The Use of Methionine Sulfoxide Reductases to Reverse Oxidized

The Use of Methionine Sulfoxide Reductases to Reverse Oxidized Methionine for Mass Spectrometry Applications Robert Cunningham1; Kratika Singhal1; Ryan T Fellers2; Luca Fornelli2; Henrique Dos Santos Seckler2; Bhavin Patel1; Egle Capkauske3; Juozas Siurkus3; Philip Compton2; Neil L Kelleher2; John C Rogers1 1Thermo Fisher Scientific, Rockford, IL; 2Northwestern University, Evanston, IL; 3Thermo Fisher Scientific, Vilnius, Lithuania

Orbitrap XL™ Hybrid Ion Trap - - + + + + + + + + + + DTT -Orbitrap MS . Targeted quantitative analysis of methionine containing peptide and phosphopeptides were done on a Thermo Scientific™ Q Exactive™ HF - + + + + + + + + + + + H2O2 The ability for both MetSR enzymes to reduce an intact protein was demonstrated using Thermo Scientific™ TurboLuc™ . Figure 5 demonstrates how oxidation is reversed by either MetSR. Figure MS using Parallel Reaction Monitoring. His WQ MSR 5A shows that 1:10 moles of methionine to moles of H2O2 can decrease the forms of oxidized Disappearance of Appearance of A Results: : TurboLuc mildly. When looking at highly oxidized samples shown in Figure 5B (1:75 ratio) significant B trx His WQ MSR Oxidized Peptide Reduced Peptide We successfully produced and purified two active MetSR enzymes, as observed by gel shift of B oxidation was able to be reversed, but some oxidation events occurred which could either be caused : from other amino acids being oxidized or creation of the sulfone that are not reversible by MetSR. an oxidized methionine rich protein, reduction of oxidized methionines in an intact protein, and MSR His in shotgun and targeted proteomics experiments at the peptide level. Parallel Reaction C: Remaining protein oxidation may be due to sulfone formation, oxidation of other amino acids, or re-

Raw file for oxidized and reduced samples using ngMsrAB-T or nmMsrAB-T. Figure 5A shows data Figure 3: Amino acid sequences of two recombinant MetSR enzymes: Amino acid sequence of ngMsrAB-T and nmMsrAB-T prior to treatment with WELQut Protease to cleave from the 1:10 moles of methionine to H2O2, whereas Figure 5B is for 1:75 moles of methionine to H2O2. MetSR treated INTRODUCTION at the WELQ site. The peaks assigned as 0.0000 is the TurboLuc protein without any methionine oxidation, and shifts by 16 Daltons are indicative of oxidation (15 Daltons are sometimes observed due to data processing). Methionine (Met) is a sulfur-containing essential amino acid that is highly susceptible to oxidation by reactive oxygen species during stress, aging, and disease in cells, and in vitro1. Met oxidation is the most common modification of purified therapeutic antibodies and other A biologics, can affect protein function, and is observed by mass spectrometry (MS) in intact Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T proteins and peptide samples as splitting the MS signal, which reduces sensitivity and CONCLUSIONS increases complexity of analysis. Methionine sulfoxides can be returned to methionine by methionine sulfoxide reductases. The product of Met oxidation is Met sulfoxide (MetO), which Methionine oxidation can be reversed in intact proteins and peptides prior to analysis by SDS-PAGE exists in two diastereomers that are reduced by two methionine sulfoxide reductases, MetSR- and intact or peptide LC-MS analysis. The Use of MethionineA and Sulfoxide MetSR-B (Figure 1).

Poster Note 64762 Disappearance of Appearance of Reversal of methionine oxidation by MetSR has benefits to MS experiments involving intact proteins, Figure 1: Schematic of Methionine Oxidation and Reduction: Methionine S-sulfoxide and C Oxidized Peptide Reduced Peptide targeted quantitative peptide analysis, and shotgun proteomics. Reductases to Reversemethionine Oxidized R-sulfoxide formation and reversal. A modified LC-MS ionization source design may alleviate the residual modifications observed at the protein and peptide level7. Methionine for Mass Spectrometry

REFERENCES Applications MetSR treated 1. Zhang, X.H. and H. Weissbach, Origin and evolution of the protein-repairing enzymes methionine sulphoxide reductases. Biol Rev Camb Philos Soc, 2008. 83(3): p. 249-57. 2. Khor, H.K., et al., Identification of methionine sulfoxide diastereomers in immunoglobulin gamma Robert Cunningham1; Kratika Singhal1; Ryan T Fellers2; Luca Fornelli2; Henrique Dos Santos Seckler2; Oxidation by Hydrogen Peroxide antibodies using methionine sulfoxide reductase enzymes. MAbs, 2010. 2(3): p. 299-308. For intact analysis, recombinant turboluciferase (TurboLuc, ~16kDa) was selected to perform an 3. Morand, K., G. Talbo, and M. Mann, Oxidation of peptides during electrospray ionization. Rapid Bhavin Patel1; Egle Capkauske3; Juozas Siurkus3; Philip Compton2; Neil L Kelleher2; John C Rogers1 initial optimization of methionine oxidation and reduction reactions due to observed sensitivity to B Commun Mass Spectrom, 1993. 7(8): p. 738-43. 1Thermo Fisher Scientific, Rockford, IL;2 Northwestern University,Methionine Evanston, oxidation can IL; also occur during sample preparation, purification , storage , and LC- oxidation, knowledge of the sequence, and ability to detect with an Orbitrap XL mass spectrometer. 4. Lao, Y.W., et al., Chromatographic behavior of peptides containing oxidized methionine residues in 2,3 All experiments for intact analysis used18µM TurboLuc. TurboLuc has three moles of methionine Control nmMsrAB-T ngMsrAB-T proteomic LC–MS experiments: Complex tale of a simple modification. Journal of Proteomics, 3 MS ionization . Variable methionine oxidation of purified proteins leads to lower and variable Control nmMsrAB-T ngMsrAB-T Thermo Fisher Scientific, Vilnius, Lithuania product quality. It also complicates liquid chromatography-mass spectrometry (LC-MS) per mole of protein and thus a 1:1 molar ratio of methionine to H2O2 would be 54µM of H2O2 for 2015. 125: p. 131-139. The Use of Methionine Sulfoxide Reductases4 to Reverse18µM of TurboLuc. A variety of oxidationOxidized conditions were assessed at theMethionine following ratios of for Mass Spectrometry Applications analysis in 4 ways: 1) Shifted and split peaks during liquid chromatography , 2) Increased 5. Peterson, A.C., et al., Parallel reaction monitoring for high resolution and high mass accuracy 1 1 2 complexity and poorer depth2 of analysis of intact proteins due to multiple oxidized forms,2 3) methionine:1 H2O2 which were 10:1,3 1:1, 1:10, 1:25, 1:75, 1:100 and3 1:500 (Figure 5 shows 1:10 2 2 1 quantitative, targeted proteomics. Mol Cell Proteomics, 2012. 11(11): p. 1475-88. Robert Cunningham ; Kratika Singhal ; Ryan T Fellers ; IncreasedLuca Fornellicomplexity & poorer; Henrique depth of analysis Dos of peptide Santos digests due Seckler to multiple oxidized; Bhavin Pateland; 1:75,Egle the restCapkauske of the data are not; shown). Juozas Siurkus ; Philip Compton ; Neil L Kelleher ; John C Rogers 6. Zeliadt, N. Moving Target: New mass spectrometry–based techniques are blurring the lines 1 2 forms, and; 4) Impaired quantitation of methionine-containing3 peptides due to the stochastic between discovery and targeted proteomics. 2014 [cited 2016 10May]; Available from: Thermo Fisher Scientific, Rockford, IL; Northwesternnature University,of oxidation of methionine Evanston, in peptides. IL; Thermo Fisher Scientific, ReductionVilnius, of Intact Lithuania Proteins by ngMsrAB and nmMsrAB Disappearance of Appearance of http://www.the-scientist.com/?articles.list/categoryNo/2921/category/Lab-Tools/. Methionine sufloxide reduction was performed by adding either ngMsrAB-T or nmMsrAB-T to the Oxidized Peptide Reduced Peptide 7. Kim, K., et al., Reducing protein oxidation in low-flow electrospray enables deeper investigation of TurboLuc control or oxidized sample at an enzyme:sample ratio of 1:4 and DTT to a final D proteoforms by top down proteomics. EuPA Open Proteomics, 2015. 8: p. 40-47. concentration of 5mM. Both enzymes were used in separate experiments. Samples were incubated Figure 2: Gel Shift Assay to Reduce MRP6: Three different MetSR genes expressed in Targeted Quantitative Peptide Analysis Figure 5: Parallel Reaction Monitoring of three methionine containing peptides: Schematic Shotgun proteomics at 37 C for 2 hours & then desalted using 3K MWCO concentrators. OVERVIEW METHODSthree different vectors were tested for their ability to reduce MRP6 after being oxidized by H O . Three heavy peptide standards (two phosphopeptides) containing an oxidized methionine (100 fmol/μL ) of parallel reaction monitoring for targeted peptide quantitation 5,6(A). Quantitative reversal of 2 2 TRADEMARKS/LICENSING were mixed with the Pierce 6 protein digest, equimolar, LC-MS grade (40 ng/μL). The mixed sample methionine oxidation in a peptide standard (B), a phosphopeptide of the same peptide standard The Pierce 6 protein LC-MS grade digest was used to assess the two MetSR enzymes using both non- Shotgun⁰ Proteomics Coupled with Methionine Oxidation Reduction Purpose ExpressionNo of Recombinant Methionine Sulfoxide Reductases was then reduced by either ngMsrAB-T or nmMsrAB-T as described earlier. The resulting peptide (C), and a second phosphopeptide (D). The bar graph area shows the cumulative area under the oxidized and mildly oxidized samples (Figure 7). Substantial methionine reversal was seen with all To synthesize two active forms of recombinant methionine sulfoxide reductase (MetSR) MsrA1 (Human) ngMsrAB-T nmMsrAB-T Thermo Scientific™ Pierce™ 6 Protein Digest, equimolar, LC-MS grade which was stored in liquid For Research Use Only. Not for use in diagnostic procedures. Reduction mixture was then injected onto a C18 column (Easy-Spray PepMap C18, 3µm, 75µm x 150 cm) and curve of individual b-ions and y-ions. Each experiment was performed in duplicate. reaction ratios less than 1:100 moles of methionine:H2O2. At 1:100 sulfones and other amino acids for over a year was used for the oxidation experiments. Control and oxidized samples were enzymes and perform proof of principle studies for mass spectrometry applications in intact Over a dozen different recombinantA B C methionine A Breductase C vectorsA B were C constructed Vector for target separated by a gradient of water and acetonitrile in 0.1% formic acid using a Thermo Scientific™ were oxidized which greatly reduced the identification of peptides due to random amino acid oxidation © 2016 Thermo Fisher Scientific Inc. All rights reserved. Eksigent is a trademark of ABSciex. All other reduced using the same procedure as intact protein analysis described above, except for C18 protein, targeted quantitative peptides, and shotgun proteomics. protein expression and optimized for E.coli ER2566. 0.1 g of biomass was lysed with cell lysis Dionex™ Ultimate™ 3000 RSLCnano system. The sample was infused into the Q Exactive HF mass events (data not shown).MetSR Intreated addition, DTT alone did not show any reversal of methionine oxidation. trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not – peptide clean up. A intended to encourage use of these products in any manner that might infringe the intellectual property buffer consisting of 50mM Tris HCl (pH 8); 100mM NaCl; 0.1% Triton X-100; 1mM EDTA spectrometer via electrospray ionization utilizing parallel reaction monitoring (Figure 4) for collection of Lastly, numerous oxidized peptides were no longer detected after treatment with MetSR. Remaining MRP6 (oxidized) rights of others. Methods (pH8) and disrupted by sonication. The soluble fraction was diluted with wash buffer targeted spectra. The resulting raw data were processed and visualized using Skyline 3.1.0.7382 Targeted Protein Analysis peptide oxidation may be due to sulfone formation, oxidation of other amino acids, or re-oxidation during LC-MS analysis Two recombinant MetSR and a methionine-rich protein were expressed, purified, and tested consisting of 250mM NaCl, 20mM imidazole, 50mM Tris-HCl, pH=8 and applied to 0.2 ml analysis software. electrospray ionization. Thefor gel mobility shiftsUse prior to use in ofMS applications. Methionine For intact and shotgun proteomics SulfoxideThermo Scientific™ HisPur Ni-NTA Reductases and washed extensively. Bound proteins were eluted to with ReverseThe protein sample was injected Oxidized onto a Thermo Scientific™ ProSwift™ Methionine RP-4H Analytical 100 μm x for Mass Spectrometry Applications Figure 6 shows the disappearance of the oxidized peptide and appearance of the corresponding 25 cm monolithic column at a flow rate of 800 nL/min over a gradient of water and acetonitrile in experiments ,hydrogen peroxide was1 added to induce mild methionine1 oxidation and 2 50mM Tris, pH8, 250mM2 NaCl, 500mM imidazole and dialyzed against 50mM Tris2 MRP6-HCl pH (reduced) 7.6, 1 RESULTS 3 3 2 reduced peptide2 after treatment with ngMsrAB or nm1 MsrAB. These data indicate that treatment Figure 6: Average intensity of 6-protein digested mix: A comparison of the average peptide intensity o 0.1% formic acid. The shotgun proteomics sample was injected onto a reversed-phase column Robertcompared to Cunningham non-oxidized controls before; Kratika and after treatment Singhal with MetSR.; Ryan Reduction T wasFellers ; Luca4 C, overnight. Fornelli Three; MetSR Henrique genes expressed Dos from Santos three different Seckler clones each ;were Bhavin tested Patel ; Egle Capkauske ; Juozas Siurkus ; Philip Compton ; Neil L withKelleher ngMsrAB or ;nm JohnMsrAB results C inRogers a >90% reduction of an oxidized methionine and resulted in of detectable peptides before and after treatment with two forms of MetSR. carried out using DTT as a cofactor and samples were analyzed by a Thermo Scientific™ (15cmIntact x 75Protein μm I.D., Analysis Prepmap C18) and separated using a gradient of water and acetonitrile in 1 2 by gel shift of an oxidized methionine rich protein 6 (Figure3 2) and two MetSR/Trx trifunctional a corresponding increase in the peptide with a reduced methionine. In addition, after treatment Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T Orbitrap XL™ Hybrid Ion Trap - - + + + + + + + + + + DTT 0.1% formic acid at a flow rate of 300 nL/min. All samples used an Eksigent™ nanoLC Ultra 2D Thermo Fisher -OrbitrapScientific, MS . Targeted Rockford, quantitative analysis IL; of methionineNorthwestern enzymes University, from Neisseria Evanston,gonorrhoeae or Neisseria IL; meningitidis Thermo were selected Fisher for MS Scientific, Vilnius, Lithuania some of the oxidized peptides were not even detectable (Figure 6 A and C for ngMsrAB-T). containing peptide and phosphopeptides were done on a Thermo Scientific™ Q Exactive™ HF applications- + (Figure + 2).+ + + + + + + + + H2O2 plusThe HPLC ability coupled for both to MetSR a Thermo enzymes Scientific to reduce™ LTQ an Orbitrap intact ™protein XL ETD was mass demonstrated spectrometer. using Thermo Scientific™ TurboLuc™ . Figure 5 demonstrates how oxidation is reversed by either MetSR. Figure MS using Parallel Reaction Monitoring. 5A shows that 1:10 moles of methionine to moles of H O can decrease the forms of oxidized His WQ MSR 2 2 Disappearance of Appearance of Shotgun proteomics Figure 2: Gel ShiftA: Assay to Reduce MRP6: Three different MetSR genes expressed in TargetedTurboLuc Quantitative mildly. When Peptide looking Analysis at highly oxidized samples shown in Figure 5B (1:75 ratio) significant Figure 5: Parallel Reaction Monitoring of three methionine containing peptides: Schematic OVERVIEWResults: B 5,6 three different vectors were testedtrx for Histheir abilityWQ to reduceMSR MRP6 after being oxidized by H O . Three heavy peptide standards (two phosphopeptides) containing an oxidized methionine (100 fmol/μL ) of parallel reactionOxidized monitoring forPeptide targeted peptide quantitation (A).Reduced Quantitative Peptide reversal of We successfully produced and purified two active MetSR enzymes, as observed by gel shift of 2 2 oxidation was able to be reversed, but some oxidation events occurred which could either be caused B: were mixed with the Pierce 6 protein digest, equimolar, LC-MS grade (40 ng/μL). The mixed sample methionine oxidation in a peptide standard (B), a phosphopeptide of the same peptide standard The Pierce 6 protein LC-MS grade digest was used to assess the two MetSR enzymes using both non- an oxidized methionine rich protein, reduction of oxidized methionines in an intact protein, and from other amino acids being oxidized or creation of the sulfone that are not reversible by MetSR. Purpose No MSR His was then reduced by either ngMsrAB-T or nmMsrAB-T as described earlier. The resulting peptide (C), and a second phosphopeptide (D). The bar graph area shows the cumulative area under the oxidized and mildly oxidized samples (Figure 7). Substantial methionine reversal was seen with all MsrA1 (Human) ngMsrAB-T nmMsrAB-T Remaining protein oxidation may be due to sulfone formation, oxidation of other amino acids, or re- To synthesizein shotgun twoand active targeted forms proteomics of recombinant experiments methionine at the sulfoxidepeptide level. reductase Parallel (MetSR) Reaction Reduction C: reaction ratios less than 1:100 moles of methionine:H O . At 1:100 sulfones and other amino acids mixtureoxidation was then during injected electrospray onto a C18ionization. column (Easy-Spray PepMap C18, 3µm, 75µm x 150 cm) and curve of individual b-ions and y-ions. Each experiment was performed in duplicate. 2 2 enzymesMonitoring and perform was used proof to demonstrate of principle studies 90-99% for reduction mass spectrometry in oxidized methionineapplications in in MetSR intact A B C A B C A B C Vector separated by a gradient of water and acetonitrile in 0.1% formic acid using a Thermo Scientific™ were oxidized which greatly reduced the identification of peptides due to random amino acid oxidation protein,treated targeted peptide quantitative and phosphopeptides. peptides, and shotgun proteomics. Dionex™Figure Ultimate™ 4: Intact 3000Protein RSLCnano MS Results: system. Spectra The samplecompared was after infused Thermo into Scientific™the Q Exactive Xcalibur™ HF mass Xtract events (data not shown). In addition, DTT alone did not show any reversal of methionine oxidation. A spectrometerRaw file for via oxidized electrospray and reduced ionization samples utilizing using parallel ngMsrAB reaction-T ormonitoring nmMsrAB (Figure-T. Figure 4) for 5A collection shows data of Lastly, numerous oxidized peptides were no longer detected after treatment with MetSR. Remaining Methods Figure 3: Amino acid sequences of two recombinant MetSR enzymes:MRP6 Amino (oxidized) acid targetedfrom thespectra. 1:10 Themoles resulting of methionine raw data to wereH O processed, whereas Figureand visualized 5B is for using 1:75 molesSkyline of 3.1.0.7382 methionine to H O . MetSR treated peptide oxidation may be due to sulfone formation, oxidation of other amino acids, or re-oxidation during Two recombinant MetSR and a methionine-rich protein were expressed, purified, and tested sequence of ngMsrAB-T and nmMsrAB-T prior to treatment with WELQut Protease to cleave 2 2 2 2 analysisThe peakssoftware. assigned as 0.0000 is the TurboLuc protein without any methionine oxidation, and shifts by electrospray ionization. for gelINTRODUCTION mobility shifts prior to use in MS applications. For intact and shotgun proteomics at the WELQ site. 16 Daltons are indicative of oxidation (15 Daltons are sometimes observed due to data processing). experiments ,hydrogen peroxide was added to induce mild methionine oxidation and Methionine (Met) is a sulfur-containing essential amino acid that is highly susceptible to MRP6 (reduced) RESULTS Figure 6: Average intensity of 6-protein digested mix: A comparison of the average peptide intensity compared to non-oxidized controls before and after treatment with MetSR. Reduction was of detectable peptides before and after treatment with two forms of MetSR. carriedoxidation out using by reactive DTT as oxygena cofactor species and samples during stress, were analyzedaging, and by disease a Thermo in cells,Scientific™ and in vitro1. Intact Protein Analysis

OrbitrapMet oxidation XL™ Hybrid is the Ion most Trap common modification of purified therapeutic antibodies and other - - + + + + + + + + + + DTT -Orbitrap MS . Targeted quantitative analysis of methionine A containingbiologics, peptide can affect and phosphopeptides protein function, andwere is done observed on a Thermoby mass Scientific™ spectrometry Q Exactive™(MS) in intact HF - + + + + + + + + + + + H2O2 The ability for both MetSR enzymes to reduce an intact protein was demonstrated using Thermo proteins and peptide samples as splitting the MS signal, which reduces sensitivity and Scientific™ TurboLuc™ . Figure 5 demonstrates how oxidation is reversed by either MetSR. Figure Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T MS using Parallel Reaction Monitoring. CONCLUSIONS increases complexity of analysis. Methionine sulfoxides can be returned to methionine by His WQ MSR 5A shows that 1:10 moles of methionine to moles of H2O2 can decrease the forms of oxidized Disappearance of Appearance of methionine sulfoxide reductases. The product of Met oxidation is Met sulfoxide (MetO), which A: TurboLuc mildly. When looking at highly oxidized samples shown in Figure 5B (1:75 ratio) significant Methionine oxidation can be reversed in intact proteins and peptides prior to analysis by SDS-PAGE Results: B Oxidized Peptide Reduced Peptide exists in two diastereomers that are reduced by two methionine sulfoxide reductases, MetSR- trx His WQ MSR oxidation was able to be reversed, but some oxidation events occurred which could either be caused and intact or peptide LC-MS analysis. We successfully produced and purified two active MetSR enzymes, as observed by gel shift of B: A and MetSR-B (Figure 1). from other amino acids being oxidized or creation of the sulfone that are not reversible by MetSR. an oxidized methionine rich protein, reduction of oxidized methionines in an intact protein, and MSR His in shotgun and targeted proteomics experiments at the peptide level. Parallel Reaction C: Remaining protein oxidation may be due to sulfone formation, oxidation of other amino acids, or re- Disappearance of Appearance of Reversal of methionine oxidation by MetSR has benefits to MS experiments involving intact proteins,

MonitoringFigure was1: Schematic used to demonstrate of Methionine 90-99% Oxidation reduction and in Reduction: oxidized methionine Methionine in MetSR S-sulfoxide and oxidation during electrospray ionization. C Oxidized Peptide Reduced Peptide targeted quantitative peptide analysis, and shotgun proteomics. treatedmethionine peptide Rand-sulfoxide phosphopeptides. formation and reversal. Figure 4: Intact Protein MS Results: Spectra compared after Thermo Scientific™ Xcalibur™ Xtract A modified LC-MS ionization source design may alleviate the residual modifications observed at the Raw file for oxidized and reduced samples using ngMsrAB-T or nmMsrAB-T. Figure 5A shows data 7 Figure 3: Amino acid sequences of two recombinant MetSR enzymes: Amino acid protein and peptide level . sequence of ngMsrAB-T and nmMsrAB-T prior to treatment with WELQut Protease to cleave from the 1:10 moles of methionine to H2O2, whereas Figure 5B is for 1:75 moles of methionine to H2O2. MetSR treated INTRODUCTION at the WELQ site. The peaks assigned as 0.0000 is the TurboLuc protein without any methionine oxidation, and shifts by 16 Daltons are indicative of oxidation (15 Daltons are sometimes observed due to data processing). REFERENCES Methionine (Met) is a sulfur-containing essential amino acid that is highly susceptible to MetSR treated 1. Zhang, X.H. and H. Weissbach, Origin and evolution of the protein-repairing enzymes methionine oxidation by reactive oxygen species during stress, aging, and disease in cells, and in vitro1. sulphoxide reductases. Biol Rev Camb Philos Soc, 2008. 83(3): p. 249-57. Met oxidation is the most common modification of purified therapeutic antibodies and other A 2. Khor, H.K., et al., Identification of methionine sulfoxide diastereomers in immunoglobulin gamma biologics, can affect protein function, and is observed by mass spectrometry (MS) in intact Oxidation by Hydrogen Peroxide antibodies using methionine sulfoxide reductase enzymes. MAbs, 2010. 2(3): p. 299-308. For intact analysis, recombinant turboluciferase (TurboLuc, ~16kDa) was selected to perform an Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T proteins and peptide samples as splitting the MS signal, which reduces sensitivity and CONCLUSIONS3. Morand, K., G. Talbo, and M. Mann, Oxidation of peptides during electrospray ionization. Rapid initial optimization of methionine oxidation and reduction reactions due to observed sensitivity to increases complexity of analysis. Methionine sulfoxides can be returned to methionine by B Commun Mass Spectrom, 1993. 7(8): p. 738-43. oxidation, knowledge of the sequence, and ability to detect with an Orbitrap XL mass spectrometer. methionineMethionine sulfoxide oxidation reductases. can also The occur product during of sample Met oxidation preparation, is Met purification sulfoxide (MetO),, storage which , and LC- Methionine4. Lao, oxidationY.W., et al., can Chromatographic be reversed in intact behavior proteins of peptides and peptides containing prior tooxidized analysis methionine by SDS-PAGE residues in 2,3 All experiments for intact analysis used18µM TurboLuc. TurboLuc has three moles of methionine – existsMS in ionization two diastereomers. Variable that methionine are reduced oxidation by two of methionine purified proteins sulfoxide leads reductases, to lower and MetSR variable- Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T and intactproteomic or peptide LC LCMS-MS experiments: analysis. Complex tale of a simple modification. Journal of Proteomics, per mole of protein and thus a 1:1 molar ratio of methionine to H O would be 54µM of H O for A andproduct MetSR quality.-B (Figure It also 1). complicates liquid chromatography-mass spectrometry (LC-MS) 2 2 2 2 2015. 125: p. 131-139. 4 18µM of TurboLuc. A variety of oxidation conditions were assessed at the following ratios of analysis in 4 ways: 1) Shifted and split peaks during liquid chromatography , 2) Increased Disappearance of Appearance of Reversal5. Peterson, of methionine A.C., et oxidation al., Parallel by MetSR reaction has monitoring benefits tofor MS high experiments resolution andinvolving high mass intact accuracy proteins, methionine: H O which were 10:1, 1:1, 1:10, 1:25, 1:75, 1:100 and 1:500 (Figure 5 shows 1:10 Figurecomplexity 1: Schematic and poorer of Methionine depth of analysis Oxidation of intact and proteinsReduction: due to Methionine multiple oxidized S-sulfoxide forms, and 3) 2 2 Oxidized Peptide Reduced Peptide targetedquantitative, quantitative targeted peptide proteomics.analysis, and Mol shotgun Cell Proteomics, proteomics. 2012. 11(11): p. 1475-88. and 1:75, the rest of the data are not shown). C – methionineIncreased R- sulfoxidecomplexity formation & poorer anddepth reversal. of analysis of peptide digests due to multiple oxidized 6. Zeliadt, N. Moving Target: New mass spectrometry based techniques are blurring the lines

forms, and; 4) Impaired quantitation of methionine-containing peptides due to the stochastic A modifiedbetween LC-MS discovery ionization and source targeted design proteomics. may alleviate 2014 the [cited residual 2016 modifications10May]; Available observed from: at the Reduction of Intact Proteins by ngMsrAB and nmMsrAB Disappearance of Appearance of nature of oxidation of methionine in peptides. protein http://www.theand peptide level-scientist.com/?articles.list/categoryNo/2921/category/Lab7. -Tools/. Methionine sufloxide reduction was performed by adding either ngMsrAB-T or nmMsrAB-T to the Oxidized Peptide Reduced Peptide 7. Kim, K., et al., Reducing protein oxidation in low-flow electrospray enables deeper investigation of TurboLuc control or oxidized sample at an enzyme:sample ratio of 1:4 and DTT to a final D proteoforms by top down proteomics. EuPA Open Proteomics, 2015. 8: p. 40-47. concentration of 5mM. Both enzymes were used in separate experiments. Samples were incubated REFERENCES METHODS at 37 C for 2 hours & then desalted using 3K MWCO concentrators. 1. Zhang, X.H. and H. Weissbach, Origin and evolution of the protein-repairing enzymes methionine MetSR treated TRADEMARKS/LICENSING ⁰ sulphoxide reductases. Biol Rev Camb Philos Soc, 2008. 83(3): p. 249-57. Expression of Recombinant Methionine Sulfoxide Reductases Shotgun Proteomics Coupled with Methionine Oxidation Reduction 2. Khor, H.K., et al., Identification of methionine sulfoxide diastereomers in immunoglobulin gamma Thermo Scientific™ Pierce™ 6 Protein Digest, equimolar, LC Oxidation by Hydrogen Peroxide -MS grade which was stored in liquid antibodiesFor Research using Use methionine Only. Not sulfoxide for use in reductase diagnostic enzymes. procedures. MAbs, 2010. 2(3): p. 299-308. Over a dozen different recombinant methionine reductase vectors were constructed for target For forintact over analysis, a year wasrecombinant used for theturboluciferase oxidation experiments. (TurboLuc, Control~16kDa) and was oxidized selected samples to perform were an 3. Morand© 2016 ,Thermo K., G. Talbo Fisher, and Scientific M. Mann, Inc. Oxidation All rights ofreserved. peptides Eksigent during electrospray is a trademark ionization. of ABSciex Rapid. All other reduced using the same procedure as intact protein analysis described above, except for C18 protein expression and optimized for E.coli ER2566. 0.1 g of biomass was lysed with cell lysis initial optimization of methionine oxidation and reduction reactions due to observed sensitivity to B MetSR treated Communtrademarks Mass are Spectromthe property, 1993. of Thermo 7(8): p. Fisher 738-43. Scientific and its subsidiaries. This information is not Methioninebuffer consisting oxidation ofcan 50mM also Trisoccur – HClduring (pH sample 8); 100mM preparation, NaCl; 0.1% purification Triton X , -storage100; 1mM , and EDTA LC- oxidation,peptide knowledge clean up. of the sequence, and ability to detect with an Orbitrap XL mass spectrometer. 4. Lao,intended Y.W., to et encourage al., Chromatographic use of these behavior products of in peptides any manner containing that might oxidized infringe methionine the intellectual residues property in MS (pH8)ionization and2,3 disrupted. Variable by methionine sonication. oxidation The soluble of purified fraction proteins was diluted leads with to lower wash and buffer variable All experiments for intact analysis used18µM TurboLuc. TurboLuc has three moles of methionine Targeted Protein Analysis Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T proteomicrights of others. LC–MS experiments: Complex tale of a simple modification. Journal of Proteomics, productconsisting quality. of It 250mM also complicates NaCl, 20mM liquid imidazole, chromatography 50mM Tris-mass-HCl, spectrometry pH=8 and applied (LC-MS) to 0.2 ml per LCmole-MS of analysisprotein and thus a 1:1 molar ratio of methionine to H2O2 would be 54µM of H2O2 for 2015. 125: p. 131-139. The protein sample was injected onto a Thermo Scientific™ ProSwift™ RP μ analysisThermo in 4 Scientific™ ways: 1) Shifted HisPur and Ni -splitNTA peaksand washed during extensively.liquid chromatography Bound proteins4, 2) Increased were eluted with 18µM of TurboLuc. A variety of oxidation conditions were assessed at the following-4H Analyticalratios of 100 m x Figure 6 shows the disappearance of the oxidized peptide and appearance of the corresponding 5. Peterson, A.C., et al., Parallel reaction monitoring for high resolution and high mass accuracy complexity50mM Tris, and pH8,poorer 250mM depth ofNaCl, analysis 500mM of intact imidazole proteins and duedialyzed to multiple against oxidized 50mM Trisforms,-HCl 3) pH 7.6, methionine:25 cm monolithic H2O2 which column were at 10:1, a flow 1:1, rate 1:10, of 8001:25, nL/min 1:75, 1:100over a and gradient 1:500 of (Figure water and5 shows acetonitrile 1:10 in reduced peptide after treatment with ngMsrAB or nmMsrAB. These data indicate that treatment quantitative, targeted proteomics. Mol Cell Proteomics, 2012. 11(11): p. 1475-88. Increased4oC, overnight. complexity Three & poorer MetSR depth genes of analysis expressed of peptidefrom three digests different due toclones multiple each oxidized were tested and0.1% 1:75, formicthe rest acid. of the The data shotgun are not proteomics shown). sample was injected onto a reversed-phase column with ngMsrAB or nmMsrAB results in a >90% reduction of an oxidized methionine and resulted in 6. Zeliadt, N. Moving Target: New mass spectrometry–based techniques are blurring the lines μ forms,by geland; shift 4) Impairedof an oxidized quantitation methionine of methionine rich protein-containing 6 (Figure peptides 2) and two due MetSR/Trx to the stochastic trifunctional (15cm x 75 m I.D., Prepmap C18) and separated using a gradient of water and acetonitrile in a corresponding increase in the peptide with a reduced methionine. In addition, after treatment Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T between discovery and targeted proteomics. 2014 [cited 2016 10May]; Available from: ™ natureenzymes of oxidation from Neisseria of methionine gonorrhoeae in peptides. or Neisseria meningitidis were selected for MS Reduction0.1% formic of Intact acid atProteins a flow rate by ngMsrABof 300 nL/min and. nmMsrAB All samples used an Eksigent nanoLC Ultra 2D some of the oxidized peptides were not even detectable (Figure 6 A and C for ngMsrAB-T). Disappearance of Appearance of http://www.the-scientist.com/?articles.list/categoryNo/2921/category/Lab-Tools/. ™ ™ applications (Figure 2). Methionineplus HPLC sufloxide coupled reduction to a Thermo was performed Scientific by LTQ adding Orbitrap either ng XLMsrAB ETD -massT or nm spectrometer.MsrAB-T to the Oxidized Peptide Reduced Peptide 7. Kim, K., et al., Reducing protein oxidation in low-flow electrospray enables deeper investigation of TurboLuc control or oxidized sample at an enzyme:sample ratio of 1:4 and DTT to a final D proteoforms by top down proteomics. EuPA Open Proteomics, 2015. 8: p. 40-47. concentration of 5mM. Both enzymes were used in separate experiments. Samples were incubated METHODS at 37 C for 2 hours & then desalted using 3K MWCO concentrators. TRADEMARKS/LICENSING ⁰ Expression of Recombinant Methionine Sulfoxide Reductases Shotgun Proteomics Coupled with Methionine Oxidation Reduction Thermo Scientific™ Pierce™ 6 Protein Digest, equimolar, LC -MS grade which was stored in liquid For Research Use Only. Not for use in diagnostic procedures. Over a dozen different recombinant methionine reductase vectors were constructed for target for over a year was used for the oxidation experiments. Control and oxidized samples were © 2016 Thermo Fisher Scientific Inc. All rights reserved. Eksigent is a trademark of ABSciex. All other reduced using the same procedure as intact protein analysis described above, except for C18 protein expression and optimized for E.coli ER2566. 0.1 g of biomass was lysed with cell lysis MetSR treated trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not buffer consisting of 50mM Tris – HCl (pH 8); 100mM NaCl; 0.1% Triton X-100; 1mM EDTA peptide clean up. intended to encourage use of these products in any manner that might infringe the intellectual property (pH8) and disrupted by sonication. The soluble fraction was diluted with wash buffer Targeted Protein Analysis rights of others. consisting of 250mM NaCl, 20mM imidazole, 50mM Tris-HCl, pH=8 and applied to 0.2 ml LC-MS analysis The protein sample was injected onto a Thermo Scientific™ ProSwift™ RP μ Thermo Scientific™ HisPur Ni-NTA and washed extensively. Bound proteins were eluted with -4H Analytical 100 m x Figure 6 shows the disappearance of the oxidized peptide and appearance of the corresponding 50mM Tris, pH8, 250mM NaCl, 500mM imidazole and dialyzed against 50mM Tris-HCl pH 7.6, 25 cm monolithic column at a flow rate of 800 nL/min over a gradient of water and acetonitrile in reduced peptide after treatment with ngMsrAB or nmMsrAB. These data indicate that treatment 4oC, overnight. Three MetSR genes expressed from three different clones each were tested 0.1% formic acid. The shotgun proteomics sample was injected onto a reversed-phase column with ngMsrAB or nmMsrAB results in a >90% reduction of an oxidized methionine and resulted in μ by gel shift of an oxidized methionine rich protein 6 (Figure 2) and two MetSR/Trx trifunctional (15cm x 75 m I.D., Prepmap C18) and separated using a gradient of water and acetonitrile in a corresponding increase in the peptide with a reduced methionine. In addition, after treatment Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T ™ enzymes from Neisseria gonorrhoeae or Neisseria meningitidis were selected for MS 0.1% formic acid at a flow rate of 300 nL/min. All samples used an Eksigent nanoLC Ultra 2D some of the oxidized peptides were not even detectable (Figure 6 A and C for ngMsrAB-T). applications (Figure 2). plus HPLC coupled to a Thermo Scientific™ LTQ Orbitrap™ XL ETD mass spectrometer.

Figure 2: Gel Shift Assay to Reduce MRP6: Three different MetSR genes expressed in Targeted Quantitative Peptide Analysis Figure 5: Parallel Reaction Monitoring of three methionine containing peptides: Schematic Shotgun proteomics OVERVIEW 5,6 three different vectors were tested for their ability to reduce MRP6 after being oxidized by H2O2. Three heavy peptide standards (two phosphopeptides) containing an oxidized methionine (100 fmol/μL ) of parallel reaction monitoring for targeted peptide quantitation (A). Quantitative reversal of were mixed with the Pierce 6 protein digest, equimolar, LC-MS grade (40 ng/μL). The mixed sample methionine oxidation in a peptide standard (B), a phosphopeptide of the same peptide standard The Pierce 6 protein LC-MS grade digest was used to assess the two MetSR enzymes using both non- Purpose No was then reduced by either ngMsrAB-T or nmMsrAB-T as described earlier. The resulting peptide (C), and a second phosphopeptide (D). The bar graph area shows the cumulative area under the oxidized and mildly oxidized samples (Figure 7). Substantial methionine reversal was seen with all To synthesize two active forms of recombinant methionine sulfoxide reductase (MetSR) MsrA1 (Human) ngMsrAB-T nmMsrAB-T Reduction mixture was then injected onto a C18 column (Easy-Spray PepMap C18, 3µm, 75µm x 150 cm) and curve of individual b-ions and y-ions. Each experiment was performed in duplicate. reaction ratios less than 1:100 moles of methionine:H2O2. At 1:100 sulfones and other amino acids enzymes and perform proof of principle studies for mass spectrometry applications in intact A B C A B C A B C Vector separated by a gradient of water and acetonitrile in 0.1% formic acid using a Thermo Scientific™ were oxidized which greatly reduced the identification of peptides due to random amino acid oxidation protein, targeted quantitative peptides, and shotgun proteomics. Dionex™ Ultimate™ 3000 RSLCnano system. The sample was infused into the Q Exactive HF mass events (data not shown). In addition, DTT alone did not show any reversal of methionine oxidation. A MRP6 (oxidized) spectrometer via electrospray ionization utilizing parallel reaction monitoring (Figure 4) for collection of Lastly, numerous oxidized peptides were no longer detected after treatment with MetSR. Remaining Methods targeted spectra. The resulting raw data were processed and visualized using Skyline 3.1.0.7382 peptide oxidation may be due to sulfone formation, oxidation of other amino acids, or re-oxidation during Two recombinant MetSR and a methionine-rich protein were expressed, purified, and tested analysis software. electrospray ionization. for gel mobility shifts prior to use in MS applications. For intact and shotgun proteomics experiments ,hydrogen peroxide was added to induce mild methionine oxidation and MRP6 (reduced) RESULTS Figure 6: Average intensity of 6-protein digested mix: A comparison of the average peptide intensity compared to non-oxidized controls before and after treatment with MetSR. Reduction was of detectable peptides before and after treatment with two forms of MetSR. carried out using DTT as a cofactor and samples were analyzed by a Thermo Scientific™ Intact Protein Analysis - - + + + + + + + + + + DTT OrbitrapThe XL™ Hybrid IonUse Trap-Orbitrap MSof . Targeted Methionine quantitative analysis of methionine Sulfoxide Reductases to Reverse Oxidized Methionine for Mass Spectrometry Applications containing peptide and phosphopeptides were done on a Thermo Scientific™ Q Exactive™ HF - + + + + + + + + + + + H2O2 The ability for both MetSR enzymes to reduce an intact protein was demonstrated using Thermo 1 1 2 2 2 Scientific™1 TurboLuc™ . Figure 5 demonstrates3 how oxidation is reversed3 by either MetSR. Figure 2 2 1 MS usingRobert Parallel ReactionCunningham Monitoring. ; Kratika Singhal ; Ryan T Fellers ; Luca Fornelli ; Henrique Dos Santos Seckler ; Bhavin Patel ; Egle Capkauske ; Juozas Siurkus ; Philip Compton ; Neil L Kelleher ; John C Rogers His WQ MSR 5A shows that 1:10 moles of methionine to moles of H2O2 can decrease the forms of oxidized Disappearance of Appearance of 1 2 A 3 Results: : TurboLuc mildly. When looking at highly oxidized samples shown in Figure 5B (1:75 ratio) significant B Thermo Fisher Scientific, Rockford, IL; Northwestern University,trx Evanston,His WQ IL;MSR Thermo Fisher Scientific, Vilnius, Lithuania Oxidized Peptide Reduced Peptide We successfully produced and purified two active MetSR enzymes, as observed by gel shift of B oxidation was able to be reversed, but some oxidation events occurred which could either be caused : from other amino acids being oxidized or creation of the sulfone that are not reversible by MetSR. an oxidized methionine rich protein, reduction of oxidized methionines in an intact protein, and MSR His in shotgun and targeted proteomics experiments at the peptide level. Parallel Reaction C: Remaining protein oxidation may be due to sulfone formation, oxidation of other amino acids, or re- Shotgun proteomics Monitoring was used to demonstrate 90-99% reduction in oxidized methionine in MetSR Figure 2: Gel Shift Assay to Reduce MRP6: Three different MetSR genes expressed in oxidationTargeted during Quantitative electrospray Peptide ionization. Analysis Figure 5: Parallel Reaction Monitoring of three methionine containing peptides: Schematic OVERVIEW μ 5,6 treated peptide and phosphopeptides. three different vectors were tested for their ability to reduce MRP6 after being oxidized by H2O2. Three heavy peptide standards (two phosphopeptides) containing an oxidized methionine (100 fmol/ L ) of parallel reaction monitoring for targeted peptide quantitation (A). Quantitative reversal of Figurewere 4:mixed Intact with Protein the Pierce MS Results:6 protein Spectradigest, equimolar, compared LCafter-MS Thermo grade Scientific™(40 ng/μL). Xcalibur™The mixed Xtract sample methionine oxidation in a peptide standard (B), a phosphopeptide of the same peptide standard The Pierce 6 protein LC-MS grade digest was used to assess the two MetSR enzymes using both non- Purpose No Figure 3: Amino acid sequences of two recombinant MetSR enzymes: Amino acid Rawwas file then for oxidized reduced and by either reduced ngMsrAB samples-T orusing nm MsrABngMsrAB-T as-T ordescribed nmMsrAB earlier.-T. Figure The resulting5A shows peptide data (C), and a second phosphopeptide (D). The bar graph area shows the cumulative area under the oxidized and mildly oxidized samples (Figure 7). Substantial methionine reversal was seen with all To synthesize two active forms of recombinant methionine sulfoxide reductase (MetSR) Reduction MsrA1 (Human) ngMsrAB-T nmMsrAB-T sequence of ngMsrAB-T and nmMsrAB-T prior to treatment with WELQut Protease to cleave frommixture the 1:10 was moles then ofinjected methionine onto a to C18 H2O column2, whereas (Easy Figure-Spray 5B PepMap is for 1:75 C18, moles 3µm, of 75µm methionine x 150 cm)to H and2O2. curve of individual bMetSR-ions and treated y-ions. Each experiment was performed in duplicate. reaction ratios less than 1:100 moles of methionine:H2O2. At 1:100 sulfones and other amino acids enzymes and perform proof of principle studies for mass spectrometry applications in intact A B C A B C A B C Vector Theseparated peaks assigned by a gradient as 0.0000 of water is the and TurboLuc acetonitrile protein in 0.1% without formic any acidmethionine using a oxidation, Thermo Scientific™ and shifts by were oxidized which greatly reduced the identification of peptides due to random amino acid oxidation INTRODUCTIONprotein, targeted quantitative peptides, and shotgun proteomics. at the WELQ site. 16 Dionex™Daltons are Ultimate™ indicative 3000 of oxidation RSLCnano (15 system.Daltons areThe sometimes sample was observed infused intodue theto data Q Exactive processing). HF mass A events (data not shown). In addition, DTT alone did not show any reversal of methionine oxidation. spectrometer via electrospray ionization utilizing parallel reaction monitoring (Figure 4) for collection of Lastly, numerous oxidized peptides were no longer detected after treatment with MetSR. Remaining MethionineMethods (Met) is a sulfur-containing essential amino acid that is highly susceptible to MRP6 (oxidized) oxidation by reactive oxygen species during stress, aging, and disease in cells, and in vitro1. targeted spectra. The resulting raw data were processed and visualized using Skyline 3.1.0.7382 peptide oxidation may be due to sulfone formation, oxidation of other amino acids, or re-oxidation during Two recombinant MetSR and a methionine-rich protein were expressed, purified, and tested analysis software. electrospray ionization. Metfor oxidation gel mobility is the shifts most prior common to use modification in MS applications. of purified For therapeutic intact and antibodiesshotgun proteomics and other A biologics, can affect protein function, and is observed by mass spectrometry (MS) in intact experiments ,hydrogen peroxide was added to induce mild methionine oxidation and Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T proteins and peptide samples as splitting the MS signal, which reduces sensitivity and MRP6 (reduced) RESULTS CONCLUSIONSFigure 6: Average intensity of 6-protein digested mix: A comparison of the average peptide intensity compared to non-oxidized controls before and after treatment with MetSR. Reduction was of detectable peptides before and after treatment with two forms of MetSR. increasescarried complexityout using DTT of analysis. as a cofactor Methionine and samples sulfoxides were can analyzed be returned by a toThermo methionine Scientific™ by Intact Protein Analysis methionineOrbitrap sulfoxideXL™ Hybrid reductases. Ion Trap The product of Met oxidation is Met sulfoxide (MetO), which - - + + + + + + + + + + DTT Methionine oxidation can be reversed in intact proteins and peptides prior to analysis by SDS-PAGE -Orbitrap MS . Targeted quantitative analysis of methionine existscontaining in two diastereomers peptide and phosphopeptides that are reduced were by two done methionine on a Thermo sulfoxide Scientific™ reductases, Q Exactive™ MetSR- HF - + + + + + + + + + + + H2O2 The ability for both MetSR enzymes to reduce an intact protein was demonstrated using Thermo and intact or peptide LC-MS analysis.

A andMS MetSR using Parallel-B (Figure Reaction 1). Monitoring. Scientific™ TurboLuc™ . Figure 5 demonstrates how oxidation is reversed by either MetSR. Figure Disappearance of Appearance of His WQ MSR 5A shows that 1:10 moles of methionine to moles of H2O2 can decrease the forms of oxidized Disappearance of Appearance of Reversal of methionine oxidation by MetSR has benefits to MS experiments involving intact proteins, A FigureResults: 1: Schematic of Methionine Oxidation and Reduction: Methionine S-sulfoxide and : TurboLuc mildly. When looking at highly oxidized samples shown in Figure 5B (1:75 ratio) significant C B Oxidized Peptide Reduced Peptide targeted quantitative peptide analysis, and shotgun proteomics. methionine R-sulfoxide formation and reversal. trx His WQ MSR oxidation was able to be reversed, but some oxidation events occurred which could either be caused Oxidized Peptide Reduced Peptide We successfully produced and purified two active MetSR enzymes, as observed by gel shift of B: an oxidized methionine rich protein, reduction of oxidized methionines in an intact protein, and from other amino acids being oxidized or creation of the sulfone that are not reversible by MetSR. A modified LC-MS ionization source design may alleviate the residual modifications observed at the MSR His 7 in shotgun and targeted proteomics experiments at the peptide level. Parallel Reaction C: Remaining protein oxidation may be due to sulfone formation, oxidation of other amino acids, or re- protein and peptide level .

Monitoring was used to demonstrate 90-99% reduction in oxidized methionine in MetSR oxidation during electrospray ionization. treated peptide and phosphopeptides. Figure 4: Intact Protein MS Results: Spectra compared after Thermo Scientific™ Xcalibur™ Xtract REFERENCES Raw file for oxidized and reduced samples using ngMsrAB-T or nmMsrAB-T. Figure 5A shows data 1. Zhang, X.H. and H. Weissbach, Origin and evolution of the protein-repairing enzymes methionine Figure 3: Amino acid sequences of two recombinant MetSR enzymes: Amino acid MetSR treated sequence of ngMsrAB-T and nmMsrAB-T prior to treatment with WELQut Protease to cleave from the 1:10 moles of methionine to H2O2, whereas Figure 5B is for 1:75 moles of methionine to H2O2. MetSR treated sulphoxide reductases. Biol Rev Camb Philos Soc, 2008. 83(3): p. 249-57. at the WELQ site. The peaks assigned as 0.0000 is the TurboLuc protein without any methionine oxidation, and shifts by 2. Khor, H.K., et al., Identification of methionine sulfoxide diastereomers in immunoglobulin gamma INTRODUCTION Oxidation by Hydrogen Peroxide 16 Daltons are indicative of oxidation (15 Daltons are sometimes observed due to data processing). antibodies using methionine sulfoxide reductase enzymes. MAbs, 2010. 2(3): p. 299-308. For intact analysis, recombinant turboluciferase (TurboLuc, ~16kDa) was selected to perform an Methionine (Met) is a sulfur-containing essential amino acid that is highly susceptible to 3. Morand, K., G. Talbo, and M. Mann, Oxidation of peptides during electrospray ionization. Rapid initial optimization of methionine oxidation and reduction reactions due to observed sensitivity to oxidation by reactive oxygen species during stress, aging, and disease in cells, and in vitro1. B Commun Mass Spectrom, 1993. 7(8): p. 738-43. oxidation, knowledge of the sequence, and ability to detect with an Orbitrap XL mass spectrometer. MethionineMet oxidation oxidation is the can most also common occur during modification sample of preparation, purified therapeutic purification antibodies , storage and , and other LC - 4. Lao, Y.W., et al., Chromatographic behavior of peptides containing oxidized methionine residues in 2,3 All experiments for intact analysis used18µM TurboLuc. TurboLuc has three moles of methionine A – MSbiologics, ionization can. Variableaffect protein methionine function, oxidation and is observedof purified by proteins mass spectrometryleads to lower (MS) and invariable intact Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T proteomic LC MS experiments: Complex tale of a simple modification. Journal of Proteomics, per mole of protein and thus a 1:1 molar ratio of methionine to H O would be 54µM of H O for productproteins quality. and Itpeptide also complicates samples as liquid splitting chromatography the MS signal,-mass which spectrometry reduces sensitivity (LC-MS) and 2 2 2 2 Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T 2015. 125: p. 131-139. 4 18µM of TurboLuc. A variety of oxidation conditions were assessed at the following ratios of CONCLUSIONS analysisincreases in 4 ways:complexity 1) Shifted of analysis. and split Methionine peaks during sulfoxides liquid chromatography can be returned to, 2) methionine Increased by 5. Peterson, A.C., et al., Parallel reaction monitoring for high resolution and high mass accuracy methionine: H O which were 10:1, 1:1, 1:10, 1:25, 1:75, 1:100 and 1:500 (Figure 5 shows 1:10 complexitymethionine and sulfoxide poorer depth reductases. of analysis The ofproduct intact proteinsof Met oxidation due to multiple is Met sulfoxideoxidized forms,(MetO), 3) which 2 2 quantitative, targeted proteomics. Mol Cell Proteomics, 2012. 11(11): p. 1475-88. and 1:75, the rest of the data are not shown). Methionine oxidation can be reversed in intact proteins– and peptides prior to analysis by SDS-PAGE Increasedexists in complexity two diastereomers & poorer thatdepth are of reduced analysis by of twopeptide methionine digests sulfoxidedue to multiple reductases, oxidized MetSR - 6. Zeliadt, N. Moving Target: New mass spectrometry based techniques are blurring the lines and intact or peptide LC-MS analysis. forms,A and and; MetSR 4) Impaired-B (Figure quantitation 1). of methionine-containing peptides due to the stochastic between discovery and targeted proteomics. 2014 [cited 2016 10May]; Available from: Reduction of Intact Proteins by ngMsrAB and nmMsrAB Disappearance of Appearance of nature of oxidation of methionine in peptides. Disappearance of Appearance of http://www.the-scientist.com/?articles.list/categoryNo/2921/category/Lab-Tools/. Methionine sufloxide reduction was performed by adding either ngMsrAB-T or nmMsrAB-T to the Reversal of methionine oxidation by MetSR has benefits to MS experiments involving intact proteins, Figure 1: Schematic of Methionine Oxidation and Reduction: Methionine S-sulfoxide and Oxidized Peptide Reduced Peptide 7. Kim, K., et al., Reducing protein oxidation in low-flow electrospray enables deeper investigation of TurboLuc control or oxidized sample at an enzyme:sample ratio of 1:4 and DTT to a final D C Oxidized Peptide Reduced Peptide targeted quantitative peptide analysis, and shotgun proteomics. methionine R-sulfoxide formation and reversal. proteoforms by top down proteomics. EuPA Open Proteomics, 2015. 8: p. 40-47. concentration of 5mM. Both enzymes were used in separate experiments. Samples were incubated

METHODS at 37 C for 2 hours & then desalted using 3K MWCO concentrators. A modified LC-MS ionization source design may alleviate the residual modifications observed at the 7 TRADEMARKS/LICENSINGprotein and peptide level .

Shotgun⁰ Proteomics Coupled with Methionine Oxidation Reduction Expression of Recombinant Methionine Sulfoxide Reductases Thermo Scientific™ Pierce™ 6 Protein Digest, equimolar, LC-MS grade which was stored in liquid For Research Use Only. Not for use in diagnostic procedures. REFERENCES for over a year was used for the oxidation experiments. Control and oxidized samples were © 2016 Thermo Fisher Scientific Inc. All rights reserved. Eksigent is a trademark of ABSciex. All other Over a dozen different recombinant methionine reductase vectors were constructed for target 1. Zhang, X.H. and H. Weissbach, Origin and evolution of the protein-repairing enzymes methionine reduced using the same procedure as intact protein analysis described above, except for C18 MetSR treated trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not protein expression and optimized for E.coli ER2566. 0.1 g of biomass was lysed with cell lysis MetSR treated sulphoxide reductases. Biol Rev Camb Philos Soc, 2008. 83(3): p. 249-57. – peptide clean up. intended to encourage use of these products in any manner that might infringe the intellectual property buffer consisting of 50mM Tris HCl (pH 8); 100mM NaCl; 0.1% Triton X-100; 1mM EDTA 2. Khor, H.K., et al., Identification of methionine sulfoxide diastereomers in immunoglobulin gamma rights of others. (pH8) and disrupted by sonication. The soluble fraction was diluted with wash buffer Oxidation by Hydrogen Peroxide Targeted Protein Analysis antibodies using methionine sulfoxide reductase enzymes. MAbs, 2010. 2(3): p. 299-308. LC-MS analysis consisting of 250mM NaCl, 20mM imidazole, 50mM Tris-HCl, pH=8 and applied to 0.2 ml For intact analysis, recombinant turboluciferase (TurboLuc, ~16kDa) was selected to perform an 3. Morand, K., G. Talbo, and M. Mann, Oxidation of peptides during electrospray ionization. Rapid Thermo Scientific™ The protein sample was injected onto a Thermo Scientific™ ProSwift™ RP-4H Analytical 100 μm x The Use of Methionine Sulfoxide HisPur Ni -NTAReductases and washed extensively. Bound proteins were eluted to with Reverseinitial optimization of methionineOxidized oxidation and reduction reactions Methionine due to observed sensitivity to Figure forB 6 shows the disappearanceMass of the oxidized Spectrometry peptide and appearance of the corresponding Applications Commun Mass Spectrom, 1993. 7(8): p. 738-43. 25 cm monolithic column at a flow rate of 800 nL/min over a gradient of water and acetonitrile in 50mMMethionine Tris, pH8, oxidation 250mM can NaCl, also 500mM occur duringimidazole sample and preparation,dialyzed against purification 50mM Tris, storage-HCl pH , and 7.6, LC - oxidation, knowledge of the sequence, and ability to detect with an Orbitrap XL mass spectrometer. reduced peptide after treatment with ngMsrAB or nmMsrAB. These data indicate that treatment 4. Lao, Y.W., et al., Chromatographic behavior of peptides containing oxidized methionine residues in o 0.1% formic acid. The shotgun proteomics sample was injected onto a reversed-phase column 1 1 2 4 C,MS overnight. ionization Three2,32. Variable MetSR methionine genes expressed oxidation from of purified three different proteins clones leads eachto2 lower were and tested variable 1 All experiments for intact analysis3 used18µM TurboLuc. TurboLuc3 has three moles of methionine2 with ngMsrAB2 or nmMsrAB results in a >90% reduction1 of an oxidized methionine and resulted in Control nmMsrAB-T ngMsrAB-T proteomic LC–MS experiments: Complex tale of a simple modification. Journal of Proteomics, Robert Cunningham ; Kratika Singhal ; Ryan T Fellers ; Luca Fornelli ; Henrique Dos Santos Seckler ; Bhavin Patel ; Egle(15cm xCapkauske 75 μm I.D., Prepmap ;C18) Juozas and separated Siurkus using a gradient; Philip of water Comptonand acetonitrile in ; Neil L Kelleher ; John C Rogers Control nmMsrAB-T ngMsrAB-T by gelproduct shift quality.of an oxidized It also complicatesmethionine richliquid protein chromatography 6 (Figure 2)-mass and two spectrometry MetSR/Trx (LC trifunctional-MS) per mole of protein and thus a 1:1 molar ratio of methionine to H2O2 would be 54µM of H2O2 for a corresponding increase in the peptide with a reduced methionine. In addition, after treatment Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T 2015. 125: p. 131-139. 0.1% formic acid at a flow rate of 300 nL/min. All samples used an Eksigent™ nanoLC Ultra 2D 1 2 enzymesanalysis from in 4 Neisseria ways: 1) gonorrhoeaeShifted and split or Neisseria peaks during3 meningitidis liquid chromatography were selected 4for, 2) MS Increased 18µM of TurboLuc. A variety of oxidation conditions were assessed at the following ratios of some of the oxidized peptides were not even detectable (Figure 6 A and C for ngMsrAB-T). 5. Peterson, A.C., et al., Parallel reaction monitoring for high resolution and high mass accuracy Thermo Fisher Scientific, Rockford, IL; Northwestern University, Evanston, IL; Thermo Fisher Scientific, Vilnius,plus HPLC Lithuania coupled to a Thermo Scientific™ LTQ Orbitrap™ XL ETD mass spectrometer. applicationscomplexity (Figure and poorer 2). depth of analysis of intact proteins due to multiple oxidized forms, 3) methionine: H2O2 which were 10:1, 1:1, 1:10, 1:25, 1:75, 1:100 and 1:500 (Figure 5 shows 1:10 quantitative, targeted proteomics. Mol Cell Proteomics, 2012. 11(11): p. 1475-88. Increased complexity & poorer depth of analysis of peptide digests due to multiple oxidized and 1:75, the rest of the data are not shown). 6. Zeliadt, N. Moving Target: New mass spectrometry–based techniques are blurring the lines forms, and; 4) Impaired quantitation of methionine-containing peptides due to the stochastic between discovery and targeted proteomics. 2014 [cited 2016 10May]; Available from: Figure 2: Gel Shift Assay to Reduce MRP6: Three different MetSR genes expressed in TargetedReduction Quantitative of Intact Peptide Proteins Analysis by ngMsrAB and nmMsrAB Figure 5: Parallel Reaction Monitoring of three methionine containing peptides: Schematic Shotgun proteomicsDisappearance of Appearance of http://www.the-scientist.com/?articles.list/categoryNo/2921/category/Lab-Tools/. OVERVIEW nature of oxidation of methionine in peptides. 5,6 three different vectors were tested for their ability to reduce MRP6 after being oxidized by H2O2. Three heavyMethionine peptide sufloxide standards reduction (two phosphopeptides) was performed by containing adding either an oxidized ngMsrAB methionine-T or nmMsrAB (100 -fmol/T to μtheL ) of parallel reaction monitoring for targeted peptide quantitation (A). Quantitative reversal of Oxidized Peptide Reduced Peptide 7. Kim, K., et al., Reducing protein oxidation in low-flow electrospray enables deeper investigation of were mixedTurboLuc with thecontrol Pierce or oxidized 6 protein sample digest, atequimolar, an enzyme:sample LC-MS grade ratio (40 of ng/1:4μ andL). DTTThe mixedto a final sample methionine oxidation in a peptide standard (B), a phosphopeptide of the same peptide standard TheD Pierce 6 protein LC-MS grade digest was used to assess the two MetSR enzymes using both non- proteoforms by top down proteomics. EuPA Open Proteomics, 2015. 8: p. 40-47. Purpose No was thenconcentration reduced by ofeither 5mM. ng BothMsrAB enzymes-T or nm wereMsrAB used-T as in describedseparate experiments. earlier. The resultingSamples peptidewere incubated (C), and a second phosphopeptide (D). The bar graph area shows the cumulative area under the oxidized and mildly oxidized samples (Figure 7). Substantial methionine reversal was seen with all To synthesize two active forms of recombinant methionine sulfoxide reductase (MetSR) Reduction MsrA1 (Human) ngMsrAB-T nmMsrAB-T METHODS mixtureat was 37 thenC for injected 2 hours onto& then a C18 desalted column using (Easy 3K- MWCOSpray PepMap concentrators. C18, 3µm, 75µm x 150 cm) and curve of individual b-ions and y-ions. Each experiment was performed in duplicate. reaction ratios less than 1:100 moles of methionine:H2O2. At 1:100 sulfones and other amino acids enzymes and perform proof of principle studies for mass spectrometry applications in intact A B C A B C A B C Vector separated by a gradient of water and acetonitrile in 0.1% formic acid using a Thermo Scientific™ were oxidized which greatly reduced the identification of peptides due to random amino acid oxidation TRADEMARKS/LICENSING protein, targeted quantitative peptides, and shotgun proteomics. ⁰ Recombinant Methionine Sulfoxide Reductases Dionex™Shotgun Ultimate™ Proteomics 3000 RSLCnano Coupled system. with Methionine The sample Oxidation was infused Reduction into the Q Exactive HF mass events (data not shown). In addition, DTT alone did not show any reversal of methionine oxidation. Expression of A spectrometerThermo via Scientific™ electrospray Pierce™ ionization 6 Protein utilizing Digest, parallel equimolar, reaction LCmonitoring-MS grade (Figure which 4) was for collectionstored in liquid of Lastly, numerous oxidized peptides were no longer detected after treatment with MetSR. Remaining For Research Use Only. Not for use in diagnostic procedures. Methods MRP6 (oxidized) targetedfor spectra. over a yearThe wasresulting used raw for thedata oxidation were processed experiments. and visualized Control and using oxidized Skyline samples 3.1.0.7382 were peptide oxidation may be due to sulfone formation, oxidation of other amino acids, or re-oxidation during © 2016 Thermo Fisher Scientific Inc. All rights reserved. Eksigent is a trademark of ABSciex. All other Two recombinant MetSR and a methionine-rich protein were expressed, purified, and tested Over a dozen different recombinant methionine reductase vectors were constructed for target analysisreduced software. using the same procedure as intact protein analysis described above, except for C18 electrospray ionization. trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not for gel mobility shifts prior to use in MS applications. For intact and shotgun proteomics protein expression and optimized for E.coli ER2566. 0.1 g of biomass was lysed with cell lysis MetSR treated – peptide clean up. intended to encourage use of these products in any manner that might infringe the intellectual property experiments ,hydrogen peroxide was added to induce mild methionine oxidation and buffer consisting of 50mM Tris HCl (pH 8); 100mM NaCl; 0.1% Triton X-100; 1mM EDTA (pH8) and disrupted by sonication. The soluble fraction was diluted with MRP6wash buffer (reduced) RESULTS Targeted Protein Analysis Figure 6: Average intensity of 6-protein digested mix: A comparison of the average peptide intensity rights of others. compared to non-oxidized controls before and after treatment with MetSR. Reduction was LC-MS analysis of detectable peptides before and after treatment with two forms of MetSR. carried Theout using DTT as aUse cofactor and samples of were analyzedMethionine by a Thermo Scientific™ Sulfoxideconsisting of 250mM NaCl, 20mM imidazole,Reductases 50mM Tris-HCl, pH=8 and applied to 0.2 mlto ReverseIntact Protein Analysis Oxidized Methionine for Mass Spectrometry Applications The protein sample was injected onto a Thermo Scientific™ ProSwift™ RP-4H Analytical 100 μm x Orbitrap XL™ Hybrid Ion Trap - Thermo- + Scientific™ + +HisPur + Ni -NTA+ and+ washed + extensively.+ + Bound+ DTT proteins were eluted with Figure 6 shows the disappearance of the oxidized peptide and appearance of the corresponding -Orbitrap MS . Targeted quantitative analysis of methionine 25 cm monolithic column at a flow rate of 800 nL/min over a gradient of water and acetonitrile in containing peptide and phosphopeptides were1 done on a Thermo Scientific™ Q1 Exactive™ HF 2 - 50mM+ Tris,+ pH8,+ 250mM+2 NaCl,+ +500mM + imidazole + and+ dialyzed+ + against H2O 50mM2 Tris2 -HCl pH 7.6, The ability1 for both MetSR enzymes to 3reduce an intact protein was demonstrated3 using Thermo 2 reduced peptide2 after treatment with ngMsrAB or nm1 MsrAB. These data indicate that treatment Robert Cunningham ; Kratika Singhal ; Ryan T Fellers ; Lucao Fornelli ; Henrique Dos Santos Seckler ; Bhavin Patel ; Egle Capkauske ; Juozas Siurkus ; Philip Compton ; Neil L Kelleher 4 C, overnight. Three MetSR genes expressed from three different clones each were tested Scientific™0.1% TurboLuc™ formic acid. . TheFigure shotgun 5 demonstrates proteomics how sample oxidation was injectedis reversed onto by a eitherreversed MetSR.-phase Figure column with ngMsrAB or nm; MsrABJohn results C inRogers a >90% reduction of an oxidized methionine and resulted in MS using Parallel Reaction Monitoring. μ 1 2 by gel shift of an oxidized methionine rich protein 6 (Figure3 2) and two MetSR/Trx trifunctional 5A shows(15cm that x 1:10 75 molesm I.D., of Prepmap methionine C18) to andmoles separated of H O usingcan decrease a gradient the of forms water of and oxidized acetonitrile in a corresponding increase in the peptide with a reduced methionine. In addition, after treatment Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T His WQ MSR 2 2 ™ Disappearance of Appearance of Thermo Fisher Scientific, Rockford, IL; Northwesternenzymes University,A from: Neisseria gonorrhoeaeEvanston, or Neisseria IL; meningitidis Thermo were selected Fisher for MS Scientific,TurboLuc Vilnius,0.1% mildly. formic When acidLithuania lookingat a flow at rate highly of 300oxidized nL/min samples. All samples shown usedin Figure an Eksigent 5B (1:75 ratio) nanoLC significant Ultra 2D some of the oxidized peptides were not even detectable (Figure 6 A and C for ngMsrAB-T). Results: ™ ™ B applications (Figuretrx 2). His WQ MSR oxidation plus was HPLC able coupled to be reversed, to a Thermo but some Scientific oxidation LTQ events Orbitrap occurred XL ETDwhich mass could spectrometer. either be caused Oxidized Peptide Reduced Peptide We successfully produced and purified two active MetSR enzymes, as observed by gel shift of B: an oxidized methionine rich protein, reduction of oxidized methionines in an intact protein, and from other amino acids being oxidized or creation of the sulfone that are not reversible by MetSR. MSR His Shotgun proteomics in shotgun and targeted proteomics experiments at the peptide level. Parallel Reaction Figure C2:: Gel Shift Assay to Reduce MRP6: Three different MetSR genes expressed in RemainingTargeted protein Quantitative oxidation may Peptide be due Analysis to sulfone formation, oxidation of other amino acids, or re- Figure 5: Parallel Reaction Monitoring of three methionine containing peptides: Schematic OVERVIEW 5,6 three different vectors were tested for their ability to reduce MRP6 after being oxidized by H2O2. oxidationThree during heavy electrospray peptide standards ionization. (two phosphopeptides) containing an oxidized methionine (100 fmol/μL ) of parallel reaction monitoring for targeted peptide quantitation (A). Quantitative reversal of Monitoring was used to demonstrate 90-99% reduction in oxidized methionine in MetSR were mixed with the Pierce 6 protein digest, equimolar, LC-MS grade (40 ng/μL). The mixed sample methionine oxidation in a peptide standard (B), a phosphopeptide of the same peptide standard The Pierce 6 protein LC-MS grade digest was used to assess the two MetSR enzymes using both non- treatedPurpose peptide and phosphopeptides. No Figurewas 4: Intactthen reduced Protein by MS either Results: ngMsrAB Spectra-T or compared nmMsrAB -afterT as Thermo described Scientific™ earlier. The Xcalibur™ resulting Xtract peptide (C), and a second phosphopeptide (D). The bar graph area shows the cumulative area under the oxidized and mildly oxidized samples (Figure 7). Substantial methionine reversal was seen with all To synthesize two active forms of recombinant methionine sulfoxide reductase (MetSR) Reduction MsrA1 (Human) ngMsrAB-T nmMsrAB-T Figure 3: Amino acid sequences of two recombinant MetSR enzymes: Amino acid Raw filemixture for oxidized was then and injected reduced onto samples a C18 using column ng MsrAB(Easy--SprayT or nm PepMapMsrAB -C18,T. Figure 3µm, 5A 75µm shows x 150 data cm) and curve of individual b-ions and y-ions. Each experiment was performed in duplicate. reaction ratios less than 1:100 moles of methionine:H2O2. At 1:100 sulfones and other amino acids enzymes and perform proof of principle studies for mass spectrometry applications in intact sequence of ngMsrAB-T andA nmBMsrAB C- T priorA to treatmentB C withA WELQut B CProtease Vector to cleave from theseparated 1:10 moles by a of gradient methionine of water to H 2andO2, acetonitrilewhereas Figure in 0.1% 5B isformic for 1:75 acid moles using ofa Thermomethionine Scientific™ to H2O2. MetSR treated were oxidized which greatly reduced the identification of peptides due to random amino acid oxidation protein, targeted quantitative peptides, and shotgun proteomics. at the WELQ site. The peaksDionex™ assigned Ultimate™ as 0.0000 3000 is RSLCnanothe TurboLuc system. protein The without sample any was methionine infused into oxidation, the Q andExactive shifts HF by mass events (data not shown). In addition, DTT alone did not show any reversal of methionine oxidation. INTRODUCTION A MRP6 (oxidized) 16 Daltonsspectrometer are indicative via electrospray of oxidation ionization (15 Daltons utilizing are sometimes parallel reaction observed monitoring due to data (Figure processing). 4) for collection of Lastly, numerous oxidized peptides were no longer detected after treatment with MetSR. Remaining Methods targeted spectra. The resulting raw data were processed and visualized using Skyline 3.1.0.7382 peptide oxidation may be due to sulfone formation, oxidation of other amino acids, or re-oxidation during MethionineTwo recombinant (Met) is a sulfur MetSR-containing and a methionine essential amino-rich protein acid that were is highlyexpressed, susceptible purified, to and tested oxidation by reactive oxygen species during stress, aging, and disease in cells, and in vitro1. analysis software. electrospray ionization. for gel mobility shifts prior to use in MS applications. For intact and shotgun proteomics Met oxidationexperiments is the ,hydrogen most common peroxide modification was added of purified to induce therapeutic mild methionine antibodies oxidation and other and A biologics, can affect protein function, and is observed by mass spectrometry (MS) in intact MRP6 (reduced) RESULTS Figure 6: Average intensity of 6-protein digested mix: A comparison of the average peptide intensity compared to non-oxidized controls before and after treatment with MetSR. Reduction was Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T of detectable peptides before and after treatment with two forms of MetSR. proteinscarried and peptide out using samples DTT as as a splittingcofactor the and MS samples signal, were which analyzed reduces by sensitivity a Thermo and Scientific™ Intact Protein Analysis CONCLUSIONS increasesOrbitrap complexity XL™ Hybrid of analysis. Ion Trap Methionine sulfoxides can be returned to methionine by - - + + + + + + + + + + DTT -Orbitrap MS . Targeted quantitative analysis of methionine methioninecontaining sulfoxide peptide reductases. and phosphopeptides The product of were Met doneoxidation on a is Thermo Met sulfoxide Scientific™ (MetO), Q Exactive™ which HF - + + + + + + + + + + + H2O2 The ability for both MetSR enzymes to reduce an intact protein was demonstrated using Thermo Methionine oxidation can be reversed in intact proteins and peptides prior to analysis by SDS-PAGE exists MSin two using diastereomers Parallel Reaction that are Monitoring. reduced by two methionine sulfoxide reductases, MetSR- Scientific™ TurboLuc™ . Figure 5 demonstrates how oxidation is reversed by either MetSR. Figure and intact or peptide LC-MS analysis. A and MetSR-B (Figure 1). His WQ MSR 5A shows that 1:10 moles of methionine to moles of H2O2 can decrease the forms of oxidized Disappearance of Appearance of A Disappearance of Appearance of Results: : TurboLuc mildly. When looking at highly oxidized samples shown in Figure 5B (1:75 ratio) significant B Reversal of methionine oxidation by MetSR has benefits to MS experiments involving intact proteins, trx His WQ MSR Oxidized Peptide Reduced Peptide FigureWe 1: Schematicsuccessfully of produced Methionine and purifiedOxidation two and active Reduction: MetSR enzymes, Methionine as observed S-sulfoxide by andgel shift of B oxidation was able to be reversed, but some oxidation events occurred which could either be caused C Oxidized Peptide Reduced Peptide targeted quantitative peptide analysis, and shotgun proteomics. methionine R-sulfoxide formation and reversal. : from other amino acids being oxidized or creation of the sulfone that are not reversible by MetSR. an oxidized methionine rich protein, reduction of oxidized methionines in an intact protein, and MSR His in shotgun and targeted proteomics experiments at the peptide level. Parallel Reaction C: Remaining protein oxidation may be due to sulfone formation, oxidation of other amino acids, or re- A modified LC-MS ionization source design may alleviate the residual modifications observed at the 7 Monitoring was used to demonstrate 90-99% reduction in oxidized methionine in MetSR oxidation during electrospray ionization. protein and peptide level . treated peptide and phosphopeptides. Figure 4: Intact Protein MS Results: Spectra compared after Thermo Scientific™ Xcalibur™ Xtract

Figure 3: Amino acid sequences of two recombinant MetSR enzymes: Amino acid Raw file for oxidized and reduced samples using ngMsrAB-T or nmMsrAB-T. Figure 5A shows data REFERENCES 1. Zhang, X.H. and H. Weissbach, Origin and evolution of the protein-repairing enzymes methionine sequence of ngMsrAB-T and nmMsrAB-T prior to treatment with WELQut Protease to cleave from the 1:10 moles of methionine to H2O2, whereas Figure 5B is for 1:75 moles of methionine to H2O2. MetSRMetSR treated treated INTRODUCTION at the WELQ site. The peaks assigned as 0.0000 is the TurboLuc protein without any methionine oxidation, and shifts by sulphoxide reductases. Biol Rev Camb Philos Soc, 2008. 83(3): p. 249-57. 16 Daltons are indicative of oxidation (15 Daltons are sometimes observed due to data processing). 2. Khor, H.K., et al., Identification of methionine sulfoxide diastereomers in immunoglobulin gamma Oxidation by Hydrogen Peroxide Methionine (Met) is a sulfur-containing essential amino acid that is highly susceptible to antibodies using methionine sulfoxide reductase enzymes. MAbs, 2010. 2(3): p. 299-308. For intact analysis, recombinant turboluciferase (TurboLuc, ~16kDa) was selected to perform an oxidation by reactive oxygen species during stress, aging, and disease in cells, and in vitro1. 3. Morand , K., G. Talbo, and M. Mann, Oxidation of peptides during electrospray ionization. Rapid initial optimization of methionine oxidation and reduction reactions due to observed sensitivity to Met oxidation is the most common modification of purified therapeutic antibodies and other B Commun Mass Spectrom, 1993. 7(8): p. 738-43. oxidation, knowledge of the sequence, and ability to detect with an Orbitrap XL mass spectrometer. A Methioninebiologics, oxidation can affect can also protein occur function, during sampleand is observed preparation, by mass purification spectrometry , storage (MS) , and in LCintact- 4. Lao, Y.W., et al., Chromatographic behavior of peptides containing oxidized methionine residues in 2,3 All experiments for intact analysis used18µM TurboLuc. TurboLuc has three moles of methionine – MS ionizationproteins and. Variable peptide methionine samples as oxidation splitting of the purified MS signal, proteins which leads reduces to lower sensitivity and variable and ControlControl nmMsrAB nmMsrAB-T ngMsrAB-T ngMsrAB-T -T ControlControl nm MsrAB nmMsrAB-T ng-TMsrAB ngMsrAB-T -T proteomic LC MS experiments: Complex tale of a simple modification. Journal of Proteomics, per mole of protein and thus a 1:1 molar ratio of methionine to H O would be 54µM of H O for CONCLUSIONS productincreases quality. It complexity also complicates of analysis. liquid Methionine chromatography sulfoxides-mass can spectrometry be returned (LC to methionine-MS) by 2 2 2 2 2015. 125: p. 131-139. 4 18µM of TurboLuc. A variety of oxidation conditions were assessed at the following ratios of analysismethionine in 4 ways: sulfoxide 1) Shifted reductases. and split peaks The product during liquidof Met chromatography oxidation is Met ,sulfoxide 2) Increased (MetO), which 5. Peterson, A.C., et al., Parallel reaction monitoring for high resolution and high mass accuracy methionine: H O which were 10:1, 1:1, 1:10, 1:25, 1:75, 1:100 and 1:500 (Figure 5 shows 1:10 Methionine oxidation can be reversed in intact proteins and peptides prior to analysis by SDS-PAGE complexityexists and in twopoorer diastereomers depth of analysis that are of reducedintact proteins by two due methionine to multiple sulfoxide oxidized reductases, forms, 3) MetSR- 2 2 quantitative, targeted proteomics. Mol Cell Proteomics, 2012. 11(11): p. 1475-88. and 1:75, the rest of the data are not shown). and intact or peptide LC-MS analysis. – IncreasedA and complexity MetSR-B & (Figure poorer 1). depth of analysis of peptide digests due to multiple oxidized 6. Zeliadt, N. Moving Target: New mass spectrometry based techniques are blurring the lines forms, and; 4) Impaired quantitation of methionine-containing peptides due to the stochastic Disappearance of Appearance of between discovery and targeted proteomics. 2014 [cited 2016 10May]; Available from: Reduction of Intact Proteins by ngMsrAB and nmMsrAB Disappearance of Appearance of Reversal of methionine oxidation by MetSR has benefits to MS experiments involving intact proteins, natureFigure of oxidation 1: Schematic of methionine of Methionine in peptides. Oxidation and Reduction: Methionine S-sulfoxide and http://www.the-scientist.com/?articles.list/categoryNo/2921/category/Lab-Tools/. Methionine sufloxide reduction was performed by adding either ngMsrAB-T or nmMsrAB-T to the C Oxidized Peptide Reduced Peptide targeted quantitative peptide analysis, and shotgun proteomics. methionine R-sulfoxide formation and reversal. Oxidized Peptide Reduced Peptide 7. Kim, K., et al., Reducing protein oxidation in low-flow electrospray enables deeper investigation of TurboLuc control or oxidized sample at an enzyme:sample ratio of 1:4 and DTT to a final D proteoforms by top down proteomics. EuPA Open Proteomics, 2015. 8: p. 40-47. concentration of 5mM. Both enzymes were used in separate experiments. Samples were incubated A modified LC-MS ionization source design may alleviate the residual modifications observed at the 7 METHODS at 37 C for 2 hours & then desalted using 3K MWCO concentrators. protein and peptide level .

TRADEMARKS/LICENSING ⁰ Recombinant Methionine Sulfoxide Reductases Shotgun Proteomics Coupled with Methionine Oxidation Reduction Expression of REFERENCES Thermo Scientific™ Pierce™ 6 Protein Digest, equimolar, LC-MS grade which was stored in liquid For Research Use Only. Not for use in diagnostic procedures. 1. Zhang, X.H. and H. Weissbach, Origin and evolution of the protein-repairing enzymes methionine for over a year was used for the oxidation experiments. Control and oxidized samples were MetSR treated © 2016 Thermo Fisher Scientific Inc. All rights reserved. Eksigent is a trademark of ABSciex. All other Over a dozen different recombinant methionine reductase vectors were constructed for target sulphoxide reductases. Biol Rev Camb Philos Soc, 2008. 83(3): p. 249-57. reduced using the same procedure as intact protein analysis described above, except for C18 trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not protein expression and optimized for E.coli ER2566. 0.1 g of biomass was lysed with cell lysis MetSR treated 2. Khor, H.K., et al., Identification of methionine sulfoxide diastereomers in immunoglobulin gamma – peptide clean up. intended to encourage use of these products in any manner that might infringe the intellectual property buffer consisting of 50mM Tris HCl (pH 8); 100mM NaCl; 0.1% Triton X-100; 1mM EDTA Oxidation by Hydrogen Peroxide antibodies using methionine sulfoxide reductase enzymes. MAbs, 2010. 2(3): p. 299-308. rights of others. (pH8) and disrupted by sonication. The soluble fraction was diluted with wash buffer For intact analysis, recombinant turboluciferase (TurboLuc, ~16kDa) was selected to perform an Targeted Protein Analysis 3. Morand, K., G. Talbo, and M. Mann, Oxidation of peptides during electrospray ionization. Rapid LC-MS analysis consisting of 250mM NaCl, 20mM imidazole, 50mM Tris-HCl, pH=8 and applied to 0.2 ml initial optimization of methionine oxidation and reduction reactions due to observed sensitivity to Commun Mass Spectrom, 1993. 7(8): p. 738-43. Thermo Scientific™ The protein sample was injected onto a Thermo Scientific™ ProSwift™ RP-4H Analytical 100 μm x B Methionine oxidationHisPur canNi-NTA also and occur washed during extensively. sample preparation, Bound proteins purification were , elutedstorage with , and LC- oxidation, knowledge of the sequence, and ability to detect with an Orbitrap XL mass spectrometer. Figure 6 shows the disappearance of the oxidized peptide and appearance of the corresponding 4. Lao, Y.W., et al., Chromatographic behavior of peptides containing oxidized methionine residues in 25 cm monolithic column at a flow rate of 800 nL/min over a gradient of water and acetonitrile in 50mMMS Tris, ionization pH8, 250mM2,3. Variable NaCl, 500mMmethionine imidazole oxidation and of dialyzed purified against proteins 50mM leads Tristo lower-HCl pHand 7.6, variable All experiments for intact analysis used18µM TurboLuc. TurboLuc has three moles of methionine reduced peptide after treatment with ngMsrAB or nmMsrAB. These data indicate that treatment Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T proteomic LC–MS experiments: Complex tale of a simple modification. Journal of Proteomics, o 0.1% formic acid. The shotgun proteomics sample was injected onto a reversed-phase column 4 C, overnight.product quality. Three It MetSR also complicates genes expressed liquid chromatography from three different-mass clones spectrometry each were (LC tested-MS) per mole of protein and thus a 1:1 molar ratio of methionine to H2O2 would be 54µM of H2O2 for with ngMsrAB or nmMsrAB results in a >90% reduction of an oxidized methionine and resulted in 2015. 125: p. 131-139. (15cm x 75 μm I.D., Prepmap C18) and separated using a gradient of water and acetonitrile in by gel analysisshift of an in oxidized4 ways: 1)methionine Shifted and rich split protein peaks 6 (Figureduring liquid2) and chromatography two MetSR/Trx4 trifunctional, 2) Increased 18µM of TurboLuc. A variety of oxidation conditions were assessed at the following ratios of a corresponding increase in the peptide with a reduced methionine. In addition, after treatment Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T 5. Peterson, A.C., et al., Parallel reaction monitoring for high resolution and high mass accuracy 0.1% formic acid at a flow rate of 300 nL/min. All samples used an Eksigent™ nanoLC Ultra 2D enzymescomplexity from Neisseria and poorer gonorrhoeae depth of analysisor Neisseria of intact meningitidis proteins weredue to selected multiple for oxidized MS forms, 3) methionine: H2O2 which were 10:1, 1:1, 1:10, 1:25, 1:75, 1:100 and 1:500 (Figure 5 shows 1:10 some of the oxidized peptides were not even detectable (Figure 6 A and C for ngMsrAB-T). quantitative, targeted proteomics. Mol Cell Proteomics, 2012. 11(11): p. 1475-88. plus HPLC coupled to a Thermo Scientific™ LTQ Orbitrap™ XL ETD mass spectrometer. applicationsIncreased (Figure complexity 2). & poorer depth of analysis of peptide digests due to multiple oxidized and 1:75, the rest of the data are not shown). 6. Zeliadt, N. Moving Target: New mass spectrometry–based techniques are blurring the lines forms, and; 4) Impaired quantitation of methionine-containing peptides due to the stochastic between discovery and targeted proteomics. 2014 [cited 2016 10May]; Available from: nature of oxidation of methionine in peptides. Reduction of Intact Proteins by ngMsrAB and nmMsrAB Disappearance of Appearance of http://www.the-scientist.com/?articles.list/categoryNo/2921/category/Lab-Tools/. Methionine sufloxide reduction was performed by adding either ngMsrAB-T or nmMsrAB-T to the Oxidized Peptide Reduced Peptide 7. Kim, K., et al., Reducing protein oxidation in low-flow electrospray enables deeper investigation of TurboLuc control or oxidized sample at an enzyme:sample ratio of 1:4 and DTT to a final D proteoforms by top down proteomics. EuPA Open Proteomics, 2015. 8: p. 40-47. concentration of 5mM. Both enzymes were used in separate experiments. Samples were incubated METHODS at 37 C for 2 hours & then desalted using 3K MWCO concentrators. TRADEMARKS/LICENSING ⁰ Expression of Recombinant Methionine Sulfoxide Reductases Shotgun Proteomics Coupled with Methionine Oxidation Reduction Thermo Scientific™ Pierce™ 6 Protein Digest, equimolar, LC-MS grade which was stored in liquid For Research Use Only. Not for use in diagnostic procedures. Over a dozen different recombinant methionine reductase vectors were constructed for target for over a year was used for the oxidation experiments. Control and oxidized samples were © 2016 Thermo Fisher Scientific Inc. All rights reserved. Eksigent is a trademark of ABSciex. All other reduced using the same procedure as intact protein analysis described above, except for C18 protein expression and optimized for E.coli ER2566. 0.1 g of biomass was lysed with cell lysis MetSR treated trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not buffer consisting of 50mM Tris – HCl (pH 8); 100mM NaCl; 0.1% Triton X-100; 1mM EDTA peptide clean up. intended to encourage use of these products in any manner that might infringe the intellectual property (pH8) and disrupted by sonication. The soluble fraction was diluted with wash buffer Targeted Protein Analysis rights of others. consisting of 250mM NaCl, 20mM imidazole, 50mM Tris-HCl, pH=8 and applied to 0.2 ml LC-MS analysis The protein sample was injected onto a Thermo Scientific™ ProSwift™ RP μ Thermo Scientific™ HisPur Ni-NTA and washed extensively. Bound proteins were eluted with -4H Analytical 100 m x Figure 6 shows the disappearance of the oxidized peptide and appearance of the corresponding 50mM Tris, pH8, 250mM NaCl, 500mM imidazole and dialyzed against 50mM Tris-HCl pH 7.6, 25 cm monolithic column at a flow rate of 800 nL/min over a gradient of water and acetonitrile in 2 reducedThe Use peptide of Methionine after treatment Sulfoxide with ngMsrAB Reductases or nmMsrAB. to ReverseThese data Oxidized indicate that Methionine treatment for Mass Spectrometry Applications 4oC, overnight. Three MetSR genes expressed from three different clones each were tested 0.1% formic acid. The shotgun proteomics sample was injected onto a reversed-phase column with ngMsrAB or nmMsrAB results in a >90% reduction of an oxidized methionine and resulted in μ by gel shift of an oxidized methionine rich protein 6 (Figure 2) and two MetSR/Trx trifunctional (15cm x 75 m I.D., Prepmap C18) and separated using a gradient of water and acetonitrile in a corresponding increase in the peptide with a reduced methionine. In addition, after treatment Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T ™ enzymes from Neisseria gonorrhoeae or Neisseria meningitidis were selected for MS 0.1% formic acid at a flow rate of 300 nL/min. All samples used an Eksigent nanoLC Ultra 2D some of the oxidized peptides were not even detectable (Figure 6 A and C for ngMsrAB-T). applications (Figure 2). plus HPLC coupled to a Thermo Scientific™ LTQ Orbitrap™ XL ETD mass spectrometer.

Raw file for oxidized and reduced samples using ngMsrAB-T or nmMsrAB-T. Figure 5A shows data The Use of Methionine Sulfoxide Reductases to ReverseFigure 3: Amino acid sequences Oxidized of two recombinant MetSR enzymes: Methionine Amino acid for Mass Spectrometry Applications from the 1:10 moles of methionine to H O , whereas Figure 5B is for 1:75 moles of methionine to H O . MetSR treated 1 1 2 2 2 1 sequence of ngMsrAB-T and nm3MsrAB-T prior to treatment with 3WELQut Protease to cleave 2 2 2 2 1 2 2 Robert Cunningham ; Kratika Singhal ; Ryan T Fellers ; LucaINTRODUCTION Fornelli ; Henrique Dos Santos Seckler ; Bhavin Patel ;at Eglethe WELQ Capkauske site. ; Juozas Siurkus ; Philip Compton ; Neil TheL Kelleherpeaks assigned as; John0.0000 is theC TurboLuc Rogers protein without any methionine oxidation, and shifts by 16 Daltons are indicative of oxidation (15 Daltons are sometimes observed due to data processing). 1 2 3 Thermo Fisher Scientific, Rockford, IL; NorthwesternMethionine University, (Met) is a sulfur Evanston,-containing essential IL;amino acidThermo that is highly Fishersusceptible to Scientific, Vilnius, Lithuania oxidation by reactive oxygen species during stress, aging, and disease in cells, and in vitro1. Met oxidation is the most common modification of purified therapeutic antibodies and other A biologics, can affect protein function, and is observed by mass spectrometry (MS) in intact Figure 2: Gel Shift Assay to Reduce MRP6: Three different MetSR genes expressed in Targeted Quantitative Peptide Analysis Figure 5: Parallel Reaction Monitoring of three methionine containing peptides: Schematic Shotgun proteomics proteins and peptide samples as splitting the MS signal, which reduces sensitivity and Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T OVERVIEW three different vectors were tested for their ability to reduce MRP6 after being oxidized by H O . Three heavy peptide standards (two phosphopeptides) containing an oxidized methionine (100 fmol/μL ) of parallel reaction monitoring for targeted peptide quantitation 5,6(A). Quantitative reversal of CONCLUSIONS increases complexity of analysis. Methionine sulfoxides can be returned to methionine by 2 2 were mixed with the Pierce 6 protein digest, equimolar, LC-MS grade (40 ng/μL). The mixed sample methionine oxidation in a peptide standard (B), a phosphopeptide of the same peptide standard The Pierce 6 protein LC-MS grade digest was used to assess the two MetSR enzymes using both non- methionine sulfoxide reductases. The product of Met oxidation is Met sulfoxide (MetO), which Purpose No was then reduced by either ngMsrAB-T or nmMsrAB-T as described earlier. The resulting peptide (C), and a second phosphopeptide (D). The bar graph area shows the cumulative area under the oxidized and mildly oxidized samples (Figure 7). Substantial methionine reversal was seen with all Methionine oxidation can be reversed in intact proteins and peptides prior to analysis by SDS-PAGE To synthesize two active forms of recombinant methionine sulfoxide reductase (MetSR) exists in two diastereomersMsrA1 (Human)that are reduced ngMsrAB by two-T methionine nmMsrAB sulfoxide-T reductases, MetSR- and intact or peptide LC-MS analysis. Reduction mixture was then injected onto a C18 column (Easy-Spray PepMap C18, 3µm, 75µm x 150 cm) and curve of individual b-ions and y-ions. Each experiment was performed in duplicate. reaction ratios less than 1:100 moles of methionine:H2O2. At 1:100 sulfones and other amino acids enzymes and perform proof of principle studies for mass spectrometry applications in intact A and MetSR-B (FigureA 1). B C A B C A B C Vector separated by a gradient of water and acetonitrile in 0.1% formic acid using a Thermo Scientific™ were oxidized which greatly reduced the identification of peptides due to random amino acid oxidation Disappearance of Appearance of protein, targeted quantitative peptides, and shotgun proteomics. Dionex™ Ultimate™ 3000 RSLCnano system. The sample was infused into the Q Exactive HF mass events (data not shown). In addition, DTT alone did not show any reversal of methionine oxidation. Reversal of methionine oxidation by MetSR has benefits to MS experiments involving intact proteins, Figure 1: Schematic of Methionine Oxidation and Reduction: Methionine S-sulfoxide and A spectrometer via electrospray ionization utilizing parallel reaction monitoring (Figure 4) for collection of C Lastly, numerousOxidized oxidized Peptide peptides were no longer detected Reducedafter treatment Peptide with MetSR. Remaining targeted quantitative peptide analysis, and shotgun proteomics. methionine R-sulfoxide formation and reversal. MRP6 (oxidized) Methods targeted spectra. The resulting raw data were processed and visualized using Skyline 3.1.0.7382 peptide oxidation may be due to sulfone formation, oxidation of other amino acids, or re-oxidation during

Two recombinant MetSR and a methionine-rich protein were expressed, purified, and tested analysis software. electrospray ionization. A modified LC-MS ionization source design may alleviate the residual modifications observed at the 7 for gel mobility shifts prior to use in MS applications. For intact and shotgun proteomics protein and peptide level . experiments ,hydrogen peroxide was added to induce mild methionine oxidation and MRP6 (reduced) Figure 6: Average intensity of 6-protein digested mix: A comparison of the average peptide intensity RESULTS compared to non-oxidized controls before and after treatment with MetSR. Reduction was of detectable peptides before and after treatment with two forms of MetSR. carried out using DTT as a cofactor and samples were analyzed by a Thermo Scientific™ Intact Protein Analysis REFERENCES

Orbitrap XL™ Hybrid Ion Trap - - + + + + + + + + + + DTT 1. Zhang, X.H. and H. Weissbach, Origin and evolution of the protein-repairing enzymes methionine -Orbitrap MS . Targeted quantitative analysis of methionine MetSR treated containing peptide and phosphopeptides were done on a Thermo Scientific™ Q Exactive™ HF - + + + + + + + + + + + H2O2 The ability for both MetSR enzymes to reduce an intact protein was demonstrated using Thermo sulphoxide reductases. Biol Rev Camb Philos Soc, 2008. 83(3): p. 249-57.

MS using Parallel Reaction Monitoring. Scientific™ TurboLuc™ . Figure 5 demonstrates how oxidation is reversed by either MetSR. Figure 2. Khor, H.K., et al., Identification of methionine sulfoxide diastereomers in immunoglobulin gamma Oxidation by Hydrogen Peroxide His WQ MSR 5A shows that 1:10 moles of methionine to moles of H2O2 can decrease the forms of oxidized antibodies using methionine sulfoxide reductase enzymes. MAbs, 2010. 2(3): p. 299-308. Disappearance of Appearance of A: ForTurboLuc intact analysis,mildly. When recombinant looking atturboluciferase highly oxidized (TurboLuc, samples shown~16kDa) in wasFigure selected 5B (1:75 to performratio) significant an 3. Morand, K., G. Talbo, and M. Mann, Oxidation of peptides during electrospray ionization. Rapid Results: B Oxidized Peptide Reduced Peptide trx His WQ MSR initialoxidation optimization was able of to methionine be reversed, oxidation but some and oxidation reduction events reactions occurred due to which observed could sensitivity either be causedto Commun Mass Spectrom, 1993. 7(8): p. 738-43. We successfully produced and purified two active MetSR enzymes, as observed by gel shift of B: B Methionine oxidation can also occur during sample preparation, purification , storage , and LC- oxidation,from other knowledge amino acids of beingthe sequence, oxidized andor creation ability toof detectthe sulfone with an that Orbitrap are not XL reversible mass spectrometer. by MetSR. 4. Lao, Y.W., et al., Chromatographic behavior of peptides containing oxidized methionine residues in an oxidized methionine rich protein, reduction of oxidized methionines in an intact protein, and MSR 2,3 His All experiments for intact analysis used18µM TurboLuc. TurboLuc has three moles of methionine – in shotgun and targeted proteomics experiments at the peptide level. Parallel Reaction MS ionization C. Variable: methionine oxidation of purified proteins leads to lower and variable Remaining protein oxidation may be due to sulfone formation, oxidation of other amino acids, or re- Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T proteomic LC MS experiments: Complex tale of a simple modification. Journal of Proteomics, per mole of protein and thus a 1:1 molar ratio of methionine to H O would be 54µM of H O for Monitoring was used to demonstrate 90-99% reduction in oxidized methionine in MetSR product quality. It also complicates liquid chromatography-mass spectrometry (LC-MS) oxidation during electrospray ionization. 2 2 2 2 2015. 125: p. 131-139. 4 18µM of TurboLuc. A variety of oxidation conditions were assessed at the following ratios of treated peptide and phosphopeptides. analysis in 4 ways: 1) Shifted and split peaks during liquid chromatography , 2) Increased 5. Peterson, A.C., et al., Parallel reaction monitoring for high resolution and high mass accuracy methionine:Figure 4: Intact H2O2 Proteinwhich were MS 10:1,Results: 1:1, Spectra1:10, 1:25, compared 1:75, 1:100 after and Thermo 1:500 Scientific™ (Figure 5 shows Xcalibur™ 1:10 Xtract complexity and poorer depth of analysis of intact proteins due to multiple oxidized forms, 3) quantitative, targeted proteomics. Mol Cell Proteomics, 2012. 11(11): p. 1475-88. andRaw 1:75, file for the oxidized rest of theand data reduced are not samples shown). using ngMsrAB-T or nmMsrAB-T. Figure 5A shows data – IncreasedFigure 3: complexity Amino acid & poorer sequences depth of analysistwo recombinant of peptide MetSRdigests enzymes:due to multiple Amino oxidized acid 6. Zeliadt, N. Moving Target: New mass spectrometry based techniques are blurring the lines forms,sequence and; of4) ngImpairedMsrAB -quantitationT and nmMsrAB of methionine-T prior to- containingtreatment withpeptides WELQut due toProtease the stochastic to cleave from the 1:10 moles of methionine to H2O2, whereas Figure 5B is for 1:75 moles of methionine to H2O2. MetSR treated between discovery and targeted proteomics. 2014 [cited 2016 10May]; Available from: INTRODUCTION natureat the of WELQ oxidation site. of methionine in peptides. ReductionThe peaks assignedof Intact Proteinsas 0.0000 by is thengMsrAB TurboLuc and protein nmMsrAB without any methionine oxidation, and shifts by Disappearance of Appearance of http://www.the-scientist.com/?articles.list/categoryNo/2921/category/Lab-Tools/. Methionine16 Daltons sufloxideare indicative reduction of oxidation was performed (15 Daltons by addingare sometimes either ng observedMsrAB-T ordue nm toMsrAB data processing).-T to the Oxidized Peptide Reduced Peptide 7. Kim, K., et al., Reducing protein oxidation in low-flow electrospray enables deeper investigation of Methionine (Met) is a sulfur-containing essential amino acid that is highly susceptible to TurboLuc control or oxidized sample at an enzyme:sample ratio of 1:4 and DTT to a final D proteoforms by top down proteomics. EuPA Open Proteomics, 2015. 8: p. 40-47. oxidation by reactive oxygen species during stress, aging, and disease in cells, and in vitro1. concentration of 5mM. Both enzymes were used in separate experiments. Samples were incubated Met oxidation is the most common modification of purified therapeutic antibodies and other METHODS at 37 CA for 2 hours & then desalted using 3K MWCO concentrators. TRADEMARKS/LICENSING biologics, can affect protein function, and is observed by mass spectrometry (MS) in intact ⁰ proteins and peptide samples as splitting the MS signal, which reduces sensitivity and Expression of Recombinant Methionine Sulfoxide Reductases Shotgun Proteomics Coupled with Methionine Oxidation Reduction Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T Thermo Scientific™ Pierce™ 6 Protein Digest, equimolar, LC CONCLUSIONS increases complexity of analysis. Methionine sulfoxides can be returned to methionine by -MS grade which was stored in liquid For Research Use Only. Not for use in diagnostic procedures. for over a year was used for the oxidation experiments. Control and oxidized samples were © 2016 Thermo Fisher Scientific Inc. All rights reserved. Eksigent is a trademark of ABSciex. All other methionine sulfoxide reductases. The product of Met oxidation is Met sulfoxide (MetO), which Over a dozen different recombinant methionine reductase vectors were constructed for target Methionine oxidation can be reversed in intact proteins and peptides prior to analysis by SDS-PAGE reduced using the same procedure as intact protein analysis described above, except for C18 trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not exists in two diastereomers that are reduced by two methionine sulfoxide reductases, MetSR- protein expression and optimized for E.coli ER2566. 0.1 g of biomass was lysed with cell lysis and intact or peptideMetSR LC-MS treated analysis. peptide clean up. intended to encourage use of these products in any manner that might infringe the intellectual property A and MetSR-B (Figure 1). buffer consisting of 50mM Tris – HCl (pH 8); 100mM NaCl; 0.1% Triton X-100; 1mM EDTA rights of others. (pH8) and disrupted by sonication. The soluble fraction was diluted with wash buffer Targeted ProteinDisappearance Analysis of Appearance of Reversal of methionine oxidation by MetSR has benefits to MS experiments involving intact proteins, LC-MS analysis Figure 1: Schematic of Methionine Oxidation and Reduction: Methionine S-sulfoxide and consisting of 250mM NaCl, 20mM imidazole, 50mM Tris-HCl, pH=8 and applied to 0.2 ml Oxidized Peptide Reduced Peptide targeted quantitative peptide analysis, and shotgun proteomics. The protein sample was injected onto a Thermo Scientific™ ProSwift™ RP-4H Analytical 100 μm x C methionine R-sulfoxide formation and reversal. Thermo Scientific™ HisPur Ni-NTA and washed extensively. Bound proteins were eluted with Figure 6 shows the disappearance of the oxidized peptide and appearance of the corresponding 25 cm monolithic column at a flow rate of 800 nL/min over a gradient of water and acetonitrile in 50mM Tris, pH8, 250mM NaCl, 500mM imidazole and dialyzed against 50mM Tris-HCl pH 7.6, reduced peptide after treatment with ngMsrAB or nmMsrAB. These data indicate that treatment A modified LC-MS ionization source design may alleviate the residual modifications observed at the 4oC, overnight. Three MetSR genes expressed from three different clones each were tested 0.1% formic acid. The shotgun proteomics sample was injected onto a reversed-phase column with ngMsrAB or nmMsrAB results in a >90% reduction of an oxidized methionine and resulted in 7 μ protein and peptide level . by gel shift of an oxidized methionine rich protein 6 (Figure 2) and two MetSR/Trx trifunctional (15cm x 75 m I.D., Prepmap C18) and separated using a gradient of water and acetonitrile in a corresponding increase in the peptide with a reduced methionine. In addition, after treatment Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T ™ enzymes from Neisseria gonorrhoeae or Neisseria meningitidis were selected for MS 0.1% formic acid at a flow rate of 300 nL/min. All samples used an Eksigent nanoLC Ultra 2D some of the oxidized peptides were not even detectable (Figure 6 A and C for ngMsrAB-T). ™ ™ applications (Figure 2). plus HPLC coupled to a Thermo Scientific LTQ Orbitrap XL ETD mass spectrometer. REFERENCES MetSR treated 1. Zhang, X.H. and H. Weissbach, Origin and evolution of the protein-repairing enzymes methionine sulphoxide reductases. Biol Rev Camb Philos Soc, 2008. 83(3): p. 249-57. 2. Khor, H.K., et al., Identification of methionine sulfoxide diastereomers in immunoglobulin gamma Oxidation by Hydrogen Peroxide antibodies using methionine sulfoxide reductase enzymes. MAbs, 2010. 2(3): p. 299-308. For intact analysis, recombinant turboluciferase (TurboLuc, ~16kDa) was selected to perform an 3. Morand, K., G. Talbo, and M. Mann, Oxidation of peptides during electrospray ionization. Rapid initial optimization of methionine oxidation and reduction reactions due to observed sensitivity to B Commun Mass Spectrom, 1993. 7(8): p. 738-43. Methionine oxidation can also occur during sample preparation, purification , storage , and LC- oxidation, knowledge of the sequence, and ability to detect with an Orbitrap XL mass spectrometer. 4. Lao, Y.W., et al., Chromatographic behavior of peptides containing oxidized methionine residues in MS ionization2,3. Variable methionine oxidation of purified proteins leads to lower and variable All experiments for intact analysis used18µM TurboLuc. TurboLuc has three moles of methionine Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T proteomic LC–MS experiments: Complex tale of a simple modification. Journal of Proteomics, product quality. It also complicates liquid chromatography-mass spectrometry (LC-MS) per mole of protein and thus a 1:1 molar ratio of methionine to H2O2 would be 54µM of H2O2 for 2015. 125: p. 131-139. analysis in 4 ways: 1) Shifted and split peaks during liquid chromatography4, 2) Increased 18µM of TurboLuc. A variety of oxidation conditions were assessed at the following ratios of 5. Peterson, A.C., et al., Parallel reaction monitoring for high resolution and high mass accuracy complexity and poorer depth of analysis of intact proteins due to multiple oxidized forms, 3) methionine: H2O2 which were 10:1, 1:1, 1:10, 1:25, 1:75, 1:100 and 1:500 (Figure 5 shows 1:10 quantitative, targeted proteomics. Mol Cell Proteomics, 2012. 11(11): p. 1475-88. Increased complexity & poorer depth of analysis of peptide digests due to multiple oxidized and 1:75, the rest of the data are not shown). 6. Zeliadt, N. Moving Target: New mass spectrometry–based techniques are blurring the lines forms, and; 4) Impaired quantitation of methionine-containing peptides due to the stochastic between discovery and targeted proteomics. 2014 [cited 2016 10May]; Available from: nature of oxidation of methionine in peptides. Reduction of Intact Proteins by ngMsrAB and nmMsrAB Disappearance of Appearance of http://www.the-scientist.com/?articles.list/categoryNo/2921/category/Lab-Tools/. Methionine sufloxide reduction was performed by adding either ngMsrAB-T or nmMsrAB-T to the Oxidized Peptide Reduced Peptide 7. Kim, K., et al., Reducing protein oxidation in low-flow electrospray enables deeper investigation of TurboLuc control or oxidized sample at an enzyme:sample ratio of 1:4 and DTT to a final D proteoforms by top down proteomics. EuPA Open Proteomics, 2015. 8: p. 40-47. concentration of 5mM. Both enzymes were used in separate experiments. Samples were incubated METHODS at 37 C for 2 hours & then desalted using 3K MWCO concentrators. TRADEMARKS/LICENSING ⁰ Expression of Recombinant Methionine Sulfoxide Reductases Shotgun Proteomics Coupled with Methionine Oxidation Reduction Thermo Scientific™ Pierce™ 6 Protein Digest, equimolar, LC-MS grade which was stored in liquid For Research Use Only. Not for use in diagnostic procedures. Over a dozen different recombinant methionine reductase vectors were constructed for target for over a year was used for the oxidation experiments. Control and oxidized samples were © 2016 Thermo Fisher Scientific Inc. All rights reserved. Eksigent is a trademark of ABSciex. All other reduced using the same procedure as intact protein analysis described above, except for C18 protein expression and optimized for E.coli ER2566. 0.1 g of biomass was lysed with cell lysis MetSR treated trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not buffer consisting of 50mM Tris – HCl (pH 8); 100mM NaCl; 0.1% Triton X-100; 1mM EDTA peptide clean up. intended to encourage use of these products in any manner that might infringe the intellectual property (pH8) and disrupted by sonication. The soluble fraction was diluted with wash buffer Targeted Protein Analysis rights of others. consisting of 250mM NaCl, 20mM imidazole, 50mM Tris-HCl, pH=8 and applied to 0.2 ml LC-MS analysis The protein sample was injected onto a Thermo Scientific™ ProSwift™ RP μ Thermo Scientific™ HisPur Ni-NTA and washed extensively. Bound proteins were eluted with -4H Analytical 100 m x Figure 6 shows the disappearance of the oxidized peptide and appearance of the corresponding 50mM Tris, pH8, 250mM NaCl, 500mM imidazole and dialyzed against 50mM Tris-HCl pH 7.6, 25 cm monolithic column at a flow rate of 800 nL/min over a gradient of water and acetonitrile in reduced peptide after treatment with ngMsrAB or nmMsrAB. These data indicate that treatment 4oC, overnight. Three MetSR genes expressed from three different clones each were tested 0.1% formic acid. The shotgun proteomics sample was injected onto a reversed-phase column with ngMsrAB or nmMsrAB results in a >90% reduction of an oxidized methionine and resulted in μ by gel shift of an oxidized methionine rich protein 6 (Figure 2) and two MetSR/Trx trifunctional (15cm x 75 m I.D., Prepmap C18) and separated using a gradient of water and acetonitrile in a corresponding increase in the peptide with a reduced methionine. In addition, after treatment Control nmMsrAB-T ngMsrAB-T Control nmMsrAB-T ngMsrAB-T ™ enzymes from Neisseria gonorrhoeae or Neisseria meningitidis were selected for MS 0.1% formic acid at a flow rate of 300 nL/min. All samples used an Eksigent nanoLC Ultra 2D some of the oxidized peptides were not even detectable (Figure 6 A and C for ngMsrAB-T). applications (Figure 2). plus HPLC coupled to a Thermo Scientific™ LTQ Orbitrap™ XL ETD mass spectrometer.

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