Isotope-Coded Affinity Tag (ICAT) Approach to Redox Proteomics: Identification and Quantitation of Oxidant-Sensitive Cysteine Thiols in Complex Protein Mixtures Mahadevan Sethuraman,†,‡ Mark E. McComb,‡ Hua Huang,‡ Sequin Huang,‡ Tyler Heibeck,†,‡ Catherine E. Costello,‡,§ and Richard A. Cohen*,†,‡ Vascular Biology Unit, Cardiovascular Proteomics Center, and Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118 Received July 9, 2004
An approach is described for the simultaneous identification and quantitation of oxidant-sensitive cysteine thiols in a complex protein mixture using a thiol-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, USA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. Applying this approach, we have identified cysteine thiols of proteins in a rabbit heart membrane fraction that are sensitive to a high concentration of hydrogen peroxide. Previously known and some novel proteins with oxidant-sensitive cysteines were identified. Of the many protein thiols labeled by the ICAT, only relatively few were oxidized more than 50% despite the high concentration of oxidant used, indicating that oxidant-sensitive thiols are relatively rare, and denoting their specificity and potential functional relevance. Keywords: oxidation • cysteine • thiol • iodoacetamide • isotope coded affinity tag (ICAT) • proteomics • posttranslational modification
Introduction Generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as their elimination by antioxidant defense mechanisms, is normally tightly regulated. Oxidative or nitrosative stress during aging and disease results from excessive generation of ROS/RNS and/or impaired antioxidant defenses. Post-translational modification of proteins is one of the major effects of these reactive species, and cysteine thiols are one of the functional groups most sensitive to oxidation.1,2,3 The pKa value of most protein cysteine thiols is ∼8.5, and only certain cysteine (Cys) residues exist as thiolate anions at physiological pH. Because the thiolate anion is more readily oxidized than is the Cys sulfhydryl group (Cys-SH),4 Cys residues with low pKa confer to some proteins the potential for regulation by reactive species such as hydrogen peroxide, nitric oxide, and peroxynitrite. Because reactions at reactive Cys thiols can alter the function of proteins whenever sulfhydryls are involved in catalysis or modulation of activity, they represent not only a major mechanism of normal cell signaling via reversible S-nitrosation5 or S-glutathiolation,6 but also a mechanism by which disease can interfere with protein function, e.g., by irreversible thiol oxidation.7 Thus, it is essential to identify the proteins containing oxidant-sensitive Cys residues, their relative sensitivity to oxidation, and their participa* To whom correspondence should be addressed. E-mail:
[email protected]. † Vascular Biology Unit, Boston University School of Medicine. ‡ Cardiovascular Proteomics Center, Boston University School of Medicine. § Mass Spectrometry Resource, Boston University School of Medicine.
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tion in physiological and pathological cell function. Analogues of iodoacetamide (IAM), a thiol-specific alkylating agent, react with cysteine thiolate anions more readily than with cysteine sulfhydryls. The existing methods for the identification of oxidant-sensitive cysteines (redox proteomics) are based on the reaction of IAM with free cysteine thiols, and the fact that thiol oxidation prevents binding.4,8,9 As a step toward the identification of reactive protein thiols, we recently developed an approach using an iodoacetamide analogue incorporated into an acid-cleavable isotope-coded affinity tag (cICAT) reagent (Applied Biosystems, USA) to identify and quantify oxidantsensitive protein thiols, and proof of principle was achieved using purified creatine kinase.10 In this report, we describe the successful proteomic identification of proteins within a complex mixture which contain oxidant-sensitive cysteines and the simultaneous quantitation of their susceptibility to oxidation by a high concentration of hydrogen peroxide. ICAT reagents have been used extensively in quantitative proteomics to evaluate the abundance of expressed proteins.11 The ICAT approach is based on an affinity tag containing an IAM moiety that reacts with free cysteines. The approach presented here measures the decrease in ICAT labeling when reactive cysteines are exposed to oxidants under nonreducing conditions. The change in labeling can then be quantified from the ratio of ICAT labeling (Figure 1), and the cysteine residue can be identified from the LC-MS/MS derived sequences of the peptides obtained by proteolysis. Hydrogen peroxide, which is generated in all aerobic organisms as a result of normal 10.1021/pr049887e CCC: $27.50
2004 American Chemical Society
ICAT Approach to Redox Proteomics
Figure 1. ICAT approach to redox proteomics identification and quantitation of oxidant-sensitive thiols. Proteins with free reactive cysteine thiols (green square) and nonreactive cysteine thiols (pink square) are exposed to oxidant stress and normal conditions before labeling with ICAT reagent. Some of the reactive cysteine thiols are oxidized depending upon the level of the oxidant stress and oxidized thiols are designated as (black square), “Ox”. Following this, labeling of the free thiols is performed, (green square) and (pink square), with light (blue keyhole) and heavy (red keyhole) ICAT reagent. ICAT-labeled samples are mixed and digested with trypsin, followed by purification through HPLC cation exchange and avidin affinity cartridges as outlined in the Methods section. Affinity-captured peptides are analyzed by LC-MS and MS/MS. As shown for the reactive cysteines, the oxidized cysteines are not susceptible to labeling by ICAT reagent, and hence there is a decreased intensity in the signal from the heavy-labeled peptide in MS of the reactive cysteine-containing peptide. For nonreactive cysteines, the peptides in samples prepared under normal and oxidant stress conditions exhibit equivalent signal intensity in MS. From the relative peak intensities of the MS of light and heavy ICAT-labeled peptide, the percent oxidation of thiols in the samples can be estimated. Identity of the peptide sequences can be derived from MS/MS analysis of these peptides.
cellular metabolism12,13, was used as the oxidant in this proof of principle that mass spectrometry could be used to identify and quantify the degree of oxidation of Cys thiols in a complex protein mixture.
Experimental Section Materials. Ammonium bicarbonate (NH4HCO3), tris(hydroxymethyl)amino-methane (Tris), potassium chloride, hydrogen peroxide (30% w/w), dithiothreitol (DTT) and phenylmethane sulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO). Sucrose was purchased from American Bioanalytical (Natick, MA). Phosphoric acid and monobasic potassium phosphate (KH2PO4), were purchased from Fisher Scientific (Fair Lawn, NJ). HPLC grade acetonitrile (ACN) and trifluoroacetic acid (TFA) were purchased from Burdick & Johnson (Muskegon, MI). Preparation of Membrane Particulate Fraction. Isolated rabbit heart was homogenized in 8% sucrose buffer (50 mM Tris pH 7.4, 2 mM DTT, 2 mM PMSF). The homogenate was then subjected to two low speed centrifugations (10 min at 1000 × g and 30 min at 10 000 × g) and the pellets were saved. The supernatant was centrifuged at 100 000 × g, 2 h (45Ti rotor, Beckman). The membranes were resuspended in 40% sucrose
research articles buffer (50 mM Tris pH 7.4, 2 mM DTT, 2 mM PMSF) and centrifuged at 80 000 × g, 2 h. The supernatant was adjusted to 28% sucrose and centrifuged at 80 000 × g, 2 h to pellet the membrane fraction. The membranes thus prepared were suspended in 50 mM Tris HCl pH 7.1 and dialyzed against 50 mM Tris HCl (pH 7.1) to remove additional reducing agent and protease inhibitor. Hydrogen Peroxide Treatment. Membranes (200 µg), in 50 mM Tris (pH 7.1), were incubated with hydrogen peroxide (10 mM) at room temperature for 10 min. The reaction was terminated by the addition of catalase from bovine liver (Sigma, St. Louis, MO) to a final concentration of 0.1 µg/mL. Preparation of ICAT-Labeled Tryptic Peptides of Membrane Proteins either Untreated or Treated with Hydrogen Peroxide. Membrane proteins (200 µg) in 100 µL of 50 mM Tris, pH 7.1 were incubated with light or heavy acid-cleavable ICAT reagent (Applied Biosystems, Foster City, CA) at 37 °C for 3 h with no reducing agent added. After labeling for 3 h, the light and heavy labeled proteins were mixed and dialyzed against ammonium bicarbonate (ABC) buffer (20 mM). The protein mixture in 20 mM ABC was digested with trypsin by incubating overnight at 37 °C. The digested peptides were dried by Speedvac, suspended in 10 mM KH2PO4, 20% ACN, pH 3.0. The peptides were HPLC fractionated using a strong cationexchange (SCX) PolySulfoethyl A (PolyLC) column with a step gradient of 0 to 350 mM KCl (in 10 mM KH2PO4, 20% ACN, pH 3.0). The individual fractions from the SCX were mixed with equivalent amounts of affinity loading buffer and loaded onto an avidin affinity cartridge. The avidin affinity purified peptides were then dried and suspended in the cleavage reagent (30% aqueous TFA) to release the ICAT-labeled peptides from the acid-cleavable linker by incubating at 37 °C for 2 h. The peptides obtained by acid cleavage were dried and then suspended in 0.2% formic acid for µLC-ESI MS/MS experiments. Capillary HPLC-ESI MS/MS of ICAT-Labeled Peptides. Capillary HPLC Electrospray mass spectrometry/tandem mass spectrometry (LC-MS/MS) was performed using a capillary HPLC system (CapLC, Waters Corp.) coupled with a quadrupole orthogonal time-of-flight mass spectrometer (Q-TOF API US, Micromass/Waters Corp.) with its ion source equipped with NanoLockSpray and Z-Spray source. Affinity-purified ICATlabeled peptides were dissolved in 0.2% formic acid at a concentration of ca. 10-6 M and injection volumes were 1 µL. Sample preconcentration and desalting were performed using a peptide trap cartridge in line with the auto-sampler and column. Separation was on a 300 µm × 15 cm Magic C18 (5 µm, 300 A) capillary column (Michrom BioResources Inc.). A linear gradient was used to elute peptides into the mass spectrometer at a flow rate of 1 µL min-1: 5% B to 65% B over 55 min (A: 95% H2O, 5% ACN, 0.1% formic acid, 0.001% TFA; B: 5% H2O, 85% ACN, 10% 2-propanol, 0.1% formic acid, 0.001% TFA). Columns were washed and reequilibrated between LC experiments. ESI was carried out at ca. 2.8 kV, with the ion source temperature at 80 °C, and a cone voltage of 22 V. Mass Spectra were acquired in the positive-ion mode over the range m/z 400-2000. For accurate mass MS and MS/MS, NanoLockSpray was used with a constant infusion of either renin substrate or glu1-fibrinopeptide (SIGMA) 10-6 M 50% ACN, 0.2% formic acid at 0.2 µL min-1. Mass accuracy was ca. 10 ppm and resolution was ca. 10 000 (fwhm). Data Directed Analysis tandem mass spectrometry (DDA-MS/MS) parameters were: MS/MS was performed for the 3 most abundant MS precursor ions with intensities > 25 counts; MS/MS collision Journal of Proteome Research • Vol. 3, No. 6, 2004 1229
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Figure 2. LC-MS/MS of ICAT-labeled tryptic peptides. (a) Total ion chromatogram (TIC) for LC-ESI-MS/MS of ICAT-labeled tryptic peptides from HPLC cation exchange fraction 2 of the membrane protein mixture. (b) the m/z 400-900 region of the mass spectrum of the peptides eluting between 16.3 and 16.8 min (labeled i in panel a). Inset: Expanded view of the mass spectrum around m/z 614. (c) LC-MS/MS spectrum of [M+2H]2+ m/z 614.31 corresponding to the peptide (Gly 134-Arg 143) of sarcomeric creatine kinase containing the nonreactive Cys 141. (d) the m/z 400-1250 region of the mass spectrum of the peptides eluting between 31.5 and 32.0 min (labeled ii in panel a). Inset: Expanded view of the mass spectrum of the peptides around m/z 953. (e) LC-MS/MS of [M+2H]2+ m/z 953.08 corresponding to the peptide (Leu 272-Arg 287) of sarcomeric creatine kinase containing the reactive Cys 278.
energies were set directly proportional to the mass and inversely proportional to the charge of the precursor (ca. 16 to 40 V). Ar was the collision gas at ca. 2 × 10-5 Torr in the cell. MS/MS spectra were recorded as the summation of up to 4 × 1-second scans over the range of m/z 100-2000. Data were processed with MassLynx 4.01 and ProteinLynx Global SERVER 2.05 (Micromass/Waters Corp.). Targeted and advanced shotgun14 expression analysis was used for quantitation and identification of ICAT-labeled peptides. In both cases LC-MS data were used for quantitative analysis. The quantitation of ICAT pairs was accomplished by estimating the peak area of 1230
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the single ion chromatogram reconstructed from the MS of the respective ICAT labeled peptides.10 LC-MS/MS data were then used for identification and were searched against user programmed rabbit protein databases and the Swiss Prot database, release date March 2004.
Results and Discussion A membrane particulate fraction was prepared from rabbit heart homogenate by sucrose gradient ultracentrifugation. The proteins dialyzed in Tris buffer (pH 7.1) were either not treated or treated with hydrogen peroxide (10 mM), and the samples
ICAT Approach to Redox Proteomics
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Figure 3. Effect of hydrogen peroxide on ICAT labeling. MS of the ICAT-labeled peptide pair (Leu 272-Arg 287) of sarcomeric creatine kinase containing reactive Cys 278 from rabbit heart particulate fraction in which the heavy ICAT-labeled sample was treated with (a) 0 mM or (b) 10 mM hydrogen peroxide.
were subjected to light or heavy ICAT labeling, respectively, as described in Experimental Section. The labeled proteins were digested with trypsin, and the tryptic peptides were subjected to strong cation exchange chromatography (SCX)15 by using a KCl (0-350 mM) step gradient. Four SCX fractions were subjected to avidin affinity purification to capture the ICATlabeled peptides followed by acid cleavage of the biotin linker, and the peptides were subjected to LC-MS/MS. The total ion chromatogram (TIC) for the LC-MS/MS analysis of ICAT-labeled tryptic peptides from one cation exchange fraction is shown in Figure 2a. Selected regions of the mass spectra of the ions eluting between 16.3 and 16.8 (i) and between 31.5 and 32.0 min (ii) are shown in Figure 2, parts b and d, respectively. The region of the mass spectrum around m/z 614 (see the insert in Figure 2b) shows the signal from a pair of ICAT-labeled peptides from the same sample, corresponding to a doubly charged, light and heavy ICAT-labeled pair of peptides. From the LC-MS/MS of the precursor ion16 (Figure 2c), the peptide was identified as being derived from sarcomeric creatine kinase, a key enzyme in energy metabolism that catalyzes reversible phosphorylation of creatine by ATP. This peptide, spanning residues Gly 134-Arg 143, contains Cys 141, and was labeled equally by light and heavy ICAT despite exposure of the sample subsequently labeled with heavy ICAT to hydrogen peroxide. In the insert in Figure 2d, a doubly charged peptide pair is shown for which there was a 42% decrease in labeling by the heavy ICAT as calculated from the area of the relevant reconstructed single ion chromatogram. From the LC-MS/MS of the precursor ion (Figure 2e), the peptide was identified as also being from sarcomeric creatine kinase, spanning residues Leu 272-Arg 287, and containing the ICAT-labeled, Cys 278. Earlier studies showed that Cys 278 of sarcomeric creatine kinase, and the homologous Cys 283 of cytosolic creatine kinase, are highly reactive Cys within the active site, and that modification of these Cys inactivates the
enzyme.8,17 In the mass spectra from a control protein sample that was not treated with hydrogen peroxide, there was no change in the intensity of the signal from the heavy ICATlabeled peptide containing this Cys residue (Figure 3). Table 1 lists the oxidant-sensitive and -insensitive Cys containing peptides and their respective proteins identified from the membrane protein mixture, as well as the apparent extent of oxidation of the Cys residues. Among the additional proteins identified with known oxidant-sensitive Cys was glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis (Figure 4a). Cys 149 of GAPDH has been identified as a critical thiol that is S-nitrosated by nitric oxide and regulates enzyme activity.18 We also identified proteins with novel oxidant-sensitive Cys residues. Among them is Cys 838 of the β-subunit of phosphorylase-B kinase (Figure 4b) which has been identified as a target of glutathiolation at unknown sites.19 Peptides from voltage-dependent anion selective channels (VDAC) 1, 2 and 3 (Figure 4c) were identified and found to contain reactive Cys that were oxidized by 45-55%. VDAC proteins play an essential role in cellular metabolism and are part of the permeability transition pore complex important in apoptosis.20 Cys oxidation has not previously been reported to regulate the activity of VDAC proteins, although oxidants are known to regulate apoptosis at multiple steps. Interestingly, likely because the heart was not flushed of blood prior to homogenization, we found hemoglobin peptides and identified Cys 93 of the hemoglobin β chain (Figure 4d) which was oxidized 49% by hydrogen peroxide. This Cys forms bioactive nitrosothiols when nitric oxide is transferred from the heme iron.21,22 Because this proteomic technique acquires information on ICAT-labeled peptides irrespective of the extent of oxidation, the prevalence of oxidant-sensitive Cys among the total number of Cys residues detected was considered. Only 10% of the thiols in cysteine-containing peptides were oxidized by more than Journal of Proteome Research • Vol. 3, No. 6, 2004 1231
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Table 1. Identification and Quantification of Cysteine Oxidation in Rabbit Heart Particulate Membranes name
KCRS_RABIT
description
G3P_RABIT
creatine kinase sarcomeric mitochondrial precursor glyceraldehyde phosphate dehydrogenase
ALFA_RABIT
fructose biphosphate aldolase
ATA2_RABIT
sarcoplasmic endoplasmic reticulum Ca2+ ATPase
PIGR_RABIT
polymeric immunoglobulin receptor precursor
KPBB_RABIT
phosphorylase B kinase beta regulatory chain phosphofructose kinase muscle type
K6PF_RABIT POR1_RABIT POR2_RABIT POR3_RABIT LCAT_RABIT RETB_RABIT FA10_RABIT HBB_RABIT PRTS_RABIT MDHM_RATb DHSA_HUMANb NUFM_RATb
voltage dependent anion selective channel protein 1 voltage-dependent anion-selective channel protein 2 voltage-dependent anion-selective channel protein 3 phosphatidylcholine-sterol acyltransferase [precursor] plasma retinol binding protein coagulation factor hemoglobin β 1 chain vitamin K dependent protein S precurson fragment malate dehydrogenase succinate dehydrogenase mitochondrial precursor NADH-ubiquinone oxidoreductase
start
end
sequence
% cys oxidizeda
134 272 232 143 69 201 331 437 372 468 353 587 595 276 832 108 630 224 120 46 64 64 172 334 88 442 92 233 7
143 287 245 159 86 207 341 451 397 476 374 595 612 287 844 128 648 235 138 64 74 77 181 344 95 452 104 246 22
GLSLPPACSR LGYILTCPSNLGTGLR VPTPNVSVVDLTCR IVSNASCTTNCLAPLAK VNPCIGGVILFHETLYQK CQYVTEK ALANSLACQGK VGEATETALTCLVEK VDGDTCSLNEFTITGSTYAPIGEVHK ANACNSVIK SPPVLKGFPGGSVTIRCPYNPK ARCPVPRRR RQWYPLSRKLRTSCPEPR QTLCSLLPRESR NIIYYKCNTHDER GITNLCVIGGDGSLTGADTFR CNENYTTDFIFNLYSEEGK YQIDPDACFSAK EHINLGCDVDFDIAGPSIR SCSGVEFSTSGSSNTDTGK VCNYGLTFTQK LDKPSVVNWMCYRK QRQEELCLSR RNVAPACLPQK LSELHCDK QKKHCLVTVEK GCDVVVIPAGVPR GVIALCIEDGSIHR TTGLVGLAVCDTPHER
9.8 42.3 26.9 72.2 12.3 24.3 24.1 32.5 15.1 28.6 24.0 99.6 13.7
