Targeted mass spectrometry-based approach for protein-ligand binding analyses in complex biological mixtures using a phenacyl bromide modification strategy

Article abstract of DOI:10.1021/acs.analchem.6b02658

The characterization of protein folding stability changes on the proteomic scale is useful for protein-target discovery and for the characterization of biological states. The Stability of Proteins from Rates of Oxidation (SPROX) technique is one of several mass spectrometry-based techniques recently established for the making proteome-wide measurements of protein folding and stability. A critical part of proteome-wide applications of SPROX is the identification and quantitation of methionine-containing peptides. Demonstrated here is a targeted mass spectrometrybased proteomics strategy for the detection and quantitation of methionine-containing peptides in SPROX experiments. The strategy involves the use of phenacyl bromide (PAB) for the targeted detection and quantitation of methionine-containing peptides in SPROX using selective reaction monitoring (SRM) on a triple quadrupole mass spectrometer (QQQ-MS). As proof-of-principle, the known binding interaction of Cyclosporine A with cyclophilin A protein in a yeast cell lysate is successfully detected and quantified using a targeted SRM workflow. Advantages of the described workflow over other SPROX protocols include a 20-fold reduction in the amount of total protein needed for analysis and the ability to work with the endogenous proteins in a given sample (e.g., stabile isotope labeling with amino acids in cell culture is not necessary).

Full text of DOI:10.1021/acs.analchem.6b02658

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Article
Targeted Mass Spectrometry-Based Approach for
Protein-Ligand Binding Analyses in Complex Biological
Mixtures using a Phenacylbromide Modification Strategy
Lorrain Jin, Dongyu Wang, David M. Gooden, Carol Ball, and Michael C Fitzgerald
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02658 • Publication Date (Web): 14 Oct 2016
Downloaded from http://pubs.acs.org on October 17, 2016
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Targeted Mass Spectrometry-Based Approach for
Protein-Ligand Binding Analyses in Complex Biological Mixtures using
a Phenacylbromide Modification Strategy
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RUNNING TITLE: Method for Targeted SPROX Analyses
Lorrain Jin1, Dongyu Wang1,2, Dave M. Gooden1, Carol Ball3,
and Michael C. Fitzgerald1*
1Department of Chemistry, Duke University, Durham, North Carolina 27708
2Current Address: Arbor Research Collaborative for Health, Ann Arbor, MI 48104
3Agilent Technologies, Cary, NC 27511
*Address reprint requests to:
Professor Michael C. Fitzgerald
Department of Chemistry, Box 90346
Duke University
Durham, North Carolina 27708ꢀ0346
Tel: 919ꢀ660ꢀ1547
Fax: 919ꢀ660ꢀ1605
Eꢀmail: michael.c.fitzgerald@duke.edu
REꢀSubmitted to Analytical Chemistry September 21, 2016
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Abstract
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The characterization of protein folding stability changes on the proteomic scale is useful
for proteinꢀtarget discovery and for the characterization of biological states. The Stability
of Proteins from Rates of Oxidation (SPROX) technique is one of several mass
spectrometryꢀbased techniques recently established for the making proteomeꢀwide
measurements of protein folding and stability. A critical part of proteomeꢀwide
applications of SPROX is the identification and quantitation of methionineꢀcontaining
peptides. Demonstrated here is a targeted mass spectrometryꢀbased proteomics
strategy for the detection and quantitation of methionineꢀcontaining peptides in SPROX
experiments. The strategy involves the use of phenacylbromide (PAB) for the targeted
detection and quantitation of methionineꢀcontaining peptides in SPROX using selective
reaction monitoring (SRM) on a triple quadrupole mass spectrometer (QQQꢀMS). As
proofꢀofꢀprinciple, the known binding interaction of Cyclosporine A with Cyclophilin A
protein in a yeast cell lysate is successfully detected and quantified using a targeted
SRM workflow. Advantages of the described workflow over other SPROX protocols
include a 20ꢀfold reduction in the amount of total protein needed for analysis and the
ability to work with the endogenous proteins in a given sample (e.g., stabile isotope
labeling with amino acids in cell culture is not necessary).
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INTRODUCTION
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The ability to characterize proteinꢀligand binding reactions in complex biological
mixtures is experimentally convenient because it eliminates the need for protein
purification. It is also of great fundamental importance because it enables analysis of
proteinꢀligand binding reactions to be made in a more biologically relevant context,
Recently, several mass spectrometryꢀbased proteomics approaches have been
developed for the analysis of proteinꢀligand binding interaction in complex biological
mixtures. These approaches have included the Stability of Protein from Rates of
Oxidation technique (SPROX),1ꢀ5 the pulseꢀproteolysis technique,6,7 and the cellular
thermal shift assay.8ꢀ10 The relative merits of these different strategies will not be
reviewed here, but have been recently summarized by Li and coꢀworkers.11
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One drawback to all of the mass spectrometryꢀbased proteomics approaches
reportedꢀtoꢀdate for the largeꢀscale and unbiased analysis of proteinꢀligand binding
interactions in complex mixtures is that they require relatively large amounts of protein.
For example, the iTRAQꢀ(and TMT)ꢀSPROX and SILACꢀSRPOX protocols, on which we
have previously reported,2,4,5 typically require ~2ꢀ3 mg of total protein for a single
proteinꢀtarget discovery experiment. This amount of protein is easily accessible in the
case of some samples (e.g., cell lysates derived from cultured cell lines). However, it
can be problematic for other samples. For example, a major hurdle in the application of
the above mass spectrometryꢀbased proteomics methods to clinical samples is the
amount of material required for an analysis.
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Here we report on the incorporation of a phenacylbromide (PAB) labeling
strategy into the SPROX technique in order to facilitate targeted SPROX analyses on
small amounts of protein. In SPROX a protein sample is diluted into different chemical
denaturant buffers. The proteins in each chemicalꢀdenaturantꢀcontaining buffer are
reacted with hydrogen peroxide under conditions that favor oxidation of the thioether
group in the side chains of solvent exposed methionine (Met) residues. Ultimately, the
denaturant dependence to the methionine oxidation reaction is determined using
quantitative proteomics methods. Previous proteomic applications of SPROX have
involved quantitative bottomꢀup shotgun proteomic methods using either SILAC or
isobaric mass tagging strategies.2,4 The proteomic readouts previously reported for
SPROX, have also exclusively involved dataꢀdependent acquisition methods.
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The soꢀcalled, PABꢀSPROX strategy reported here involves reaction of the tryptic
peptide mixtures generated in proteomeꢀwide SPROX analyses with PAB to specifically
label nonꢀoxidized methionineꢀcontaining peptides derived from the proteins in each of
the denaturant containing buffers used in SPROX. The use of light and heavyꢀlabelled
PAB makes it possible to identify ligandꢀinduced stability changes observed in SPROX,
in an analogous fashion to that previously described for SILACꢀSPROX.2,4 The
described PABꢀSPROX strategy takes of advantage of the high sensitivity of selected
reaction monitoring (SRM). Thus, it is especially useful for targeted SPROX analyses.
As part of this work, the PABꢀSPROX technique is used to detect the binding of
cyclosporine A (CsA) to cyclophilin A (CypA) in a complex yeast cell lysate. CsA is a
powerful immunosuppressive agent that is known to directly bind CypA.12ꢀ16 The binding
affinity of CsA to CpyꢀA has been previously established using traditional
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techniques,12,13 and using SPROX.1,4,17,18 Here we demonstrate the ability of the PABꢀ
SPROX technique developed here to detect and quantify the binding affinity of CsA to
CypA.
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EXPERIMENTAL SECTION
Materials. The following materials were purchased from SigmaꢀAldrich (St. Louis,
MO): phenacylbromide (PAB), hydrogen peroxide solution (30% w/w), sodium
phosphate monobasic, sodium phosphate dibasic, guanidine hydrochloride, methionine,
sodium lauryl sulfate (SDS), triethylammonium bicarbonate buffer (TEAB), trifluoroacetic
acid (TFA), tris(2ꢀcarboxyethyl) phosphine hydrochloride (TCEP—HCl), Sꢀmethyl
methanethiosulfonate (MMTS), and trypsin from porcine pancreas (proteomic grade).
CsA was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Trichloroacetic
acid (TCA) and acetic acid (Glacial) were purchased from EMD Chemicals, Inc.
(Gibbstown, NJ). Dimethyl sulfoxide (DMSO) and acetonitrile were purchased from
Fisher Scientific (Pittsburgh, PA). Ethyl alcohol (completely denatured) was purchased
from Avantor Performance Materials (Center Valley, PA). Formic acid (FA) was
purchased from Thermo Scientific (Rockford, IL). The reagents used in the 13C6ꢀPAB
synthesis (including aluminum chloride, magnesium sulfate,13C6ꢀbenzene, bromoacetyl
bromide, carbon disulfide, dichloromethane) were purchased from SigmaꢀAldrich (St.
Louis, MO).
Cell Culture and Cell Lysate Preparations. The Saccharomyces cerevisiae
cells were cultured as previously described.2 Cell pellets were lysed by mechanical
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disruption using glass beads (400ꢀ600 ꢁm in diameter) and a Disruptor Genie (Scientific
Industries Inc., Bohemia, NY). The cell lysis buffer (pH 7.4) included 20 mM phosphate
and a cocktail of protease inhibitors including: 1 mM AEBSF, 500 ꢂM Bestatin, 15 ꢂM Eꢀ
64, 20 ꢂM Leupeptin, and 10 ꢂM Pepstatin A (Thermo Scientific, Rockford, IL). The
sample was centrifuged at 15000 X g for 15 min at 4 °C, and the supernatant was used
in the PABꢀSPROX experiments. The total protein concentrations in the cell lysates
used in these studies were typically 5ꢀ10 mg/mL.
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Synthesis for 13C6-PAB. 13C6H5ꢀphenacylbromide (13C6ꢀPAB) was synthesized
according to a previously established procedure.19 Briefly, anhydrous AlCl3 (11.8 g, 89.3
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mmol) was added in small portions to a stirred solution of C6ꢀbenzene (500 mg, 6.0
mmol) and bromoacetyl bromide (646 ꢁL, 7.4 mmol) in CS2 (60 mL). The reaction was
refluxed for 30 min at 45°C and then cooled to room temperature. The product was
poured onto ice, extracted into dichloromethane (once with 75 mL and three times with
25 mL), and washed with water (once with 45 mL and three times with 15 mL). The
organic layer was extracted, dried over anhydrous MgSO4, filtered, and evaporated
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under reduced pressure to give the product C6ꢀPAB (BrCH2CO13C6H5) as light yellow
solid.
PAB-SPROX Protocol. In Experiment 1, a yeast cell lysate (5 ꢁg/ꢁL) was
divided into two 180 ꢁL portions. A 20 ꢁL aliquot of a 10 mM CsA solution prepared in
DMSO was added to one portion of the cell lysate to generate the (+) ligand sample,
and 20 ꢁL of DMSO was added to the other portion of the cell lysate to generate the (ꢀ)
ligand sample. The (+) and (ꢀ) ligand samples were equilibrated for 1 hr at room
temperature before 20 ꢁL aliquots of the (+) and (ꢀ) ligand samples were each
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combined with 25 ꢁL of a series of GdmClꢀcontaining buffer solutions (pH 7.4)
containing 20 mM phosphate and concentrations of GdmCl ranging from 0 to 8 M. The
final GdmCl concentrations in the 9 GdmCl buffers used in Experiment 1 were 0, 0.5,
1.0, 1.4, 1.8, 2.0, 2.5, 3.0, and 3.5 M. The final concentration of CsA in each GdmClꢀ
containing buffer was 400 µM. The protein samples in the GdmClꢀcontaining buffers
were equilibrated for 1 hr at room temperature before the methionine oxidation reaction
in SPROX was initiated with the addition of 5 ꢁL of 9.8 M H2O2. The oxidation reactions
were quenched with 1 mL of 300 mM methionine solution after 3 min. The proteins in
each GdmCl containing buffer were precipitated upon addition of 250 ꢁL of TCA and
overnight incubation on ice. The samples were centrifuged at 8000 rcf for 30 min at 4 °C,
and each supernatant was decanted. The protein pellets were each washed three times
with 300 ꢁL of iceꢀcold ethanol. The protein pellets were each dissolved in 35 ꢁL of 0.5
M TEAB with 0.1 % final concentration of SDS. The samples were vortexed, heated at
60 °C, and sonicated for 10 min at a time, for 2ꢀ3 cycles. The disulfide bonds were
reduced with a final concentration of 5 mM TCEP for 1 hr at 60 °C. Cysteine residues
were reacted in the presence of 10 mM MMTS for 10 min at RT.
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The proteins were digested overnight with 1.0 ꢁL of 1 mg/mL trypsin at 37 °C.
After addition of 20 ꢁL HOAc and 30 ꢁL ACN, the tryptic peptides generated in the (ꢀ)
and (+) ligand samples were derivatized with light and heavy PAB (respectively) by
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adding 10 ꢁL of 1 M solutions of 12C6ꢀPAB or C6ꢀPAB to (ꢀ) and (+) ligand samples
(respectively). The 12C6ꢀPAB or 13C6ꢀPAB solutions were freshly prepared in acetonitrile
(ACN). After 24 hr, the (ꢀ) and (+) ligand samples at the same denaturant concentration
were pooled together (Figure 1). The PAB reaction conditions were identical to those
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previously reported in reference 21. The selectivity of the PAB labelling reaction for
unoxidized methionineꢀcontaining peptides was confirmed in control experiments
performed on a model peptide (see Supporting Information).
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The PABꢀSPROX protocol used in Experiment 2 (i.e., the control experiment)
was identical to that described above for Experiment 1, except that 20 ꢁL of DMSO was
added to both 180 ꢁL portions of lysate. The PABꢀSRPOX protocol used in Experiment
3 (i.e., the high sensitivity analysis) was also the same as that described in Experiment
1, with the following exceptions. The initial concentration of the cell lysate was 0.5
mg/mL instead of 5 mg/mL. The final GdmCl concentrations in the denaturantꢀ
containing buffers were 0, 1.4, 1.8, and 3.5 M. After the H2O2 oxidation reaction was
quenched by methionine, 90 ꢁg of an MCFꢀ7 cell lysate was spiked in each sample to
facilitate the TCA precipitation.
LC-MS/MS Analysis. The PABꢀlabelled tryptic peptides were desalted using C18
resin according to the manufacturer’s protocol and analyzed by LC−MS/MS using an
Agilent 6460 series triple quadrupole mass spectrometer (QQQꢀMS) equipped with a
ChipCube (Agilent Technologies, Inc., Santa Clara, CA). Peptides were concentrated
and desalted on a trapping column (100 ꢂm ID × 20 mm) and ultimately separated on
an analytical column (75 ꢂm ID × 150 mm) using the following nanoflow pump gradient:
3−35% B in 52 min, 35−85% B in 3 min, and 85% B for 2 min. Solvent A contained
0.1% formic acid (v/v) in H2O; and solvent B contained 0.1% formic acid in CH CN. The
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flow rate used for the separation was 0.40 ꢁL/min. The QQQꢀMS was operated in a
targeted SRM mode using 4ꢀ6 selected transitions (see Table 1) depending on the
experiment. The dwell time of each transition was 40 ms. The fragmentor and cell
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acceleration voltages were set at 135 V and 7 V, respectively. Tandem MS was
performed by CID fragmentation using a collision energy of 5 V. For each LCꢀMS/MS
run, approximately 200ꢀ500 ng of material were injected.
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Proteomic Data Analysis. SRM transitions of targeted methionineꢀcontaining
peptides were determined by adding the mass of the tag, 119.1 Da and 125.1Da for
12C6ꢀPAB and 13C6ꢀ PAB (respectively), and subtracting the mass of the neutral loss
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fragment, 166.0 Da and 172.0 Da for C6ꢀPAB and C6ꢀ PAB (respectively). Also, the
PAB modification incorporates a preꢀformed positive charge on the peptide (Figure 2),
which was taken into consideration for the m/z calculations of the sulfonium ionꢀ
containing precursors. For the SRM transitions determined in each experiment (see
Table 1), an extracted ion chromatogram was generated using Agilent MassHunter
Quantitative Analysis Software Version B. 06. 00 (Agilent Technologies, Inc., Santa
Clara, CA). Ultimately, the areas under the extracted ion chromatogram (EIC) peaks
were used to generate L/H ratios. Hits were selected (i.e., ligand binding was detected)
based on the evaluation of L/H ratios that were significantly departed from 1 (Figure 3).
The L/H ratios with significant departures from 1 were taken to be those that were
changed by greater than 1.7ꢀfold. This 1.7ꢀfold threshold was the same as that
previously utilized in our SILACꢀSPROX and SILACꢀpulse proteolysis protocols for
protein folding and ligand binding analysis.4ꢀ6 In those protocols, the selection of a 1.7ꢀ
fold cutꢀoff values was based on global analyses of all the L/H values recorded in each
experiment where greater than 90% of all the measured L/H ratios were typically within
1.7ꢀfold of the median.
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RESULTS AND DISCUSSION
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Experimental Workflow. The PABꢀSPROX protocol developed here (Figure 1)
involves incubation of a protein sample (e.g., cell lysate) with and without ligand, dilution
of the (ꢀ) and (+) ligand samples into a series of different chemical denaturant buffers,
and subsequent reaction of the thioether group in solvent exposed side chains of
methionine (Met) residues with hydrogen peroxide. The oxidation reaction, the
conditions of which are specifically tuned to selectively oxidize the thioether group in
exposed methionine resides, is quenched with excess methionine. The proteins in each
denaturant containing buffer are precipitated and digested with trypsin. The tryptic
peptides generated in the (ꢀ) and (+) samples are reacted with light and heavy labeled
PAB to incorporate a fixedꢀcharge sulfonium ion into the thioether sideꢀchain of
unoxidized methionine residues in peptides (Figure 2).20,21 The tryptic peptide mixtures
generated from the (ꢀ) and (+) ligand samples at the same denaturant concentrations
are combined, and each of the combined samples are submitted to quantitative LCꢀ
MS/MS analysis using a SRM method.
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In the LCꢀMS/MS analysis the PABꢀlabeled methionine side chain undergoes
gasꢀphase fragmentation during CID, and produces a fixed mass neutral loss and a
specific product ion (Figure 2). Thus, it is possible to use an SRM method to detect the
transition between the sulfonium precursor ion and the postꢀfragmentation product ion.
Ideally, the collision energy is tuned to optimize the yield of the product ion (Figure 2)
and minimize cleavage of the peptide backbone. The same transitions are scheduled
for the lightꢀ and heavyꢀPABꢀlabeledꢀmethionineꢀcontaining peptides of interest. This
enables the resulting light to heavy ratios to report on the relative extent to which the
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target methionineꢀcontaining peptide was oxidized in the SPROX analysis of the (ꢀ) and
(+) ligand samples (Figure 3). Methionineꢀcontaining peptides from proteins with
ligandꢀinduced changes in their chemical denaturation behavior will yield L/H ratios that
depart from 1, whereas proteins not effected by the ligand will yield L/H ratios that are 1
at each denaturant concentration (Figure 3). As in iTRAQꢀ and SILACꢀSPROX, a
methionineꢀcontaining peptide probe used in PABꢀSPROX need not map to back to the
specific ligand binding site in a protein to be useful for the detection of proteinꢀligand
binding. This is because one methionineꢀcontaining peptide probe can report on the
stability and ligand binding properties of the entire protein folding domain to which it
maps.
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CsA Binding Experiment. The PABꢀSPROX protocol (Figure 1) was initially
evaluated in a SPROX experiment (Experiment 1) that involved the use of 9 denaturant
concentrations and 1.8 mg of total protein from a yeast cell lysate (i.e., ~100 ꢁg of total
protein at each denaturant concentration in the (ꢀ) and (+) ligand samples. This amount
of protein was comparable to that typically utilized in the iTRAQꢀSPROX and SILACꢀ
SPROX protocols on which we have previously reported.2,4 The +2 and +3 charge
states of the VIPDFMLQGGDFTAGNGTGGK peptide from CypA and the +3 charge
state of the VETGVIKPGMVVTFAPAGVTTEVK peptide from translation elongation
factor protein were targeted in the LCꢀMS/MS analysis.
The L/H ratios evaluated in Experiment 1 for the methionine containing peptide
from CypA showed significantly altered L/H ratios (>1.7ꢀfold change) at 5 consecutive
GdmCl concentrations, 1.0, 1.4, 1.8, 2.0 and 2.5 M (Figure 4A). The data are consistent
with a CsAꢀinduced stabilization of CypA. In contrast, the L/H ratios evaluated in
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Experiment 1 for the methionine containing peptide from TEF1 were not significantly
changed at any two consecutive denaturant concentrations (Figure 4A). The data are
consistent with TEF1 not interacting with CsA.
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Quantitation of Ligand Binding. In the case of ligandꢀinduced stabilization
reactions detected by PABꢀSPROX, a dissociation constant (Kd value) can be
calculated from the observed denaturant dependence of the measured L/H ratios. The
calculation of Kd values from PABꢀSPROX data is directly analogous to that which we
have previously described for SILACꢀSPROX.17 It involves the calculation of a binding
free energy (∆∆Gf) using equation 1.
∆∆ꢀꢁ = −ꢂ × ∆ꢃꢉ/ꢊ
Equation 1
ꢄꢅꢆꢇꢈ
In equation 1, m is the δ∆Gf/δ[GdmCl] and ∆C1/2 SPROX is the SPROX transition midpoint
shift upon ligand binding. The m value used for cyclophilin in these calculations was 3.7
kcal molꢀ1 Mꢀ1, which was previously determined by experiment.22 The ∆C1/2
from
SPROX
the experiment was 1.5 M (1.0 M to 2.5 M) (Figure 4A).
The dissociation constant (Kd value) is ultimately calculated using equation 2.
[ꢍ]
ꢑ
ꢒꢓꢔ
ꢋꢌ =
Equation 2
∆∆ꢐ
ꢏ
(ꢎ
ꢕꢉ)
In equation 2, [L] is the molar concentration of the free ligand, n is the number of
independent binding sites (in all cases, it was assumed that n =1), R is the universal gas
constant and T is the reaction temperature in Kelvin (K). The CsA concentration in this
experiment, 400 ꢁM, was assumed to be in large excess over the CypA concentration in
the protein samples. Therefore, the free ligand concentration was estimated to be 400
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ꢁM. The Kd value calculated from the PABꢀSPROX data in Figure 4A was 34 nM, which
is within the range of Kd values (30ꢀ200 nM) previously reported for the CsAꢀCypA
complex.12,13,16
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Control Experiment. A control experiment (Experiment 2) was also performed in
which two identical protein samples from a yeast cell lysate were subjected to the
protocol outlined in Figure 1. In the control experiment approximately 100 ꢁg of total
protein was distributed into each of 9 chemical denaturantꢀcontaining buffers. Two
charge states of the VIPDFMLQGGDFTAGNGTGGK peptide from CypA (the known
target of CsA) were targeted in the LCꢀMS/MS readout. Also targeted was the
VETGVIKPGMVVTFAPAGVTTEVK peptide from TEF1, which (based on the above
results) was not found to be a target of CsA. The L/H ratios observed for both of the
targeted peptides were relatively constant and close to 1 (Figure 4B), as would be
expected for the control (i.e., no differential stability changes were detected).
High Sensitivity Analysis. The primary motivation for developing the PABꢀ
SPROX protocol was to reduce the amount of protein material required for SPROX. To
this end, we investigated the minimal amount of yeast lysate that could be used in the
CsAꢀCypA binding experiment. The total amount of yeast cell lysate protein per
denaturant concentration was reduced from 100 ꢁg in Experiment 1 to 10 ꢁg in
Experiment 3. In Experiment 3, a minimum number of denaturant concentrations was
also used. The PABꢀSPROX protocol described here is meant to be used in validation
studies of protein hits identified in proteinꢀtarget discovery experiments using the
iTRAQꢀSPROX protocol. Thus, the specific methionineꢀcontaining peptide(s) and the
specific denaturant concentrations at which there are significant differences between
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the (ꢀ) and (+) ligand samples in the SPROX experiment will generally be known for
most PABꢀSPROX applications. For example, previous SPROX analyses of CypA in
the presence and in the absence of CsA revealed that the SPROX transition midpoints
shift from ~1 M to 2.5 M GdmCl in the presence of ligand.4,17 Thus, in Experiment 3,
we performed the PABꢀSPROX experiment using only 4 denaturant concentrations, 0,
1.4, 1.8, and 3 M GdmCl. In theory, one carefully chosen denaturant concentration that
is between the SPROX transition midpoints of the (ꢀ) and (+) ligand samples could be
used to detect ligand binding in the PABꢀSPROX experiment. In practice, it is
advantageous to use at least two denaturant concentrations, including one that is and
one that is not between the SPROX transition midpoints of the (ꢀ) and (+) ligand
samples. This helps confirm that there is a denaturant dependence to the altered L/H
ratio. Such a denaturant dependence is important to establish, as any endogenous
oxidation or oxidation due to sample handling could also produce altered L/H ratios, but
such oxidation is not expected to be denaturant dependent. The results obtained from
targeting the +2 charge state of the VIPDFMLQGGDFTAGNGTGGK peptide from CypA
in Experiment 3 are shown in Figure 4C. The decreased L/H ratios observed at 1.4 and
1.8 M GdmCl are consistent with the CsAꢀinduced stabilization expected for CypA.
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The successful application of the PABꢀSPROX protocol to the analysis of small
amounts of protein (i.e., 10 ꢁg of total protein per denaturant concentration) in
Experiment 3, required addition of ~90 ꢁg of total protein from an MCF 7 cell lysate to
the yeast cell lysate samples in the denaturantꢀcontaining buffers after they were
reacted with H2O2 and quenched with excess methionine. Addition of the MCFꢀ7 cell
lysate proteins was necessary to enhance the yield of yeast cell lysate proteins in the
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TCA precipitation step. The TCA precipitation step in SPROX is necessary to remove
the denaturant and excess methionine in each of the protein samples before they are
submitted to a bottomꢀup proteomics analysis. Using 10 ꢁg of total protein in each
denaturantꢀcontaining buffer, we found that the yield of precipitated protein was low, as
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judged
VIPDFMLQGGDFTAGNGTGGK peptide from cyclophilin A) in the SRM experiment.
Addition of the MCFꢀ7 lysate made it possible to detect the
by
our
inability
to
detect
target
peptides
(such
as
the
VIPDFMLQGGDFTAGNGTGGK peptide starting from as little as 10 ꢁg of total protein
from our yeast cell lysates.
Addition of the MCF7 cell lysate to the yeast cell lysate samples increases the
complexity of the mixture to be analyzed by LCꢀMS/MS. However, at least in the
example highlighted in this proofꢀofꢀprinciple study, it did not compromise our ability to
detect and quantify the VIPDFMLQGGDFTAGNGTGGK peptide in the SRM experiment.
Clearly, an important consideration in the selection of the cell lysate used to facilitate
the protein precipitation step in high sensitivity PABꢀSPROX experiments is that the
methionineꢀcontaining peptide targeted in PABꢀSPROX must be unique to the test
lysate (i.e., not found in the lysate that is spiked into the test lysate). This was indeed
the case for the yeast CsA binding experiment described here. The human CypA
protein
in
the MCFꢀ7
lysate
does
not
have
the
tryptic
peptide
VIPDFMLQGGDFTAGNGTGGK in its sequence.
Characteristics of PAB-modified peptides. In the course of these studies we
found that PABꢀmodified peptides have a range of different retention times compared to
the unmodified peptides. This makes it difficult to utilize scheduled SRMs in the PABꢀ
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SRPOX experiment unless the retention times of the PABꢀmodified peptides are preꢀ
determined. One disadvantage of not being able to predict the retention time of PABꢀ
modified peptides is that there can be a background of peaks in the EIC. However, use
of the light and heavyꢀlabeled PAB in the described protocol makes it relatively straight
forward to select the appropriate peaks in the EIC, as the correct peaks will have nearly
identical retention times (Figure 5).
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The neutral loss observed with PABꢀlabeled methionineꢀcontaining peptides is
generated using a lower collision energy than is typically utilized for CID fragmentation
of the peptide backbone. On the one hand, this means that only one
transition/peptide/charge state can be probed. On the other hand, this is advantageous
because it significantly reduces the noise in the SRM experiment. Another attractive
feature of the PAB modification is that the sulfonium ion remaining after the neutral loss
holds a positive charge (Figure 2). This charged modification has the potential to
increase the ionization efficiency of methionine containing peptides.
CONCLUSIONS
The results of the proofꢀofꢀprinciple study described here demonstrate the utility
of a new PABꢀSPROX protocol for detection and quantitation of proteinꢀligand binding
interactions in complex biological mixtures. The described protocol, which involves the
PABꢀderivatization of methionineꢀcontaining peptides for their targeted detection and
quantitation in SPROX experiments, has important advantages over previously reported
iTRAQꢀ and SILACꢀSPROX protocols. Unlike SILACꢀSPROX, the PABꢀSPROX
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protocol can be directly applied to the endogenous proteins in a sample (i.e., cell
culturing methods to generated isotopically labeled material are not required).
Compared to iTRAQꢀSPROX, the PABꢀSPROX protocol requires significantly less
protein material. Demonstrated here is the successful detection of the CsA binding to
CypA in a yeast cell lysate using just 80 ꢁg of total yeast cell lysate protein. This
amount of material is ~20ꢀfold less than that required in a typical iTRAQꢀSPROX
experiment. The PABꢀSPROX protocol relies on an SRM workflow. The ultimate
sensitivity gains realized in any given application will largely depend on the relative
abundance of targeted methionineꢀcontaining peptide and the sensitivity of the mass
spectrometry instrumentation employed for the analysis.
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ASSOCIATED CONTENT
Additional information as noted in the text. This material is available free of charge via
the Internet at http://pubs.acs.org
ACKNOWLEDGEMENT
This work was supported by a grant from the US National Science Foundation
(CHEꢀ1308093) to M.C.F. The authors also thank Ameritox for providing access to their
mass spectrometry instrumentation during the early stages of this project.
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REFERENCES
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9
(1) West, G. M.; Tucker, C. L.; Xu, T.; Park, S. K.; Han, X. M.; Yates, J. R.; Fitzgerald, M.
C. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 9078ꢀ9082.
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(2) Strickland, E. C.; Geer, M. A.; Tran, D. T.; Adhikari, J.; West, G. M.; DeArmond, P.
D.; Xu, Y.; Fitzgerald, M. C. Nat. Protoc. 2013, 8, 148ꢀ161.
(3) Xu, Y. R.; Strickland, E. C.; Fitzgerald, M. C. Anal. Chem. 2014, 86, 7041ꢀ7048.
(4) Tran, D. T.; Adhikari, J.; Fitzgerald, M. C. Mol. Cell. Proteomics 2014, 13, 1800ꢀ1813.
(5) Adhikari, J.; West, G. M.; Fitzgerald, M. C. J. Proteome Res. 2015, 14, 2287ꢀ2297.
(6) Adhikari, J.; Fitzgerald, M. C. J Am Soc Mass Spectr 2014, 25, 2073ꢀ2083.
(7) Na, Y. R.; Park, C. Protein Sci. 2009, 18, 268ꢀ276.
(8) Savitski, M. M.; Reinhard, F. B. M.; Franken, H.; Werner, T.; Savitski, M. F.;
Eberhard, D.; Molina, D. M.; Jafari, R.; Dovega, R. B.; Klaeger, S.; Kuster, B.; Nordlund,
P.; Bantscheff, M.; Drewes, G. Science 2014, 346, 1255784ꢀ1 ꢀ1255784ꢀ10.
(9) Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundback, T.; Nordlund,
P.; Molina, D. M. Nat. Protoc. 2014, 9, 2100ꢀ2122.
(10) Franken, H.; Mathieson, T.; Childs, D.; Sweetman, G. M. A.; Werner, T.; Togel, I.;
Doce, C.; Gade, S.; Bantscheff, M.; Drewes, G.; Reinhard, F. B. M.; Huber, W.; Savitski,
M. M. Nat. Protoc. 2015, 10, 1567ꢀ1593.
(11) Li, J.; Xu, H.; West, G. M.; Jones, L. H. Med. Chem. Comm. 2016, 7, 769ꢀ777.
(12) Handschumacher, R. E.; Harding, M. W.; Rice, J.; Drugge, R. J. Science 1984, 226,
544ꢀ547.
(13) Harding, M. W.; Handschumacher, R. E. Transplantation 1988, 46, S29ꢀS35.
(14) Schreiber, S. L. Science 1991, 251, 283ꢀ287.
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(15) Schreiber, S. L.; Crabtree, G. R. Immunol. Today 1992, 13, 136ꢀ142.
(16) Liu, J.; Albers, M. W.; Chen, C. M.; Schreiber, S. L.; Walsh, C. T. Proc. Natl. Acad.
Sci. U.S.A. 1990, 87, 2304ꢀ2308.
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(17) Graham M. Wewst, L. T., Michael C. Fitzgerald. Anal. Chem. 2008, 80, 4175ꢀ4185.
(18) West, G. M.; Thompson, J. W.; Soderblom, E. J.; Dubois, L. G.; DeArmond, P. D.;
Moseley, M. A.; Fitzgerald, M. C. Anal. Chem. 2010, 82, 5573ꢀ5581.
(19) Froelich, J. M.; Kaplinghat, S.; Reid, G. E. Eur. J. Mass Spectrom. 2008, 14, 219ꢀ
229.
(20) Reid, G. E.; Roberts, K. D.; Kapp, E. A.; Simpson, R. I. J. Proteome Res. 2004, 3,
751ꢀ759.
(21) Amunugama, M.; Roberts, K. D.; Reid, G. E. J. Am. Soc. Mass Spectrom. 2006, 17,
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(22) Wang, M. Z.; Shetty, J. T.; Howard, B. A.; Campa, M. J.; Patz, E. F.; Fitzgerald, M.
C. Anal. Chem. 2004, 76, 4343ꢀ4348.
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Table 1. Summary of SRM transitions used in the experiments summarized here.
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Protein
Name
Precursor Precursor Product
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Peptide Sequence
z
m/z
m/z
CPR1 VIPDFM(12C6ꢀPAB)
2
1101.0
1018.0
LQGGDFTAGNGTGGK
CPR1 VIPDFM (13C6ꢀPAB)
2
3
3
3
3
1104.0
734.4
736.4
850.1
852.1
1018.0
679.0
679.0
794.8
794.8
LQGGDFTAGNGTGGK
CPR1 VIPDFM(12C6ꢀPAB)
LQGGDFTAGNGTGGK
CPR1 VIPDFM(13C6ꢀPAB)
LQGGDFTAGNGTGGK
TEF1 VETGVIKPGM(12C6ꢀPAB)
VVTFAPAGVTTEVK
TEF1 VETGVIKPGM(13C6ꢀPAB)
VVTFAPAGVTTEVK
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Figure Legends
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Figure 1. Schematic representation of experimental workflow used in the PABꢀSPROX
protocol developed in this work.
Figure 2. Schematic representation of PAB labeling reaction and gas phase
fragmentation behavior of resulting PABꢀlabeled methionineꢀcontaining peptides that do
not undergo oxidation in the PABꢀSPROX protocol.
Figure 3. Schematic representation of PABꢀSPROX data expected for methionineꢀ
containing peptides in a ligand binding experiment using PABꢀSPROX. Shown are the
SPROX data (top panels) and the corresponding PABꢀSPROX data (bottom panels) of
a targeted methionineꢀcontaining peptide probe from a nonꢀinteracting protein (A), a
protein displaying a ligandꢀinduced stabilization (B), and a protein displaying a ligandꢀ
induced destabilization (C).
Figure 4. PABꢀSPROX data generated in Experiments 1, 2, and 3. Denaturant
dependence of the L/H ratios measured in Experiment 1, 2, and 3 are shown in (A), (B),
and (C) (respectively). The filled circles represent data from the +2 charge state of the
VETGVIKPGMVVTFAPAGVTTEVK peptide from TEF1. The open and filled squares
represent data from the +2 and +3 charge state (respectively) of the
VIPDFMLQGGDFTAGNGTGGK peptide from CypA.
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Figure 5. Representative extracted ion chromatograms (EICs) used to generate the
PABꢀSPROX data shown in Figure 4A and 4C for the +3 charge state of the
VIPDFMLQGGDFTAGNGTGGK peptide from CypA. The EICs used to generate the 0
and 1.4 M GdmCl data points in Figure 4A are shown in (A) and (B), respectively. The
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EICs used to generate the 0 and 1.4 M GdmCl data points in Figure 4C are shown in (C)
and (D), respectively. The shaded regions were included in the area under the curve
calculations. The peaks marked with a “*” are likely due to the presence a deamidated
forms of the targeted peptide VIPDFMLQGGDFTAGNGTGGK.
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Cell Lysate
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(ꢀ) Ligand (+) Ligand
1a. Distribute Aliquots
1b. Distribute Aliquots
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2a. React
w/ H2O2
2b. React
w/ H2O2
3a. Quench
3b. Quench
w/ Met
w/ Met
4. TCA Precipitation
5. BottomꢀUp Proteomics
Sample Preparation
6. PABꢀLabeling
Heavy
PABꢀLabeled
Tryptic Peptides
Light
PABꢀLabeled
Tryptic Peptides
7. Combine
[Denaturant]
LCꢀMS/MS using SRM to Evaluate L/H Ratios of
Targeted MethionineꢀContaining Peptides
Figure 1
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SolutionꢀPhase Labeling
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H3C
R
H3C
S
Pathway 1
S
Pathway 2
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O
O
PAB
O
O
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N
N
H
H
HN
HN
ꢀCH3SR
O
O
C
C
O
O
N
C
N
H
C
H
HN
HN
O
PAB (RꢀBr) =
R = CH2COC6H5
Br
Figure 2
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A. No Interaction B. Stabilization C. Destabilization
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(ꢀ)
(+)
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0
[Den]
[Den]
[Den]
[Den]
[Den]
[Den]
Heavy = (+)
Light = (ꢀ)
1
0
Figure 3
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A.
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[GdmCl] (M)
B.
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[GdmCl] (M)
C.
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-1
-2
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[GdmCl] (M)
Figure 4
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Analytical Chemistry
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Time(min)
Time(min)
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D.
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Time(min)
Time(min)
Figure 5
ACS Paragon Plus Environment

Analytical Chemistry
Page 28 of 28
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ACS Paragon Plus Environment