Enrichment and Six-Plex Profiling of the DNA Damage Response
Transcription
Enrichment and Six-Plex Profiling of the DNA Damage Response
Enrichment and Six-Plex Six Plex Profiling g of the DNA Damage g Response p Pathway y Using g AmineAmine and Cy t i R Cysteine-Reactive ti T Tandem d M Mass Tag T g Reagents R g t Ryan y D D. Bomgarden Bomgarden, g Michael M. M Rosenblatt, Rosenblatt John C. C Rogers g Th Thermo Fisher Fi h Scientific, S i tifi Rockford, R kf d IL, IL USA Overview Purpose: To identify and characterize the DNA damage damage-response response proteins in pulmonary carcinoma A549 cells in response to five different anti anti-cancer cancer agents agents. Methods: Six-plex, amine-reactive Tandem Mass Tag (TMT) reagents were used for total proteome p labeling g and relative quantitation q among g ttreatments. eat e ts S Six-plex p e cyste cysteine-reactive e eact e Tandem a de Mass ass Tag ag (cys (cysTMT)) reagents were used for subproteome peptide labeling followed by enrichment using a novel immobilized anti anti-TMT TMT antibody resin resin. Results: Using both amine- and cysteine-reactive TMT reagents, we identified over 4700 peptides p p (FDR ( < 1%)) and q quantitated the relative abundance abu da ce o of o over e 1000 000 proteins p o e s of o A549 5 9 cells ce s treated ea ed with DNA-damaging da ag g agents. However, only changes in the most abundant DNA damage damageregulated proteins were observed in un-enriched un enriched samples labeled with TMT CysTMT reagent TMT. reagent-enriched enriched samples resulted in the identification and quantitation of additional proteins whose levels were altered after drug t t treatment. t I t d ti Introduction The DNA damage-response pathway is critical in maintaining genome stability, and proteins within this pathway are commonly mutated or misregulated g in cancer cells (Figure ( g 1). ) 1 We used a mass spectrometry p y ((MS)) based p proteomic oteo c approach app oac to identify de t y and a d characterize c a acte e the t e DNA da damageage response proteins in pulmonary carcinoma A549 cells in response to five anti-cancer anti cancer agents: camptothecin, camptothecin etoposide, etoposide actinomycin D D, hydroxyurea hydroxyurea, and paclitaxel (Figure 2) 2). Each of these drugs has been used clinically to treat cancer and has distinct mechanisms of action. action Camptothecin and etopiside t i id are ttopoisomerase i II and d I iinhibitors, hibit respectively, ti l and d preventt unwinding i di off DNA d during i replication li ti in i S S-phase. h A Actinomycin ti i D, D an RNA polymerase II inhibitor, and hydroxyurea, a dNTP synthesis inhibitor, are also S-phase inhibitors. Paclitaxel is a microtubule stabilizer that arrests cells in mitosis. We used TMT reagents g to determine relative changes g in overall o e a protein p ote abundance abu da ce after a te each eac drug d ug ttreatment. eat e t Tandem T d M Mass Tag T reagents t enable bl concurrentt id identification tifi ti and d multiplexed quantitation of proteins in different samples using tandem MS.2 Isobaric TMT reagents share identical structures, including a peptidereactive labeling g group, g p, a spacer p arm and an MS/MS reporter. p During g MS/MS S/ S analysis, a a ys s, each eac isobaric soba c tag ag produces p oduces a unique u que reporter epo e ion o that a enables quantitation. Current amine amine-reactive reactive NHS NHS-ester ester duplex or six-plex six plex isobaric TMT reagents covalently attach to the free amino termini and lysine residues of peptides and proteins proteins. Our cysTMT reagents and an anti TMT antibody resin provide an approach of thiol labeling, anti-TMT labeling affinity enrichment, i h t and d quantitation tit ti similar i il to t isotope-coded i t d d affinity ffi it tags t (ICAT™) 3 Overall, (ICAT™). O ll targeted t t d cysteine t i labeling l b li and d affinity ffi it enrichment i h t using i cysTMT reagents results in reduced sample complexity and improved proteome depth compared to the amine-labeled proteome. Methods Sample Preparation Cell treatment and Western blot: A549 cells grown in DMEM with 10% FBS were treated with 10 μM μ camptothecin, p , 200 μM μ etoposide, p , 2 μM μ actinomycin act o yc D,, 10 0 mM hydroxyurea, yd o yu ea, o or 10 0 μg/ μg/ml paclitaxel pac ta e for o 20 0 hours ou s at 37 ºC C. A549 cell lysates (10 μg) were separated by SDS-PAGE SDS PAGE and blotted using Thermo Scientific p53 antibodies diluted 1:10,000. 1:10 000 TMT® reagent g labeling: g Control or treated A549 cells were sonicated in lysis ys s buffer bu e (6 M urea/ u ea/ 50 mM Tris/ s/ 5 mM EDTA)) and a d reduced educed with t 5 mM DTT for 1 hour at 50 ºC and alkylated with 375 mM iodoacetamide for 15 minutes at room temperature. temperature Each sample was acetone precipitated and enzymatically digested at 37 ºC C overnight overnight. Peptides were labeled with the appropriate TMTzero® or TMTsixplex® reagent (126 (126-131) 131) for 2 hours at room temperature, t t quenched h d with ith hydroxylamine, h d l i and d combined bi d before b f li id chromatography liquid h t h (LC)-MS/MS (LC) MS/MS analysis. l i cysTMT® reagent labeling and enrichment: Treated A549 cells were lysed and reduced as above. above Reductant was removed using Thermo Scientific Zeba 7K Desalting Columns Columns, and 100 μg of each sample was immediatel combined with immediately ith 20 μL L of 25 mM ccysTMT sTMT reagent reagent. Each sample was labeled l b l d with ith the th appropriate i t cysTMTzero® TMT ® or cysTMTsixplex® TMT i l ® reagentt (126-131) for 2 hours at room temperature, combined, and separated by 6%-12% SDS-PAGE. Eight gel bands were separately enzymatically digested at 37 ºC overnight g before extraction and lyophylization. y p y Labeled peptides p p were resuspended p in TBS and incubated with 50 μL μ of immobilized anti-TMT antibody resin per gel band overnight with end end-over-end over end shaking at 4 ºC. C. After collecting the unbound sample sample, the resin was washed four times with 4 M urea/TBS urea/TBS, four times with 0 0.05% 05% CHAPS CHAPS, four times with TBS TBS, and four times with water water. Peptides were eluted four times with 50% acetonitrile acetonitrile, 0 4% TFA and vacuum-dried 0.4% ac m dried before LC LC-MS/MS MS/MS anal analysis. sis LC/MS A NanoLC-2D™ high-pressure liquid chromatograph (HPLC) (Eksigent) with a Proteopep™II C18 column, 75 µm ID x 20 cm (New Objective) was used to separate p peptides p p using g a 4%-40% g gradient ((A: water,, 0.1% formic acid;; B: acetonitrile,, 0.1% % formic acid)) at a flow rate of 250 nL/min for 60 min. A Thermo Scientific LTQ Orbitrap XL ETD mass spectrometer was used to detect peptides using a top 2 x 3 experiment consisting of single-stage single stage MS, MS followed by acquisition of three MS/MS spectra with higher-energy C-trap dissociation (HCD) fragmentation, fragmentation followed by three MS/MS with collision ind ced dissociation (CID) for protein identification induced identification. Parameters in this mode were: Isolation I l ti width: idth 3.0 3 0 m/z; / C Collision lli i energy: 50% (10% with ith two t steps). t ) Only doubly- and triply-charged peptides were selected for fragmentation. Dynamic exclusion parameters were set to: Repeat count = 1; Repeat duration = 30;; Exclusion list size = 500;; Exclusion duration = 20. Target g values were as follows: MS = 5 e5;; MS/MS (HCD) ( ) = 1 e5. Ion transfer times were set to 500 for FTMS and 300 for MS/MS (HCD). Two microscans were required for HCD spectra spectra. To determine relative changes g in abundance of other p proteins affected by y drug treatment, we used both amine amine- and cysteine-reactive cysteine reactive TMT reagents (Figure 3). 3) Six-plex Six plex amine-reactive amine reactive TMT reagents were used for total proteome labeling of peptides derived from each treatment treatment. A set of six sixplex cysTMT reagents were used to label the cysteine cysteine-containing containing s bproteome In contrast to amine subproteome. amine-reactive reacti e TMT reagents reagents, these tags were used d to t label l b l denatured d t d proteins t i before b f sample l mixing i i and d enzymatic digestion. Because of the relatively low abundance of cysteine-containing peptides, approximately 6% of peptides are labeled, allowing g for potential p sampling p g of lower abundant proteins. p Enrichment of cysTMT y reagent-labeled g peptides p p was facilitated byy an immobilized antiTMT antibody resin, which has affinity for the TMT reagent reporter region (Figure 3) 3). Using these parallel workflows, we were able to identify over 4700 peptides p p and q quantifyy over 1000 proteins p (Figure ( g 2C and 4). ) Onlyy 541 proteins were similar between the amine- and cysteine-labeled p y samples, p , demonstrating increased sample diversity after enrichment. Unique to the cysTMT enriched sample, sample were some known and novel DNA damagedamage response proteins, proteins which resulted in changes in relative abundance after drug treatment treatment. Specifically, Specifically two DNA damage signaling targets targets, ASC ASC-1 1 and aslin 1 1, were ere red reduced ced o over er ttwo-fold o fold in response to camptothecin camptothecin, etoposide t id and d actinomycin ti i D (Figure (Fi 5A, 5A 5B). 5B) A similar i il reduction d ti occurred for the DNA helicase MCM complex, with subunits 2-6 being quantified after enrichment (Figure 5C and data not shown). Some proteins such as the HUWE1,, a p p p53 E3 ubiquitin q ligase g like MDM2,, had increased expression p upon p drug g treatment (Figure ( g 5D). ) Others such as the small GTPase guanidine exchange factor RalBP1, had drug-specific drug specific down regulation (Figure 5E). 5E) FIGURE 5. Identification and quantitation q of Thermo Scientific cysTMT y reagent-labeled g peptides. p p A ASC-1 complex subunit P100 HDFcYR B Aslin 1 sFcTGKSDSR F TGKSDSR Data analysis: Mixed CID and HCD data were processed with Thermo Scientific Proteome Discoverer™ 1.2 software against g the IPI human database using g a 20 pp ppm mass tolerance. Static modifications were set to the TMTsixplex (229.16 Da) or cysTMTsixplex reagent (304.18 Da) and dynamic modifications included phosphorylation and methionine oxidation. oxidation C R Results lt D MCM3 YVLcTAPR E3 ubiquitin biq itin ligase HUWE1 KVIcTGPNDtSPGsPR Multiple genomic and proteomic studies have identified f roles for f p53 in regulation of cell cycle arrest, DNA repair, and apoptosis. Activation of the p53 DNA damage-response p g p pathway p y causes p53 p stabilization through g phosphorylation, p p y , leading g to transcription p of various p53-regulated p g p proteins (Figure 1). Treatment of A549 cells with five anti anti-cancer cancer agents, camptothecin etoposide camptothecin, etoposide, actinomycin D D, hydroxyurea hydroxyurea, and paclitaxel resulted in varying degrees of p53 activation (Figure 2B) 2B), which is consistent with their potency and mechanism of action. action E RalBP1 MELcGATRLGyFGR (A-E) MS spectra of enriched cysTMT-labeled cysteine-containing peptides and TMT reporter quantitation showing changes in relative abundance of peptides after each drug treatment. FIGURE 3. Schematic of TMT and cysTMT reagent workflows. A C Conclusions l i FIGURE 1. 1 Model M d l off p53 53 DNA damage d response pathway. th • AmineA i and d cysteine-reactive t i ti T Tandem d M Mass T Tags are complementary reagents that ffacilitate more in-depth proteomic analysis. Mdm2 • cysTMT reagents combined with immobilized anti-TMT anti TMT resin enrichment allow for relative quantitation of lower abundant proteins. t i Apoptosis • Six plex TMT reagents enabled simultaneous relative quantitation Six-plex of five different anti-cancer drugs drugs. • Combined CID and HCD spectra p processing p g facilitated enhanced quantitative peptide q p p identification. DNA damaging agents (Campto/Etop/ActD) p B p53 p53 protein levels Cell survival Cell cycle arrest & genetic repair Activation of the p53 DNA damage-response g pathwayy results in p53 phosphorylation y and stabilization through interference with MDM2-mediated degradation. Increased p53 levels result in increased expression of stress response genes that promote cell survival through cell cycle arrest or DNA damage repair. p53 activation also induces expression of pro-apoptotic genes and the MDM2 ubiquitin E3 ligase which in turn modulates the down-regulation of the pathway. pathway (A) Si Six-plex l TMT reagentt workflow kfl ffor peptide tid llabeling b li after ft sample l treatment t t t and d digestion. di ti (B) SixSi plex cysTMT reagent workflow for protein labeling followed by digestion and labeled peptide enrichment using immobilized anti-TMT antibody resin. FIGURE 2. A549 cell drug treatments and response. A FIGURE 4. Comparison of TMT and cysTMT reagent reagent-enriched enriched peptides and proteins. C Cell C ll cycle l phase TMT reporter [] No treatment - - 126 Camptothecin 10 μM S 127 Etoposide Actinomycin D Hydroxyurea Paclitaxel 200 μM S 128 2 μM S 129 10 mM S 130 10 μg/ml M 131 Fold change c e Drug 10 127/126 128/126 129/126 130/126 131/126 B TMT Total cysTMT Enriched 3604 Peptides p 1 0 100 200 300 935 Peptides p TMT Total cysTMT Enriched 823 Proteins 781 Proteins 400 0.1 Protein Number B A 3522 82 1152 282 541 240 p53 p - Capto Etop ActD HU Pax (A) Table of different anti-cancer agents and concentrations used for A549 cells treatment, the cell cycle phase of drug action, action and the TMTsixplex or cysTMTsixplex reagents used to label each sample sample. (B) Western blot of p53 after drug treatment. (C) Log graph of relative quantitation of 400 cysTMT reagentlabeled proteins normalized to no treatment control control. 4756 Total Peptides 1063 Total Proteins Venn diagrams g depicting p g the number of unique q labeled peptides p p (A) ( ) and proteins p (B) ( ) identified using g Scaffold 3 Q+ (Proteome Software) for TMT reagent-labeled samples and cysTMT reagent sample post enrichment of A549 cell extract using immobilized anti-TMT anti TMT resin. References 1 Vousden 1. Vousden, K K.H. H and Prives C C. (2009) (2009). Blinded by the light: The growing complexity of p53 p53. Cell 137(3):413-431. 137(3):413-431 2. Thompson, p A., et al. ((2003). ) Tandem mass tags: g a novel quantification strategy q gy for comparative p analysis y of complex p protein p mixtures by MS/MS. Anal. Chem. 75: 1895-204. 1895 204. 3 G 3. Gygi, i S.P., S P ett al. l (1999). (1999) Q Quantitative tit ti analysis l i off complex l protein t i mixtures using isotope-coded affinity tags. Nat. Biotech. 17: 994999. Acknowledgements The authors would like to thank Eugene Cichon for help with additional sample preparation. preparation Tandem Mass Tag, g, TMT,, TMTzero,, TMTsixplex, p , cysTMT, y , cysTMTzero, y , and cysTMTsixplex y p are registered g trademarks of Proteome Sciences plc. ICAT is a trademark of the University of Washington. Eksigent is a registered trademark and NanoLC is a trademark of AB SCIEX LLC. LLC Proteopep is a trademark of New Objective Objective. All other trademarks are the property of Thermo Fisher Scientific Inc Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property t rights i ht off others. th