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Sirtuin-1 (SIRT1) is an NAD+-dependent protein deacetylase that is sensitive to oxidative signals. Our purpose was to determine whether SIRT1 activity is sensitive to the low molecular weight nitrosothiol, S-nitrosoglutathione (GSNO), which can transduce oxidative signals into physiological responses. SIRT1 formed mixed disulfides with GSNO-Sepharose, and mass spectrometry identified several cysteines that are modified by GSNO, including Cys-67 which was S-glutathiolated. GSNO had no effect on basal SIRT1deacetylase activity, but inhibited stimulation of activity by resveratrol (RSV) with an IC50 of 69μM. These observations indicate that S-glutathiolation of SIRT1 by low concentrations of reactive glutathione can modulate its enzymatic activity.
Antioxid. Redox Signal. 13, 1023–1032.
Sirtuin-1 (SIRT1) is a highly conserved NAD+-dependent deacetylase that plays an instrumental role in cell cycle progression, energy metabolism, and aging. In the vascular endothelium, SIRT1 promotes cellular homeostasis through deacetylating a wide variety of targets, including eNOS, FOXO1, and p53 (20). SIRT1 activity depends upon the availability of NAD+ such that increases in the [NAD+]/[NADH] ratio during caloric restriction activate the enzyme (13) and by doing so can regulate gene expression (6) and apoptosis (1). Recent evidence suggests that SIRT1 may be sensitive to other cellular redox couples. In neural progenitor cells, GSH depletion caused by buthionine sulfoximine shifted terminal cell differentiation in a SIRT1-dependent manner, indicating that SIRT1 may be sensitive to intracellular GSH levels (21).
SIRT1 activity is stimulated by the polyphenol, resveratrol (RSV), a component of red wine, which has a variety of cardioprotective effects, including suppression of NF-κB-dependent endothelial inflammation caused by TNFα (5) or cigarette smoke extract (4). Furthermore, RSV increases eNOS expression and activity, and in turn, improves bioavailability of nitric oxide (•NO) (12, 27). At present, potent small molecule activators of SIRT1 are highly sought after for their therapeutic potential (15).
SIRT1 activity is regulated by post-translational modifications including sumoylation at lysine-734 that increases its activity. UV radiation and hydrogen peroxide decreases SIRT1 sumoylation and deacetylase activity, and therefore increases apoptosis (29). Likewise, SIRT1 activity is stimulated by cyclinB/Cdk1-dependent phosphorylation at threonine-530 and serine-540 that regulates cell cycle progression (23). Direct post-translational modification of SIRT1 by oxidants has not yet been reported.
•NO is an important signaling molecule that mediates a wide variety of cellular effects by either stimulating guanylyl cyclase to increase cyclic GMP or by inducing reversible adducts on cysteine thiols that include S-sulfenation, S-nitrosation, and S-glutathiolation (25). S-nitrosothiols play a prominent role as intermediates in •NO signaling. •NO reacts with GSH, the prevalent low molecular weight thiol in the cell, to generate S-nitrosoglutathione (GSNO) which can then transnitrosate proteinyl thiols, eliciting a change in protein function (14). S-nitrosated thiols can undergo further oxidation to form S-thiolated proteins that are covalently modified by glutathione, cysteine, or homocysteine (7). An increasing body of evidence implicates GSNO as mediating S-glutathiolation of protein thiols in addition to its role in S-transnitrosation. Exogenous and endogenous application of •NO results in S-glutathiolation of hsp70, actin, and vimentin in rat vascular smooth muscle cells (28). In vitro GSNO can S-glutathiolate aldose reductase, glyceraldehyde-3-phosphate dehydrogenase, caspase-3, c-jun and cathepsin-K on specific cysteine thiols (3, 9, 17, 19, 30).
The goal of this work was to determine whether SIRT1 is subject to direct modification and regulation by a cellular oxidant. Our findings clearly demonstrate that GSNO attenuates stimulation of enzymatic activity of purified SIRT1 by RSV without affecting its basal activity. In addition, reduction of GSNO-treated protein with dithiothreitol (DTT) restored SIRT1 responsiveness to RSV stimulation, indicating that GSNO induces a reversible thiol modification. Mass spectrometry analysis conclusively identified several SIRT1 thiols that are sensitive to GSNO-induced oxidation, and S-glutathiolation of Cys-67 was definitively demonstrated by LC-MS/MS. This work presents novel findings that indicate that SIRT1 is directly modified by GSNO, providing a novel redox regulatory mechanism of SIRT1 activity.
The SIRT1-Flag deacetylation activity assay was tested for specificity in the presence and absence of a specific SIRT1 inhibitor or the activator, RSV. In the presence of 5μM EX-243 ((S)-6-chloro-2,3,4,9-tetrahydro-1-H-carbazole-1-carboxamide), but not its inactive (R) stereoisomer, EX-242, basal SIRT1 activity was blocked (Supplemental Fig. 1; see www.liebertonline.com/ars) indicating that SIRT1 deacetylase activity measurement was specific. In the presence of RSV (50μM), SIRT1 activity was stimulated approximately 2-fold, as reported previously (18). Prior to characterizing the effects of GSNO, SIRT1-Flag isolated on Flag Sepharose beads was treated with DTT (2mmol/l) to ensure global cysteine reduction. After removing DTT by buffer exchange, SIRT1 was incubated with GSNO at concentrations between 156nM and 500μM. After an additional buffer exchange, activity was assayed in the presence or absence of RSV (50μM). Under these conditions, there was no statistically significant effect of GSNO on basal SIRT1 activity. However, GSNO caused a concentration-dependent inhibition of SIRT1 stimulation by RSV (Fig. 1A, white bars). The concentration of GSNO required for half-maximal inhibition of RSV stimulation was 69μM, with complete inhibition at 500μM (Fig. 1B). Following GSNO, an additional reduction step with DTT restored RSV stimulation to levels equivalent to that of SIRT1 that was not treated with GSNO (Fig. 1A, dark gray bars). These data demonstrate one or more SIRT1 cysteine residues are susceptible to reversible oxidation by GSNO which inhibits stimulation of enzyme activity by RSV.
In order to determine the reactivity of SIRT1 cysteines with low molecular weight thiols, GSH-Sepharose was used in either unmodified form or as GSNO-Sepharose after nitrosation with acidified nitrite. As evidenced by the absence of SIRT1 in the supernatant and wash fractions, SIRT1 bound covalently to GSNO-Sepharose, but not to GSH-Sepharose (Fig. 2). BSA, as a negative control, did not bind to either column material (results not shown). The formation of a covalent disulfide adduct with GSNO was demonstrated by elution of the protein from GSNO-Sepharose with DTT. These results indicate that SIRT1 efficiently forms mixed disulfides in vitro with GSNO, but not GSH.
In order to identify the specific cysteines modified by GSNO, SIRT1-Flag was subjected to differential cysteine alkylation by NEM and IAM, followed by MALDI-TOF MS and LC-MS/MS. Differential alkylation labels cysteines based on whether the thiols are reduced or oxidized (24). After initial reduction with DTT, SIRT1 was first exposed to GSNO and alkylated with IAM to alkylate remaining free cysteine thiols. These cysteines were recognized during analysis of the mass spectra by the increased mass conferred by IAM (+57Da). Cysteines that were reversibly oxidized by GSNO were then reduced with DTT and alkylated with NEM, and were recognized by an increase in mass of 125Da. In all, SIRT1-Flag was subjected to four conditions: GSNO-IAM-DTT-NEM, IAM-DTT-NEM, GSNO-NEM-DTT-IAM, and NEM-DTT-IAM. Analysis of the spectra and comparison between each condition allowed us to identify GSNO-reactive cysteines on SIRT1.
By MALDI-TOF MS, sequence coverage of differentially alkylated mouse SIRT1 samples varied between 56% and 65%. Peaks were assigned to known cysteine-containing peptides of SIRT1 (Table I). In a representative mass spectrum from SIRT1 not previously treated with GSNO (IAM-DTT-NEM), a peak was observed at m/z 2678.277 that corresponds to the SIRT1 peptide containing amino acids 46–74 in which Cys-61 (human Cys-67) is alkylated with IAM (+57Da) (Fig. 3B). A peak corresponding to the NEM-alkylated form of the same peptide (+125Da) at m/z 2746.313 in the GSNO-IAM-DTT-NEM-treated sample confirmed that the site was modified by GSNO. These results indicate that cysteine-61 was reversibly oxidized by GSNO, reduced by DTT, and alkylated with NEM (Fig. 3A). The peak observed in the sample not treated with GSNO (m/z 2678.277) did not appear in the GSNO-IAM-DTT-NEM sample, indicating that the oxidation of Cys-61 was nearly quantitative and confirming that GSNO forms a reducible modification on Cys-61. Cys-61 was confirmed to be alkylated with IAM by LC-MS/MS in the IAM-DTT-NEM sample, but not in the GSNO-IAM-DTT-NEM treated sample (Supplemental Fig. 2; see www.liebertonline.com/ars). Analysis of all spectra yielded a list of putative GSNO-reactive cysteines on SIRT1 that followed a similar pattern of differential alkylation as Cys-61 that included Cys-C245, Cys-C260, Cys-C318, and Cys-C613 (Table I). These five cysteines identified on mouse SIRT1 are potential sites at which reversible oxidation of SIRT1 by GSNO may regulate SIRT1 function.
In order to confirm the specific modification induced by GSNO that was detected by differential alkylation, human SIRT1 was treated with GSNO, followed by alkylation of remaining free thiols with NEM. SIRT1 then was subjected to in-solution digest and analyzed by LC-MS/MS. An [M+3H]3+ ion corresponding to m/z 483.55 (Fig. 4A) was identified as the SIRT1 peptide containing amino acids 66–77 with an additional mass of 305Da, consistent with the addition of a single glutathione adduct. The fragmentation ion spectrum clearly demonstrated that human SIRT1 is S-glutathiolated on cysteine 67 (Fig. 4B).
The lack of a definitive crystal structure of SIRT1 and its large size makes it otherwise difficult to determine which of its 19 total cysteines might be reactive surface cysteines. Therefore, identifying a small subset of SIRT1 cysteines that are reactive to GSNO is an important advance in determining how intracellular SIRT1 might be redox regulated.
Using MALDI-TOF MS and LC-MS/MS in combination with differential alkylation, five cysteines were identified on mouse SIRT1 that were S-glutathiolated after exposure to GSNO, and S-glutathiolation of Cys-67 was confirmed by LC-MS/MS on human SIRT1. All five SIRT1 reactive cysteines identified through MALDI-TOF MS are candidates for OPTMs that regulate enzymatic activity. Autiero et al. recently utilized modeling to predict the structure of SIRT1 (2). Via personal correspondence, Autiero confirmed that human SIRT1 Cys-67, Cys-268, and Cys-623 were solvent-exposed in their computer-generated model, and may therefore be susceptible to thiol modification. SIRT1 is regulated by post-translational modifications, including phosphorylation and sumoylation, which are localized to the N- and C-terminal regions of the enzyme (2). S-glutathiolation by GSNO at Cys-67 and Cys-623 are consistent with post-translational regulation at these terminal regions of SIRT1. Cys-268 is of interest because it lies in the NAD+ binding region identified by modeling of SIRT1 and other members of the SIRT family, in which the catalytic core is highly conserved (2, 16). Binding of NAD+ results in changes in SIRT1 conformation that allow catalysis to proceed. It is possible that S-glutathiolation of this cysteine might reduce binding affinity of NAD+.
S-glutathiolation of SIRT1 has no effect on basal activity, but does inhibit SIRT1 activation by RSV with an IC50 of 69μM. This effect was reversed following reduction with DTT, demonstrating that GSNO induces a reversible modification on SIRT1. This was confirmed by demonstrating that SIRT1 formed mixed disulfides with GSNO-Sepharose. Considering the normal millimolar intracellular GSH concentrations and the low IC50 of 69μM for GSNO to inhibit SIRT1, our findings strongly suggest that SIRT1 stimulation by RSV may be regulated in vivo by direct redox modification(s) of SIRT1.
By demonstrating that SIRT1 can be regulated by S-glutathiolation of specific Cys residues, our results have uncovered the potential for SIRT1 to be directly regulated by oxidative modifications that may link its activity to GSH redox and production of cellular reactive oxygen and nitrogen species. These novel findings also indicate that the GSH redox state may affect the response of SIRT1 to polyphenols. These results imply that antioxidants might significantly potentiate the response to small molecule activators of SIRT1. Our findings open an exciting new avenue of SIRT1 regulation that will be important in understanding SIRT1 as a therapeutic target for the treatment of several chronic diseases.
pAd-track SIRT1-Flag containing full-length mouse SIRT1 was obtained from Addgene, Cambridge MA (plasmid 8438) (22). pAd-track SIRT1-Flag was linearized and recombined with pAdeasy in BJ5183 cells to generate cosmids. Suitable cosmids were digested with PAC-1 and transfected into HEK293 cells utilizing calcium phosphate. Adenoviral colonies were isolated and propagated in HEK293A cells. Adenovirus was isolated using double cesium chloride gradient centrifugation. Titers were determined by OD260 assay and expressed as OPU/ml (optical particle units per milliliter).
Plasmids containing human GST-SIRT1 fusion protein were supplied by Dr. Tony Kouzarides at The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge (11). SIRT1 protein was cloned into pcDNA cmv-6x His and prepared in transformed BL21 bacteria by standard methods. SIRT1-His E. Coli Rosetta-2 DE3 cells (Novagen, Darmstadt, Germany) were transformed with a plasmid containing a construct for 6x His-tagged SIRT1 (human) protein. Transformed bacterial cells were then grown up to an OD600 of 0.6–1.0, induced with 1mmol/L IPTG (AppliChem, Darmstadt, Germany) and protein expression proceeded at 30°C for 4h. Bacterial cells were then spun down, and the bacterial pellet was incubated in lysis buffer (50mmol/L Tris pH 8.0, 10mmol/L NaCl, 1mmol/L DTT, 1mmol/L PMSF) containing 1mg/ml lysozyme. Lysed bacteria were sonicated, 25 Units benzonase (BD Biosciences, San Jose, CA) were added, and the mixture was incubated on ice for 15min. The lysed bacteria were spun down at 17000 g for 10min at 4°C. The supernatant was collected and subjected to ammonium sulfate (Acros Organics, Geel, Belgium) precipitation at a concentration of 40% saturation. The mixture was then spun down at 17000 g for 5min at 4°C and the supernatant was discarded. The pellet was resuspended in 50mmol/L Tris pH 8.0, 500mmol/L NaCl, 0.1% Triton, 300mmol/L sucrose, 1mmol/L PMSF, 1mmol/L DTT, and 100μM zinc sulfate, and dialyzed overnight at 4°C in lysis buffer. The dialyzed sample was then purified via the 6X His tag using a His60 Nickel column (ClonTech, Mountain View, CA).
HEK293T cells (ATCC, Manassas, VA) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin-streptomycin. Bovine aortic endothelial cells (BAEC, Cell Systems Corporation, Kirkland, WA) were cultured in DMEM supplemented with 10% FCS, 1% penicillin-streptomycin, 0.1% DMSO, and 1mmol/L ascorbic acid in dishes pre-coated with 0.1% gelatin. Media was refreshed every 48h.
Infected HEK293T cells or BAECs were lysed in RIPA buffer (50mmol/L Tris pH 7.4, 150mmol/L NaCl, 1% NP40, 0.1% SDS, 0.5% deoxycholine, 1mmol/L PMSF, 1X protease inhibitor cocktail (Roche)). Lysates were subjected to sonication, then rotated at 4°C for 30min, followed by centrifugation for 10min at maximum speed to remove insoluble material. Anti-Flag M2 agarose affinity gel (Sigma-Aldrich, St. Louis, MO) was prepared, 40μl gel suspension was pipetted into microcentrifuge tubes, and the suspension was washed three times with 1ml ice-cold wash buffer (PBS, 150mmol/L NaCl). Processed cell lysates were then added to the affinity gel and rotated at 4°C for at least 2h. The protein was incubated with 2mmol/L DTT in wash buffer (PBS, 150mmol/L NaCl) and incubated at 37°C for 30min, followed by buffer exchange to remove DTT.
S-nitrosoglutathione, GSH- and GSNO-Sepharose were prepared as previously described (8, 10, 14). To make GSNO, 0.75g reduced L-glutathione was dissolved in 5.25ml ice-cold 0.5 N HCl. 170mg of sodium nitrite was then added to generate nitrous acid (HNO2). The resulting red mixture was stirred at 4°C for 40min. 2ml chilled acetone was then added and the mixture stirred for an additional 10min at 4°C to allow precipitation of the GSNO. The mixture was then centrifuged at 2000 g for 5min. The supernatant was carefully decanted, and the product was washed three times with acetone and three washes with ether. The GSNO was then dried via SpeedVac and stored in the dark at −20°C. GSNO was prepared freshly for each experiment and stored on ice. GSNO was diluted to indicated concentrations and samples were treated for 15min at 37°C.
GSH- and GSNO-Sepharose were prepared and used as previously described (10). All solutions were degassed with nitrogen prior to use. One gram of activated Sepharose 4B (Amersham Biosciences, Piscataway, NJ) was reconstituted in 10ml PBS at room temperature for 15min. The beads were then washed twice with 10ml PBS. Thiol reduction was performed by incubating the beads in 50mmol/L Tris pH 8.5, 0.3 M NaHCO3, 1mmol/L EDTA, and 1% (w/v) DTT for 45min on a shaker. The beads were then rinsed with several column volumes of wash buffer (50mmol/L Tris pH 7.4, 250mmol/L NaCl, 1mmol/L EDTA). The activated thiol Sepharose 4B (GSH-Sepharose) was stored at 4°C in wash buffer. Free thiols were determined by Ellman's reagent. Half of the bead volume was reserved to generate GSNO-Sepharose. These beads were rinsed in several bead volumes of 10mmol/L HCl, followed by resuspension in 2ml 10mmol/L HCl. 2ml 10mmol/L NaNO2 was added to form nitrous acid. The mixture was incubated on a shaker for 15min in buffer A (50mmol/L Tris pH 8.0, 250mmol/L NaCl, 1mmol/L EDTA, 0.01% NP40). The resulting GSNO-Sepharose was rinsed in several bead volumes of buffer A and stored in the dark at 4°C. For experiments, 100μl beads were rinsed three times in buffer A and then incubated with 100μg recombinant SIRT1 on a shaker for 30min at room temperature. The beads were spun down for 1min at 8000 g and the beads containing bound SIRT1 were washed three times with Buffer A. All fractions were reserved for analysis by SDS-PAGE, followed by transfer to nitrocellulose membranes and Memcode reversible protein stain (Pierce, Rockford, IL).
SIRT1 activity was measured according to modified published methods (26). The assay is a two-step process in which the purified SIRT1 is first incubated with a peptide substrate corresponding to the p53 acetylation target site spanning amino acids 379–382 (Arg-His-Lys-Lys(Ac)-AMC). This step involves a 30min incubation at 37°C with 1mmol/L NAD+ and 50μM substrate peptide to allow SIRT1 to freely deacetylate the peptide in activity assay buffer (50mmol/L Tris-Cl, pH 8.0, 137mmol/L NaCl, 2.7mmol/L KCl, 1mmol/L MgCl2). Deacetylation of the peptide exposes the lysine to tryptic cleavage. In the second step, 1mmol/L nicotinamide (NAM) is added to block further SIRT1 activity, and trypsin is then added to cleave any deacetylated substrate. Lysine cleavage by trypsin releases the fluorescent AMC fluorophore allowing the quantification of the amount of substrate deacetylated by SIRT1 during the first step. The amount of fluorescence is directly proportional to the level of SIRT1 activity in the sample. The trypsin reaction was allowed to proceed for 60min. The cleaved fluorescent tag was excited at 375nm, and fluorescence emission was recorded at 465nm in a Fluoroskan Ascent microplate fluorometer (Thermo Scientific, Waltham, MA). Total protein content was determined by SDS-PAGE, followed by GelCode blue staining. Densitometry of the SIRT1 bands was performed using ImageJ (NIH) and quantified according to a BSA standard curve run on the same gel.
SIRT1-Flag protein was immuno-precipitated with anti-Flag M2 affinity gel (Sigma-Aldrich) and divided into four equal aliquots. Two of the aliquots were treated with 2mmol/L GSNO at 37°C for 10min to react with any reactive surface cysteines. The other two samples were left untreated to serve as controls. All four samples were then washed with wash buffer (PBS, 150mmol/L NaCl). One GSNO-treated sample and one control sample were treated with iodoacetamide (IAM, 40mmol/L, Sigma) for 1h on ice in the dark to alkylate free cysteines. The other set of GSNO-treated and control samples were alkylated in parallel under the same conditions, but with N-ethylmaleimide (NEM, 40mmol/L, Sigma) instead of IAM. All samples were washed 3x with wash buffer and boiled at 95°C for 5min in reducing Laemmli buffer. Samples were then subjected to SDS-PAGE using PAGEr Gold Precast 4–12% Tris-Glycine Gels (Lonza, Basel, Switzerland). The gel was then washed three times in MilliQ water for 5min each time, followed by GelCode Blue (Pierce) staining overnight. The gel was again washed three times in MilliQ water for 5min each time to destain the gel. The protein bands were excised and placed in separate, fresh 1.5mL SafeLock tubes (Eppendorf, Hamburg, Germany). The gel pieces were washed to remove SDS, salts, and other contaminants from the gel pieces before digestion, and then underwent reduction of reversibly oxidized thiols with DTT and alkylation with either IAM or NEM, whichever one was not used previously on the sample.
An in-solution digestion method was employed without reduction to preserve and identify S-glutathiolated cysteine residue(s) using mass spectrometry. Purified human SIRT1-His (20μg) at a protein concentration of 0.22μg/μl, was treated with GSNO (2mmol/L) and incubated for 15min at 37°C. The sample was then alkylated with 40mmol/L NEM dissolved in 100mmol/L ABC pH 8.0. The samples were alkylated in the dark, at room temperature for 30min. Untreated SIRT1-His was also alkylated with 40mM NEM and run in parallel as a negative control.
To remove the alkylating reagent and buffer salts, both samples were subjected to a ReadyPrep 2D Cleanup Kit (BioRad, Hercules, CA). After precipitation, the protein pellet was resuspended in 5μl of 100mM ABC pH 8.0. 20μL of acid-labile RapiGest™ SF Surfactant (Waters, Milford, MA) solution was added to each sample, along with 200ng of trypsin. The samples were digested overnight at 37°C. TFA (1μl) was added to degrade the detergent and the samples were then divided in half in preparation for MALDI-TOF MS and Orbitrap LC-MS/MS. All samples were desalted using micro-C18 ZipTip™ (Millipore, Temecula, CA) columns. Approximately 1–5μg of peptide samples were loaded onto the ZipTip™ column and eluted into 10μl of 50% ACN, 0.1% TFA.
MALDI-TOF MS was performed on Reflex IV™ mass spectrometer (Bruker-Daltonics, Billerica, MA) and LC-MS/MS was acquired on an LTQ-Orbitrap™ hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a TriVersa NanoMate ion source (Advion, Ithaca, NY). A washing regimen consisted of adding 100μl of 100mM ammonium bicarbonate (ABC) (Fluka) at pH 8.0, then 100μl of 100mM ABC, 50% HPLC-grade acetonitrile (ACN) (Fisher Scientific), then 100μl of 100% ACN with light vortexing in a Thermomixer (Eppendorf) for 15min for each step. The washing regimen was repeated a total of three times. After the washing regimen was completed, the gel pieces were dried using the SpeedVac evaporator for 15min with no heat. Gel pieces were rehydrated in fresh reduction buffer, consisting of 30μl of 20mM DTT (Thermo Fisher Scientific, San Jose, CA), 100mM ABC, 5% CAN, and incubated for 1h at 55°C. The supernatant was then removed and the gel pieces were washed in 100μl of 100mM ABC, followed by dehydration using 100μl of 100% ACN for 20min with light vortexing. The supernatant was removed and the samples that were initially alkylated with IAM were now alkylated with 40mM NEM for 30min in the dark, at room temperature. The samples that were initially alkylated with NEM were alkylated a second time with 40mM IAM. After in-gel alkylation, the previously described washing regimen was used again to clean the gel samples in preparation for digestion. MS-grade Trypsin Gold (Promega Corporation, Madison, WI) was prepared in 50mM ABC, 5% ACN buffer to obtain a 1:10 enzyme to sample weight ratio per 25μl of trypsin buffer. The dried gel pieces were then rehydrated in 25μl of trypsin buffer and incubated at 37°C overnight. Excess trypsin was washed off the exterior of the gel pieces by quickly adding then decanting 20mM ABC (pH 8.0). An extraction regimen was then used to extract and collect the peptides from the gel pieces. ABC (100μl of 20mM) was added to the gel pieces, lightly vortexed for 20min, and the supernatant was collected in a fresh 1.5mL SafeLock tube, followed by 100μl of 1% trifluoroacetic acid (TFA) (Thermo Fisher Scientific), 50% ACN, followed by 100μl of 100% ACN. The extraction regimen was repeated once more, pooling all the supernatants. The supernatants were then dried down in the SpeedVac evaporator. The remaining salt pellet was then resuspended in 500μl of 50% ACN and dried down in the SpeedVac evaporator to eliminate the remaining volatile salts from the sample. The peptides were resuspended in 20μl of 0.1% TFA. Ten μl of the sample was used for MALDI-TOF MS while the retaining the remaining sample for LC-MS/MS analysis. 0.5μl of the desalted peptide sample was then crystallized onto the AnchorChip™ target (Bruker-Daltonics, Billerica, MA) with 2,5-dihydroxybenzoic acid (DHB) as the matrix. Spectra were taken on samples using a Reflex IVTM matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker-Daltonics). Spectra were taken over the range m/z 300–4000. Each output spectra was taken from an average of the approximately 300–500 laser shots of each sample.
All MS spectra obtained from MALDI-TOF MS were analyzed using the program “M over Z” (Genomic Solutions, Ann Arbor, MI). Each spectrum was smoothed to improve the isotopic peak resolution and decrease background noise. The spectrum was internally calibrated against three known, unique, and unmodified SIRT1 peptide masses. The peaks in the spectrum were then auto-labeled and a mass list of monoisotopic peak masses was then generated and searched using the MS-Fit program on Protein Prospector (UCSF; http://prospector.ucsf.edu/prospector/mshome.htm, accessed June 6, 2010). Search settings allowed for carbamidomethylation, N-ethylmaleimide alkylation, and methionine oxidation. Two missed cleavages and an error tolerance of 50ppm were allowed for the database search.
Any cysteine-containing peptides from the search results were compiled in a list and the masses were manually searched through each spectrum. Peaks for cysteine-containing peptides showing differential labeling between the four samples were recorded. This search culminated in the identification of five specific cysteine residues of interest. These cysteines showed reactivity toward the alkylating agents, were labeled differentially following modification by GSNO, and are cysteines that likely accessible on the surface of the native protein and are potential sites for regulation.
For LC-MS/MS, peptides were subjected to purification using ZipTip™ (Millipore) micro reversed-phase extraction. LC-MS was performed on the resulting peptides using a nanoAcquity UPLC™ capillary high-performance liquid chromatography system (Waters Corp., Milford, MA) coupled to an LTQ-Orbitrap™ hybrid mass spectrometer (ThermoFisher Scientific) equipped with a TriVersa NanoMate ion source (Advion, Ithaca, NY). Sample concentration and desalting were performed online using a nanoAcquity UPLC™ trapping column. (180μm x 20mm, packed with 5μm, 100 Å Symmetry C18 material, Waters Corp.) at a flow rate of 15μl/min for 1min, while separation was accomplished across a nanoAcquity UPLC™ capillary column (100μm x 100mm, packed with 1.7μm, 130 Å BEH C18 material, Waters Corp.) A linear gradient of A and B buffers. (buffer A: 3% CAN/0.1%FA; buffer B: 97% CAN/0.1% FA) over 240min was used at a flow rate of 0.5μl/min to elute peptides into the mass spectrometer. Columns were washed and re-equilibrated between LC-MS experiments. Electrospray ionization was carried out with the NanoMate at 1.75kV, with the LTQ heated capillary set to 225°C. Mass spectra were acquired in the Orbitrap in the positive ion mode over the range m/z 300–2000 at a resolution of 60,000 (approximately 1 mass spectrum/ s). Mass accuracy after internal calibration (using lock mass on siloxane ions) was typically within +/−2ppm. Simultaneously, tandem MS (MS/MS) were acquired using the LTQ in data-dependent, inclusion list-directed mode: Inclusion lists (parent ion lists) were generated for cysteine-containing SIRT1 peptides through in silico tryptic digestion of the human SIRT1 protein sequence, including up to four missed cleavages and glutathiolation, di- or tri-oxidation, NEM or IAM-modification of each cysteine alone and in combination, calculating m/z values for charge states 2+, 3+, and 4+. The five most abundant, multiply-charged species from the list that were found in the mass spectrum with signal intensities >8000NL were chosen for MS/MS. Alternatively, if the mass spectrum contained no entries from the list, then the most abundant multiply charged detected ions were chosen for MS/MS. MS/MS collision energies were set at 35% using helium as the collision gas, and MS/MS were acquired over a range of m/z values dependent on the precursor ion. Dynamic exclusion was set such that MS/MS for each species was acquired maximally twice. All spectra were recorded in profile mode for further processing and analysis. Xcalibur™ and Proteome Discoverer™ software (ThermoFisher Scientific) were used for MS and MS/MS data analysis, while peptide and protein assignments were conducted with Mascot™ to search against the human and mouse species subsets of the IPI database, employing an error window of 8ppm on the precursor ions and 1.2Da on the fragment ions. The error tolerant search function of Mascot™ was employed. Alternatively, variable modifications were set to include methionine oxidation, cysteine carbamidomethylation, N-ethyl maleimide modification, di- and tri-oxidation, and glutathiolation, asparagines and glutamine deamidation, and glutamate and glutamine pyroglutamic acid formation. Potential assignments by Mascot™ were verified and refined by manual interpretation of the original spectra. Scaffold™ (Proteome software) was used for meta-analysis of the Mascot™ search results.
To further confirm the identification of peptides of SIRT1 that was not treated with GSNO, but alkylated in the IAM-DTT-NEM treated samples, we subjected samples to LC-MS/MS. We detected a [M+2H]2+ ion corresponding to m/z 1339.661 in the control spectrum (Supplemental Fig. 2). The ion was then subjected to collision-induced fragmentation in the mass spectrometer. The fragmentation ion spectrum of this ion identified the peptide fragment as amino acids 46–74 containing a carbamidomethyl group (+57Da) on Cys-61. This observation indicates that under control conditions in which SIRT1 is not treated with GSNO, the residue is modified by the initial alkylation with IAM. Furthermore, this peptide does not appear in the GSNO-IAM-DTT-NEM sample because GSNO induces a reversible modification that blocks alkylation by IAM. The peptide containing Cys-61 should instead be modified by NEM after DTT reduction, and will therefore have a different mass. This peptide (Cys-61+125Da) was not identified in spectra derived from LTQ-Orbitrap, but was positively identified in the MALDI-TOF spectra (Fig. 3). These results indicate that GSNO treatment of SIRT1 results in a site-specific S-glutathiolation. Although we identified five putative reactive cysteines by analysis with MALDI-TOF MS, only mouse SIRT1 Cys-61 was identified as being modified by GSNO using both MALDI-TOF MS and LC-MS/MS methods.
This work was presented and published in abstract form at annual meetings of the Society of Free Radical Biology and Medicine, Free Rad Biol Med 45 Suppl. 1 S76, 2008, 47 Suppl. 1 S83, 2009. This work was supported by NIH Grant PO1 HL068758, the National Heart, Lung, and Blood Institute sponsored Boston University Cardiovascular Proteomics Center (Contract No. N01-HV-28178, NIH NCRR P41-RR10888, S10-R15942, S10-RR20946) and NIH pre-doctoral training grant HL007969-06A1. We thank Dr. Mengwei Zang for expert assistance and for providing valuable reagents. The authors would also like to thank Dr. Jessica Fry for providing a critical reading of the manuscript and helpful scientific discussion.