Equal amounts
of the protein were alkylated with heavy- and light-labeled CAR
reagents and mixed in differing proportions of heavy- to light-
CAR for Quantitative Proteomics Analyses
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Figure 3. Summary of stability study for the CAR reagent/D-Pen conjugate. The disulfide moiety was stable to reduction by DTT in
buffer A (alkylation buffer containing urea and SDS) under conditions used for protein derivatization. Reduction was achieved under
mild conditions in buffer B (ammonium bicarbonate containing 20% methanol) using TCEP. Data shown are the average of triplicate
experiments. Error bars denote standard deviation.
labeled material. Samples were run on a gradient SDS-PAGE
gel and the bands excised. After in-gel tryptic digestion and
purification on immobilized avidin beads, three cysteinecontaining peptides adducted with light or heavy cleaved CAR
tags were quanitified by LC/MS analysis (Supplementary Figure
1, Supporting Information). The heavy-to-light ratios accurately
reflected expected ratios within 10% based on relative amounts
of material mixed at the protein level, and linearity was
maintained over 2 orders of magnitude. The identities of
recovered peptides were confirmed by sequencing in a separate
LC-MS/MS analysis.
Comparison of Protein Expression in HeLa and HEK-293
Cell Lines using CAR Reagents. Two widely studied and
distinct cell lines were chosen for comparison by CAR analysis
in order to ensure a broad diversity of relative protein expression levels (Figure 4a). Identical amounts of protein (40 µg)
extracted from HeLa and HEK-293 cells were labeled with light
and heavy CAR reagents, respectively. Labeled cell extracts were
then combined and fractionated on a gradient SDS-PAGE gel.
The entire lane was divided into 10 fractions of equal size, and
each section was subjected to in-gel tryptic digestion. Recovered peptides were enriched for those containing cysteine using
immobilized avidin beads. Fractions were treated in parallel
by transferring peptide-bound avidin beads to 1 mL polypropylene filtration tubes (Supelco, Bellefonte, PA) after binding.
All washing and recovery steps were performed in these tubes
using a tabletop centrifuge (200 × g) to collect effluent in plastic
culture tubes (13 × 100 mm). Resulting peptides were freed of
polar impurities using the Stage tip method,10 and subjected
to LC-MS/MS analysis on a hybrid linear ion trap Fourier
transform ion cyclotron resonance (FTICR) mass spectrometer
using a TOP10 method as described.11 The vast majority of
peptide ions from all 10 fractions were detected as signals split
into pairs of light- and heavy-labeled material in the FTICR
survey scans.
Labeled peptides were identified from their MS/MS spectra
throughout each chromatographic run. More than 62 000 MS/
MS spectra were acquired during the analysis of the ten gel
fractions. Tandem mass spectra were searched with the SE-
QUEST algorithm12 using a 100 ppm precursor ion tolerance
against a composite database containing both the forward and
reversed IPI human sequences.13 The use of this target/decoy
database allowed us to determine an estimated false-positive
rate for our dataset as described.14 A final list of peptide
identifications was selected based on several factors including
mass accuracy, tryptic state, Xcorr, ∆Cn, internal tryptic sites,
and the presence of cysteine. The combined use of these factors
resulted in an estimated false positive rate of <0.5% (8
estimated incorrect hits in 3420 total) for the entire dataset at
the peptide level. When data derived from all 10 gel fractions
obtained from the SDS-PAGE gel were combined, 1847 individual proteins (including seven peptides that matched to the
reversed database) were identified from 5615 unique peptide
species (Supplementary Tables 1 and 2, Supporting Information) with an estimated false positive protein identification rate
of 0.4%. Following identification and validation, chromatograms were extracted in an automated fashion for each
precursor ion of a peptide pair and the peaks integrated for
quantification. In all, 1620 proteins were successfully quantified.
The distribution of expression ratios for all quantified proteins
is shown in Figure 4b,c.
A representative example of the quantification procedure is
shown for a peptide derived from elongation factor 2 (EF2,
Figure 5) demonstrating this protein to be approximately 1.5fold more abundant in HEK-293 relative to HeLa cells. This
result was confirmed by Western blot analysis using an
antibody specific for human EF2 (Figure 5b, inset). As demonstrated in Figure 5c and Supplementary Figure 2 (see
Supporting Information), the reagent-modified peptides yield
high-quality MS/MS spectra that are readily interpretable
through automated database searching.
Expression ratio measurements were found to be very
reproducible. For example, filamin A was detected and quantified via 14 peptides (Figure 6). The observed peptide ratios
showed excellent agreement with one another, and an approximately 20-fold higher expression level in HeLa versus
HEK-293 cells was determined. These results were confirmed
via Western blot experiments.
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Figure 4. Summary of the CAR analysis strategy. (a) Overview of procedural steps for profiling protein expression ratios from HeLa
and HEK-293 cells. (b) Protein expression ratio distribution of log2-transformed ratios (heavy:light, HEK293:HeLa) for all quantified
proteins (Supplementary Table 1, Supporting Information). Large differences, often >10 fold, were identified. (c) Examples of protein
classes that are differentially expressed relative to overall protein ratio distribution. Of 1620 protein identifications for which quantitative
data was recorded, 1396 mapped to biological processes according to the PANTHER classification system (https://panther.appliedbiosystems.com). Four of these processes were selected to represent classes that suggest differential expression between 293 and HeLa
cells. Each data point represents a protein’s measured ratio as a function of the fraction of proteins with ratios less than (lower axis)
this value within the depicted classification. Points that lie above zero on the y-axis indicate greater expression in 293 cells, whereas
points that lie below indicate greater expression in HeLa cells.
In addition to the examples of EF2 and filamin A, the relative
expression levels of five other proteins were confirmed by
Western blot analysis (Supplementary Figure 2, Supporting
Information). All examples were chosen based solely on the
availability of appropriate antibodies.
Discussion
Catch-and-release reagents represent an important technological advance in relative quantification of protein expression
levels. Here we demonstrate the effectiveness and simplicity
of the CAR method yielding abundant data from small amounts
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of starting material. The key chemical feature of this new tool
is a cleavable disulfide moiety that was designed to be
extraordinarily inert to reduction by DTT under conditions of
cysteine alkylation, yet maintain a susceptibility to cleavage by
phosphines (Figure 2). Steric hindrance provided by the disulfide-flanking carbon framework on the linker imparts upon
the reagent the ability to easily withstand the reductive conditions employed during labeling of protein cysteine residues by
the iodoacetyl terminus of the reagent. The two methyl carbons
on the terminal side of the disulfide bridge also serve to carry
isotopic label in the heavy CAR reagent counterpart. No
CAR for Quantitative Proteomics Analyses
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Figure 5. Analysis of peptides recovered from immobilized avidin by reduction with TCEP as demonstrated for peptide E581-K594
from human elongation factor 2. All steps shown were performed via automated procedures as described in the text. (a) Base peak
chromatogram showing the peak corresponding to the tagged peptide. (b) Extracted ion chromatograms corresponding to light (HeLa)
and heavy (HEK-293) tagged peptides. Peak integration results agree with Western blot data (Inset, left). The mass spectrum of the
chromatographic peak is included (Inset, right). (c) Peptide identity is derived from its tandem mass spectrum. The MS/MS spectrum
of the heavy labeled peptide is shown.
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Andover, MA) to bypass a synthetic step. The result was a heavy
CAR reagent with five 13C labels instead of six. However, a total
of seven positions are available for isotopic substitution if one
also considers the amide nitrogen derived from nitromethane.
(iii) Use of disposable conventional immobilized avidin- Lysis
can be performed in biocompatible buffers under mild conditions allowing for the linker to be cleaved liberating labeled
targets directly from immobilized avidin. Separate steps for
removal of cleaved biotin are therefore not necessary since this
portion of the molecule remains with the avidin resin and is
discarded. The use of a disposable avidin source also allows
for easy parallel sample handling and broadens the potential
for high-throughput techniques. (iv) Small mass tags remaining
with cysteine-containing peptides leading to easily interpretable
MS/MS spectra- In no case has ligand-induced peptide fragmentation been observed to degrade the quality of CID spectra.
This leads to MS/MS spectral data that are highly amenable to
computer-based peptide sequence identification (for examples,
see Supplementary Figure 2, Supporting Information).
Figure 6. Relative quantification of filamin A (FLNA) in HeLa and
HEK-293 cells by two independent methods. A total of 22 unique
cysteine-containing peptides served to confidently identify the
protein, while 14 of these were used for quantification by the
CAR method. Western blot analysis strongly supported the
results.
evidence of disulfide reduction or cysteine-mediated sulfur
exchange has been observed under alkylation conditions.
Naturally, excessive stability to any reduction would render the
reagent ineffective for its intended purpose, the recovery of
tagged target molecules directly from conventional immobilized
avidin beads. We found that the disulfide linker was readily
cleaved, however, using TCEP as reductant in the presence of
20% methanol (Figure 2d). The critical importance of cosolvent
or denaturant during reduction was discovered when 10 mM
TCEP failed to cleave any detectable amount of CAR reagent/
D-Pen conjugate in buffer lacking methanol, even after 30 min
at 50 °C (data not shown).
Key features of the CAR strategy are summarized below. (i)
Chromatographic equivalence of labeled peptides- The chromatographic coelution of light- and heavy-labeled peptides on
LC-MS/MS analysis was achieved by the use of 13C labels in
the heavy counterpart. (ii) Relatively low financial expense for
isotope incorporation- In the current synthetic scheme, every
position in the molecule that carries isotopic density is derived
from readily available and relatively inexpensive 13C-labeled
starting materials: acetone, nitromethane, and acetic acid
(Supplementary Figure 3, Supporting Information). Although
labeling of all six terminal carbon positions is preferred, we
used a commercially available monoisotopically substituted
iodoacetic acid source (Cambridge Isoptope Laboratories,
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To assess the scale of experiments that might be performed
using the reagents, we compared protein expression ratios from
two widely studied human cancer cell lines, HeLa and HEK293 cells. These samples are derived from vastly different cell
types, epithelial and embryonic kidney cells, respectively, and
were expected to show great differences in abundance of a large
number of proteins. Just 40 µg of starting material from each
cell type were utilized. After labeling in the presence of DTT,
the protein samples were mixed and fractionated via SDSPAGE. Ten gel regions were digested with trypsin followed by
avidin capture of cysteine-containing peptides. In parallel, all
samples were simultaneously and vigorously washed, followed
by gentle elution of target peptides with TCEP which allowed
for greatly reduced levels of polymers commonly found in
affinity isolation experiments. Nearly 2000 proteins were identified and more than 1600 of these quantified from a small
amount of starting material suggesting that the CAR strategy
will be applicable to proteome studies involving samples
derived from limited amounts of primary tissue. Multiple
ongoing investigations in our laboratory are already highlighting the value of this technique in tissue studies.
To ensure correct identifications, researchers often exclude
all proteins identified by a single peptide from further consideration,15 because most incorrect protein identifications fall into
this category. As we have previously shown,13 the majority of
these identifications are correct and represent a large fraction
of all protein identifications. One strength of the CAR strategy
is its ability to simplify complex peptide mixtures making a
greater number of proteins available for identification. However, this necessarily reduces the number of tryptic peptides
one can expect from any given protein, and renders a large
portion of the data unusable if a multiple-peptides-per-protein
standard is applied. Rather than dismissing this potentially
valuable subset of proteins, we applied several lines of evidence
in addition to algorithm-assigned scoring to adjust confidence
in protein identification. These additional measurements include specific protease cleavage, ppm mass accuracy, the
number of unique peptide spectral matches per proteins, and
the signal-to-noise ratio of the ionized peptide in the precursor
MS spectrum. Guided by the distribution of decoy database
hits, we designed filtering constraints that excluded nearly all
false positive protein identifications while retaining the majority
of estimated true positives including those identified by single
peptides. Ultimately, 1840 proteins were selected from 3427
CAR for Quantitative Proteomics Analyses
unique peptide sequences with estimated false-positive rates
of 0.4 and 0.2%, respectively.
Results of automated protein quantification measurements
using the VISTA algorithm are summarized (Figures 4b and 4c).
Figure 4b shows the number of proteins identified from all
fractions as a function of their 293-to-HeLa cell abundance
ratios expressed in log(2). Large differences in protein expression were identified (for example, see Supplementary Figure
2, Supporting Information). The same data are expressed in
Figure 4c via an alternative reprentation in which the y-axis
represents the ratio of HEK-293 to HeLa cell derived proteins
in log(2). The x-axis denotes the fraction of total quantified
proteins with HEK-293:HeLa ratios lower than an individual
protein being considered. For example, the protein with the
highest HEK-293:HeLa ratio (y-value of approximately 6 in log(2)) was found at an x-value of 1.0, because its HEK-293:HeLa
ratio was larger than 100% of other proteins quantified. Data
corresponding to all proteins quantified are plotted in this
manner as the sigmoidal black curve in Figure 4c. A few
attributes of this data are worthy of note. For example, the
y-value corresponding to the median lies just above 0.0, as
described for Figure 4b. Upon careful consideration, it is not a
surprise that the average ratio of quantified proteins lies closer
to 1.2:1.0 than to the expected 1:1 ratio, as the two cell lines
were purposely chosen for their expected marked protein
expression differences. Samples were carefully combined after
CAR labeling in a 1:1 ratio according to measurement of total
protein performed in triplicate for each sample (mg protein/
mL). However, relative quantification reflects relative abundance (µmol protein/mL) such that small deviations from a 1:1
ratio at the abscissa median could be expected in samples as
different as these. For example, a single protein that is very
highly expressed in one cell line, but less so in the other, would
be expected to shift the median significantly. Further support
for the precision of the determined ratios lies in the very close
agreement of ratios determined from different peptides within
the same protein (Supplementary Table 2, Supporting Information). Still more evidence for this assessment rests in the finding
that ratios derived for a subset of proteins annotated according
to the PANTHER classification system (https://panther.appliedbiosystems.com) form general trends in Figure 4c indicating
that certain pathways are more abundant in one cell line than
the other. Four such subsets are included on the graph
demonstrating that DNA repair proteins and those involved in
oxidative phosphorylation are expressed to a higher degree in
HEK-293 cells. Conversely, cell adhesion proteins and surface
receptor mediated signal transduction components are expressed to a higher degree in HeLa cells. Analysis of such trends
will certainly be facilitated in the future when coupled with
CAR analysis.
In conclusion, we have presented a novel reductively cleavable reagent for large-scale quantitative proteomics experiments. We demonstrated its use by identifying nearly 2000
proteins from HeLa and HEK-293 cells and determining relative
expression levels for more than 1600 proteins in a matter of
days. With its utility and simplicity, it is anticipated that this
technique will facilitate quantitative proteomics experiments
on a large scale.
Methods
Preparation of CAR Reagents. General steps to the chemical
synthesis of CAR reagents are shown in Supplementary Figure
3 (see Supporting Information).
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Disulfide Stability Testing. The iodoacetyl moiety of the
reagents was first quenched with D-Pen in order to study the
reactivity of the disulfide itself. In separate tubes, 1.0 mM lightor heavy-labeled CAR reagents were prepared in 50 mM
ammonium bicarbonate buffer (pH ) 8.3) from a 35 mM stock
in storage solution (50% acetonitrile in 0.01% TFA). In a
separate tube, a 10 mM stock of D-Pen was prepared in 50 mM
ammonium bicarbonate (pH ) 8.3). The CAR reagent solutions
were combined 1:1 with the D-Pen solution in separate tubes
and incubated for 2 h in the dark at room temperature. The
final concentration of each CAR/D-Pen conjugate stock solution
was 0.5 mM.
Conjugate stability to reduction by DTT was tested by
diluting light- and heavy-labeled CAR/D-Pen to 10 µM in 100
mM Tris, (pH ) 8.3) containing 6 M urea, 5 mM EDTA, and
0.05% SDS. A stock solution of DTT was prepared in this buffer
at double its target concentration. To a separate 0.5 mL tube
was transferred 5 µL of light-labeled conjugate (25 pmol)
measured with a syringe. An equal volume of DTT solution
freshly prepared at double its target concentration in the
alkylation buffer was then added to initiate reaction. Reactions
were stopped at the appropriate time points using 185 µL of
7.5% acetonitrile containing 5% formic acid. The heavy-labeled
CAR/D-Pen conjugate was then added (5 µL measured with a
syringe, 25 pmol) as an internal standard. The entire sample
was then shaken for 10 min with 10 µL MonoQ resin previously
washed with 7.5% acetonitrile containing 5% formic acid to
remove SDS. A 30 µL portion of the sample was removed and
dried on a centrifugal evaporator. Nonvolatile buffer components were removed by the Stage tip method10 and the
remaining material resuspended in 30 µL 7.5% acetonitrile
containing 5% formic acid. Analysis by microcapillary LC/MS
was performed on a LTQ mass spectrometer using 2 µL of each
sample (approximately 300 fmol internal standard).
The same procedure was employed to test cleavage conditions using TCEP as reductant, except that 50 mM ammonium
bicarbonate (pH ) 8.3) containing 20% methanol was used as
solvent. In cases where the reaction was performed at 50 °C,
both reaction components were preincubated at that temperature for 5 min prior to reaction initiation. As neither SDS nor
urea were used in the cleavage experiments, the MonoQ and
Stage tip purification steps were omitted and 2 µL sample were
used directly for LC/MS analysis immediately after reaction
quenching and internal standard addition.
It is important to note that the highest purity of TCEP
(BioVectra dcl, Oxford CT) was necessary to avoid the appearance of excessive polymeric impurities upon mass spectral
analysis. In every case described here, TCEP stock solutions
were freshly prepared at a concentration of 100 mM in 350 mM
ammonium hydroxide and diluted to its target concentration
in an appropriate buffer. The pH of each solution was tested
with pH strips to ensure it was within the proper range.
Validation of Relative Protein Abundance Measurements
using Bovine R-Lactalbumin. Bovine R-lactalbumin, 40 µg, was
dissolved in 50 µL of a cysteine alkylation buffer previously
described (100 mM Tris, pH ) 8.3, 6 M urea, 5 mM EDTA,
0.05% SDS).16 The sample was degassed at reduced pressure
(0.3 Torr) on a centrifugal evaporator for 30 s. It was then
brought to 1 mM DTT from a 25 mM stock freshly prepared in
water and divided in half. After reducing at 50 °C for 30 min,
the samples were brought to room temperature and light or
heavy CAR reagent added to 5 mM final concentration from a
35 mM stock (stored in 50% acetonitrile containing 0.01% TFA).
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Samples were alkylated in the dark for 2.5 h at room temperature and quenched with 30 mM D-Pen from a 300 mM stock
freshly prepared in water. After 1 h of quenching in the dark
at room temperature, the samples could be stored at 4 °C
overnight if desired. Light- and heavy-labeled proteins were
then mixed in appropriate ratios and 2 µg total protein per lane
were applied to a 1 mm 12% SDS-PAGE gel. No DTT was used
in the sample buffer as all cysteines were considered alkylated.
After Coomassie staining and band excision, in-gel tryptic
digestion was performed as previously described17 and the
peptides extracted. After drying each sample by vacuum
centrifugation, peptides were redissolved in 10 µL of 7.5%
acetonitrile containing 5% formic acid. They were then diluted
with 150 µL of avidin binding buffer (250 mM sodium phosphate [pH ) 6.5] containing 10% glycerol). A blank sample was
also prepared in parallel to ensure that the resulting sample
pH was between 5 and 6. In a separate tube, immobilized avidin
resin (Pierce, Rockford, IL) was washed twice with excess avidin
wash buffer (50 mM sodium phosphate [pH ) 6.0] containing
10% glycerol) and the resin distributed into 0.5 mL tubes, 20
µL packed resin per sample. The wash buffer was removed from
each avidin-containing tube with a syringe and peptide samples
added. Tubes were shaken at room temperature for 1 h, at
which point they were centrifuged and buffer removed with a
syringe. The resin was washed twice with constant shaking
using 250 µL avidin wash buffer each time. Each sample was
then washed three times with 250 µL avidin final wash buffer
(50 mM ammonium bicarbonate [pH ) 8.3] containing 5 mM
EDTA and 20% methanol) before incubating the beads with
50 µL biotin cleavage buffer (avidin final wash buffer containing
5 mM TCEP). The beads were then incubated at 50 °C for 90
min in cleavage buffer. Samples were agitated briefly every 30
min to ensure accessibility of immobilized peptides to the
TCEP. After cooling to room temperature, the overlays were
removed with a syringe and saved in fresh tubes. Resin from
each sample was washed twice with 75 µL of avidin final wash
buffer at room temperature for 15 min with shaking. Washes
were combined with their corresponding recovered samples
and acidified using 10 µL acetic acid. After evaporation to
dryness on a vacuum centrifuge, peptides were freed of
contaminants using the Stage tip method10 and evaporated
again before analysis by LC-MS/MS using the LTQ FT mass
spectrometer.
Comparison of Protein Expression Levels from Two Cell
Lines using CAR Reagents. E1A-transformed Human Embryonic Kidney (HEK) 293 cells (gift from Edward Harlow, Massachusetts General Hospital, Boston, MA) and HeLa cells
(American Type Culture Collection, Manassas, VA) were maintained in Dulbecco’s Modified Eagle’s Medium (Mediatech,
Herdon, VA) supplemented with 10% fetal bovine serum
(Hyclone, Logan, UT), penicillin (20 inhibitory units/mL) and
streptomycin (20 µg/mL). Upon reaching confluence cells were
washed once with ice-cold PBS and lysed to approximately 2
mg/mL protein in 50 mM Tris buffer (pH ) 7.4) containing
150 mM sodium chloride, 2 mM EDTA, 1% octyl glucoside, 0.2%
SDS, 0.5% cholate, and EDTA-free Complete Mini protease
inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)
included according to manufacturer’s instructions. Protein
concentration was measured in triplicate by the BCA assay
(Pierce, Rockford, IL). Samples were diluted 1:1 with cysteine
alkylation buffer (100 mM Tris [pH ) 8.3] containing 6 M urea,
5 mM EDTA, and 0.05% SDS) and dialyzed into 1 L of that
buffer for 3 h using Slide-A-Lyzer MINI dialysis cups (Pierce,
1490
Journal of Proteome Research • Vol. 6, No. 4, 2007
Gartner et al.
Rockford, IL). Protein concentration was again measured and
40 µg of each sample placed into fresh tubes. After a 30 s degassing period on a vacuum centrifuge (0.3 Torr), samples were
brought to 1 mM DTT from a 25 mM stock freshly prepared in
water. Reduction was carried out for 30 min at 50 °C and the
samples cooled to room temperature. Light-labeled CAR reagent was added to the HeLa cell lysate at 5 mM final concentration from its 35 mM stock in storage solvent, whereas
HEK 293 lysate was alkylated with the heavy-labeled reagent.
Reaction was allowed to proceed for 2.5 h in the dark at room
temperature before quenching with 30 mM D-Pen added from
a 500 mM stock freshly prepared in water. After 1 h of
quenching at room temperature, samples were stored at 4 °C
overnight.
Samples were combined and fractionated by SDS-PAGE on
a 1.5 mm gradient gel (NuPage 4-12% Bis-Tris, Invitrogen,
Carlsbad, CA) and stained with Coomassie Brilliant Blue. The
entire lane was divided into 10 sections of equal size and ingel trypsin digestion performed on each fraction. After peptide
extraction and drying, biotinylated fragments were isolated and
purified on immobilized avidin resin exactly as described for
bovine R-lactalbumin analysis. However, all washing and
elution steps were done in parallel using empty solid phase
extraction tubes (Supelco, Bellefonte, PA). The entire portion
of each sample recovered was then analyzed by microcapillary
LC-FTICR-MS/MS analysis as described below.
LC-MS and LC-MS/MS Analyses. Liquid chromatography
tandem mass spectrometry (LC-MS/MS) was performed using
the LTQ FT, which is a hybrid linear (2-D) ion trap-Fourier
transform ion cyclotron resonance (FTICR) mass spectrometer
(7 T, ThermoElectron, San Jose, CA) as described.11 Briefly, the
entire volume of each reconstituted sample was loaded onto a
125 µm × 18 cm fused silica C18 (Magic C18-AQ, 200 Å pore
size, 5 µm diameter, Michrom BioResources, Auburn, CA)
microcapillary column using a FAMOS capillary autosampler
(LC Packings, Sunnyvale, CA) and an Agilent 1100 series binary
HPLC pump (Agilent Corporation, Palo Alto, CA) with an inline flow splitter. Peptides were transferred from the autosampler directly to the resolving column for 20 min at a pressure
of 120 bar in Buffer A (3% acetonitrile, 0.15% formic acid),
followed by gradient elution at 60 bar from 7 to 33% Buffer B
(97% acetonitrile, 0.15% formic acid) over 55 min. Effluent was
directed into a nanospray source of the mass spectrometer
operating at a 2.1 kV source potential. During gradient elution,
ten ion-trap MS/MS spectra were acquired per data-dependent
cycle from a high-resolution (R > 70 000 @ 500 m/z) FTICR
survey spectrum.
Database Searching and Data Processing. Accurate precursor ion masses, MS/MS spectra, and chromatographic information were extracted from the raw file output of the LTQ FT with
in-house software as described.18 Tandem mass spectra, represented in the dta file format, were searched with the SEQUEST algorithm (version 27, revision 12)12 against a concatenated target (forward) and decoy (reversed) human IPI protein
database (ftp.ebi.ac.uk/pub/databases/IPI/current, downloaded on July 4, 2005) with the following restrictions: mass
tolerance of (100 parts-per-million; at least one tryptic terminus per peptide; up to two internal cleavage sites per peptide;
dynamic modifications of 15.99492 for methionine to account
for oxidation; static modifications of 145.05613 on all cysteines
to account for the mass addition of the cleaved light reagent;
dynamic modifications of 5.01677 on cysteines to account for
the potential mass difference of the heavy reagent. Search
research articles
CAR for Quantitative Proteomics Analyses
results and chromatographic data were stored in a PostgreSQL
database.
The SEQUEST-derived scores XCorr, ∆Cn as well as partsper-million accuracy were used to derive filtering cutoffs of
non-redundant top-ranked peptide-spectral matches. An inhouse algorithm similar in principle to one previously described19 adjusted threshold values for these measurements to
maximize sensitivity while maintaining a < 1% false positive
rate, as estimated by the number of decoy database hits.13,14
All peptide matches were secondarily required to have two
tryptic termini, and to contain at least one cysteine residue.
This score-independent method for enriching for correct
identifications allowed for the use of fairly non-stringent scorebased filtering criteria13: Charge ) 2+: XCorr g 1.3, ∆Cn g
0.01; Charge ) 3+: XCorr ) 1.7, ∆Cn g 0.03; Charge ) 4+:
XCorr g 1.8, ∆Cn g 0.01. Analysis was performed on data
collected from each gel fraction independently. Protein identifications were assembled from their constituent identified
peptides in Microsoft Excel. A given protein was selected as
being confidently assigned if at least one uniquely identified
peptide from the protein exceeded filtering criteria described
above, had zero internal cleavage sites, and had two tryptic
ends.
All top-ranked cysteine-containing peptides returned by
SEQUEST were submitted for quantitative analysis by an
automated software suite, VISTA, which we have used previously11,20,21 (Bakalarski et al., manuscript in preparation). Briefly,
the theoretical masses of the heavy- and light-labeled peptides
were calculated from their sequence composition information
and these masses used to extract precursor ion chromatogram
intensities of each variant from FTICR MS spectra. Extracted
candidate peaks were filtered for a mass accuracy of better than
(20 ppm and for the presence of the predicted isotopic
distribution for the peptide. For each isotopic variant, the area
under the curve was separately determined as a function of
elution time, and the relative peptide abundance between the
two samples reported as a ratio of their respective areas.
Quantified peptides were scored for quality using empirically
determined test conditions. Proteins were considered successfully quantified if they met the identification criteria described
above, met minimal ratio quality test conditions, and agreed
with measured ratios across independent identifications of the
protein. Manual validation of many hundreds of chromatograms was also performed.
Acknowledgment. Support for this work was provided
by NIH grants (HG003456 and GM67945). We thank members
of the Gygi lab for helpful discussions and S. Gerber for
technical assistance.
Supporting Information Available: Supplementary
Figures 1-3 and Tables 1 and 2. This material is available free
of charge via the Internet at http://pubs.acs.org.
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