HDX-MS Overview. Pat Boyce Pharmaceutical and Life Sciences Marketing Team Manager Waters Europe Waters Corporation 1

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1 HDX-MS Overview Pat Boyce Pharmaceutical and Life Sciences Marketing Team Manager Waters Europe 211 Waters Corporation 1

2 Acknowledgements Scott Berger Asish Chakraborty Joomi Ahn Jing Qing Yu Rose Lawler Pat Young Michael Eggertson Jing (Susan) Fang 211 Waters Corporation 2

3 Overview HDX-MS basics Basic theory Types of experiment Example applications Epitope mapping Structural biology Protein characterization Protein-ligand binding HDX-MS at Waters 211 Waters Corporation 3

4 Deuterium Exchange at Amide Hydrogen for LC/MS studies R 1 Peptide bond R 2 Peptide bond R 3 Back Exchange during LC/MS Amide HDX rate affected by local protein structure NH 3 + C CO H NH C CO Amide H D H NH C CO 2 - H No exchange unfolded: 1 s or faster rigid, inaccessible structures: months D 2 O (solution) H s at backbone amide positions H s & D s Diagram Credit: Engen, J. R. (29) Anal. Chem. 81(19), 787 at backbone amide positions - Measure mass difference to determine uptake 211 Waters Corporation 4

5 Workflow for HDX-MS Analysis Sample at defined ph and temperature (e.g. 25 C, ph 7) Add D 2 O, ph 7 D 2 O D 2 O D 2 O D 2 O Labeling at defined ph and temperature (e.g. 25 C, ph 7) At various times, move an aliquot to quench buffer D 2 O D 2 O D 2 O D 2 O Quenched C, ph 2.5 Online Pepsin Digest Isotope pattern Deuterium content ESI Q-TOF HDMS E UPLC, C, ph 2.5 Peptides Relative Deuterium Level (Da) APO HOLO Determine H/D exchange rate in uptake curve Time (min) 211 Waters Corporation 5

6 Typical HDX experiments Mass Spectrometry Reviews 26, 25, (Engen & Wales) 211 Waters Corporation 6

7 Example of Global (Intact Protein) Level HDX-MS Experiment Anal. Biochem. 42 (212) Continous labeling protein oligomer dissociation study Global picture of overall flexibility & rigidity This publication is usng HDX-MS to study insulin oligomer dissociation 211 Waters Corporation 7

8 Example of Global (Intact Protein) Level HDX-MS Experiment Anal. Biochem. 42 (212) Continous labeling protein oligomer dissociation study Global picture of overall flexibility & rigidity This publication is usng HDX-MS to study insulin oligomer dissociation 211 Waters Corporation 8

9 Example of Global (Intact Protein) Level HDX-MS Experiment Cell 157, , May 8, 214 Pulse-labeling protein re-folding experiment Global picture of overall flexibility & rigidity Protein re-folding and analysis of folded/unfolded populations 211 Waters Corporation 9

10 APO APO APO APO APO HOLO HOLO HO LO HO LO HO LO APO APO APO APO APO HOLO HOLO HO LO HO LO HOLO APO APO APO APO APO HOLO HOLO HOLO HOLO HOLO APO APO APO APO APO HO LO HO LO HO LO HO LO HO LO HDX: Local (Peptide) Level HDX-MS Relative Deuterium Level (Da) Time (mi n) Relative Deuterium Level (Da) Time (mi n) Relative Deuterium Level (Da) Time (min ) Relative Deuterium Level (Da) Time (min) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Time (min ) Time (min ) Time (min ) Time (min) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Time (min) Time (min) Time (min ) Time (min) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Time (min) Time (min) Time (min ) Time (min) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Relative Deuterium Level (Da) Time (min) Time (min ) Time (min ) Time (min) No change Uptake changed Protein HDX Measures the relative rates of deuterium uptake of a protein Determines global differences in protein structure Peptide-Digest HDX Measures the relative rates of deuterium uptake at peptide level Determines LOCAL differences in protein structure. 211 Waters Corporation 1

11 Structural Biology Examples 211 Waters Corporation 11

12 Recent HDX-MS Paper, March 214 Protein Science 214 vol 23: Patterns of structural dynamics in RACK1 protein retained throughout evolution: A hydrogen-deuterium exchange study of three orthologs Krzysztof Tarnowski, Kinga Fituch, Roman H. Szczepanowski, Michal Dadlez, and Magdalena Kaus-Drobek 211 Waters Corporation 12

13 Recent HDX-MS Paper, March 214 Protein Science 214 vol 23: HDX-MS studies of 3 RACK1 orthologs Seven-bladed β-propeller structure D exchange after 1 seconds overlaid on crystal structures Which parts of the structure are flexible and which are rigid Peptide level HDXMS 97% sequence coverage. Incubation times in D2O: 1s, 1 min, 2 min, 1 hour Displayed 1s incubation time point HDX-MS also used to study RACK1 dimer 211 Waters Corporation 13

14 Recent HDX-MS Paper, March 214 Protein Science vol 23: In conclusion, we mapped the stiff and plastic regions of RACK1 in solution and compared patterns of hydrogen deuterium exchange among three RACK1 orthologs and between monomeric and dimeric yrack1. Our results reveal an evolutionarily retained building plan for RACK1, in which large regions of plasticity are precisely positioned by the intertwining small stable core. This supports a more fluidic structure of external regions of the polypeptide chain than previously implicated by the crystal structures, in which A and D strands are structurally well defined. By contrast, we found that in solution, their stabilizing hydrogen bonds are broken much more frequently than in the neighboring B and C strands. This could be an important determinant that enables this relatively small molecule to accommodate numerous diverse molecular partners 211 Waters Corporation 14

15 Recent HDX-MS Paper, May 214 Cell 157, , May 8, 214 GroEL/ES Chaperonin Modulates the Mechanism and Accelerates the Rate of TIM-Barrel Domain Folding Florian Georgescauld, Kristina Popova, Amit J. Gupta, Andreas Bracher, John R. Engen, Manajit Hayer-Hartl, and F. Ulrich Hartl 211 Waters Corporation 15

16 Recent HDX-MS Paper, May 214 Cell 157, , May 8, 214 Use of HDX-MS to study how GroEL/ES promotes proteinfolding Purified GroEL-DapA complex Folding initiated by adding GroES/ATP Comparisons with spontaneous folding and SREL/ES assisted folding Folding time periods from 3 s to 6 mins Pulse-labeling with D 2 O for 12 s Peptide-level HDX- MS 211 Waters Corporation 16

17 Recent HDX-MS Paper, May 214 Cell 157, , May 8, 214 Biomodal isotope distributions Either folded or unfolded Chaperonin accelerates DapA folding 3-fold over spontaneous rate 211 Waters Corporation 17

18 Recent HDX-MS Paper, May 214 Cell 157, , May 8, 214 Peptide level pulselabeling HDX-MS used to compare DapA refolding Different peptides have different half-times 211 Waters Corporation 18

19 Epitope Mapping Example 211 Waters Corporation 19

20 Typical Epitope Mapping HDX-MS Experiment Drug Discovery Today Vol 19, 1, January Waters Corporation 2

21 Typical Epitope Mapping HDX-MS Experiment Proc. Natl. Acad. Sci. USA 11, , January 213 Two samples, continuous labeling experiment fhbp (factor H-binding protein) o 97% sequence coverage fhbp + mab 7 peptides, 4 discontinuous segments, had reduced D exchange HDX-MS was the most effective method to rapidly supply nearcomplete information about epitope structure 211 Waters Corporation 21

22 Biopharma Protein Characterization Examples 211 Waters Corporation 22

23 HDX-MS Used in Interferon Comparability Studies J. Pharm. Sci June 1;1(6): Interferon peptides from four different treatment pairs Same peptides were repeatedly generated in all conditions. There were no difference between two different batches (Contrl and Exp batches were in 8 years apart). Control 8-yr old batch Control Pegylated There were no difference between IFN vs. PEG-IFN There were no difference between two different media conditions Significant difference in deuteration was found in some peptides (local regions) upon oxidation. Control Diff media condition Control Oxidized Houde, et. al. J. Pharm. Sci June 1;1(6): Waters Corporation 23

24 Example Protein Characterization HDX-MS Experiment J. Am. Soc. Mass Spectrom. (212) 23 Two samples, continuous labeling local (peptide) level experiment (1 s 4 hr), triplicate 91% sequence coverage G-CSF PEGylated G-CSF (2 Kda PEG) 65% of C-GSF sequence showed no detectable change in HDX Peptides containing residues known to be involved in receptor binding showed only minor differences Conformational stability of receptor binding regions not significantly affected by PEGylation 211 Waters Corporation 24

25 mab Comparability Study with HDX- MS Drug Discovery Today, Vol 19, Number 1, January 214 Global level Global level 3 batches of mab 3 different processes Continuous labeling experiment Intact protein (global) and peptide level measurements 211 Waters Corporation 25

26 Comparing HDX-MS with DSC Localized Information From HDX-MS Poster presented at WCBP 212 (Ahn, Lu) 3 mab Non-mutated and mutated IgG1 Continuous labeling experiment Peptide-level measurement DSC establishes that a conformational change has occurred affecting a specific domain HDX-MS tells which peptides in sequence are affected 211 Waters Corporation 26

27 Protein-Ligand Binding Example 211 Waters Corporation 27

28 Example Protein-Ligand Binding HDX-MS Experiment Nature Communications 3, 1288 (Dec. 212) Two samples, continuous labeling local (peptide) level experiment (1 s 1 hr) 98% sequence coverage DOT1L DOT1L complexed with inhibitor Some regions demonstrated reduction in D uptake after inhibitor binding 211 Waters Corporation 28

29 DOT1L with no compound Relative % deuterium <2% % undetermined Slide courtesy of John Engen Inhibitors affect DOTL1 differently Changes to deuteration with inhibitors all data shown are after 1 minutes of deuteration DOT1L+SAH DOT1L+EPZ DOT1L+FED2 No change.5-.8da >.8Da PDB:1NW3 Yu, et al. (212) Nature Communications 3, Waters Corporation 29

30 HDX-MS Supporting Protein Crystallography and Protein NMR Studies 211 Waters Corporation 3

31 Using HDX-MS to Identify Regions and Produce Modified Constructs Biotechnology and Genetic Engineering Reviews, Vol 23, Dec. 26 and Proteins 29; 76: HDX-MS rapidly identifies highly flexible regions Optimized constructs then designed Significantly improved NMR spectra Minimal perturbation of the ordered regions of the protein structure About 2%-4% of crystallography failures addressed by this strategy 211 Waters Corporation 31

32 HDX-MS at Waters 211 Waters Corporation 32

33 Hydrogen Deuterium Exchange MS systems Dedicated standards and UPLC BEH separation chemistries nanoacquity UPLC with HDX technology Xevo G2-S QTof MS and Synapt G2-Si HDMS ProteinLynx Global Server (PLGS) and DynamX data processing 211 Waters Corporation 33

34 Waters System Solution for Automated HDX nanoacquity UPLC with HDX Automation technology Xevo G2-S QTof MS ProteinLynx Global SERVER (PLGS) DynamX TM Fast chromatography at C Automates HDX experiments Accurate measurement Reliable peptic peptide ID by MS E Automated HDX data Processing for deuteration determination 211 Waters Corporation 34

35 Waters System Solution for Automated HDX nanoacquity UPLC with HDX Automation technology Synapt G2-Si HDMS ProteinLynx Global SERVER (PLGS) DynamX TM Fast chromatography at C Automates HDX experiments Accurate measurement ETD capability Ion mobility Reliable peptic peptide ID by MS E Automated HDX data Processing for deuteration determination 211 Waters Corporation 35

36 Waters HDX Workflow at the Peptide Level Relative Deuterium Level (Da) peptide APO HOLO Time (min) Undeuterated / H2O Deuterated / D2O Protein in H 2 O, ph 7.4 at room temp Add 2-fold excess D 2 O Protein labeling occurs For various times Quench ph to 2.5, temp to C OPTIONAL AUTOMATION Inside HDX Manager at C Quenched protein is injected into HDX manager at ph 2.5 Local Analysis at Peptide Level Online Pepsin Digestion ph 2.5 nanoacquity UPLC using 1.7 µm BEH13 C18 column for fast separation Peptide map PLGS and IDENTITY E for peptide ID ESI Q-Tof MS E Or ESI HDMS E Deuterium Uptake DynamX Processing Determination 211 Waters Corporation 36

37 Measurement of average mass change over time Labeling time 24 m 6 m 1 % % Uptake of deuterium from solution for different regions 1 vary according to time % % 1 m 1 m 1 % % % 1 % Changes in mass (distribution) correlated to conformational changes s % % s 1 % Time % m/z Waters Corporation 37

38 Waters M-Class ACQUITY UPLC with HDX Technology Waters next generation UPLC platform for nano to microscale separations True UPLC separations for protein and peptide-level HDX-MS measurements Reproducible, rapid and robust separations at degrees C up to 15K psi 211 Waters Corporation 38

39 Waters nanoacquity UPLC with HDX Technology UPLC provides major advantages for HDX studies: Exceptional Reproducibility Minimize back-exchange by operating at C Rapid separations with high resolution to reduce time spent on column HPLC Example shown is a 52 kda protein digested with pepsin at physiological ph UPLC High-Speed and High-Resolution UPLC Separation at Zero Degrees Celsius; Anal. Chem. 28, 8, ; Thomas E. Wales et al; 1.121/ac Waters Corporation 39

40 ACQUITY UPLC M-Class with HDX Technology 211 Waters Corporation 4

41 Complex HDX Data can be Resolved by enabling Ion Mobility Iacob et al; Ion Mobility adds an additional dimension to mass spectrometric analysis of solutionphase hydrogen/ deuterium exchange; Rapid Commun. Mass Spectrom. 28; 22: Waters Corporation 41

42 IMS separates co-eluting labeled peptides UCA64_191_77_bsa_2m 287 (3.54) Cm (278:36) 1 1: TOF MS ES+ 9.8e4 2 m Labeling No IMS separation % m/z Ion Mobility Enabled 2 minl, 75 pmol BSA, IMS Res Mode, 5-2k, optimized cond(4min trap).3s sc UCA64_191_77_bsa_2m_rt_4_JA2 291 (3.567) Cm (278:294) 1: TOF MS ES e4 ASIQKFGERALKA 2+ % with IMS-enabled separation m/z UCA64_191_77_bsa_2m_rt_4_JA 284 (3.472) Cm (278:294) 1: TOF MS ES e3 % AVEGPKL 1+ m/z UCA64_191_77_bsa_2m 287 (3.54) Cm (278:36) 1: TOF MS ES+ 211 Waters Corporation 42

43 Waters HDX software: DynamX TM Automated uptake calculation Easy view Convenient interpretation 211 Waters Corporation 43

44 DynamX: IMS Support 1 min Time Course Changes in Uptake 1 min Peptide Identified Interfering Peptide separated by Mobility 1 min 1 sec Ref 211 Waters Corporation 44

45 Understanding the Difference Plot Difference plots represent uptake (Da) differences between ref and exp. A black vertical bar represents a sum of the mass differences observed for each peptide in all time points. Ref (IFN) Confidence limit Black dotted line at y-axis values +1.1 Da Time-course 1 sec 1 min 12 min 6 min 24 min Blue dotted line at y-axis values +.5 Da Vertical bar Indicates that this peptide showed higher uptake in Oxidized IFN. Exp (Oxidized IFN) What it means These displays are available in DynamX software (except confidence limit). In this data, certain peptides with the significant differences can be easily visualized. In this case, oxidized IFN revealed significant conformational changes in several peptides. Houde, et. al. J. Pharm. Sci June 1;1(6): Waters Corporation 45

46 HDX for Comparability: Experimental Difference Chart Is my protein different from a reference compound? Greater deuteration in APO Yes. Difference chart reveals locations of different deuteration. Four regions in Apo CaM displayed greater difference. APO HOLO No difference btw Apo & Holo 211 Waters Corporation 46

47 HDX for Comparability: Experimental Butterfly Chart How different are the two conditions? Butterfly Chart reveals the exchange rate and deuteration incorporation in comparison for all peptides in all time points. APO Where is the peptide #43? Residues AA, localising the contribution to the difference HOLO 211 Waters Corporation 47

48 Waters HDX software: Efficiency of data processing 1 kda protein typically generates ~2 reproducible peptic peptides Number of spectra to process in entire HDX study was calculated to be: 2 peptides x 6 labeling time points x 2-set comparison (bound vs. unbound) x 2 (N=2 duplicates) = 48 spectra to determine deuterium uptake Manual Manual Calculation of deuterium uptake Semi- Automated Help via export to Excel Macro etc. Fully Automated DynamX Full Month 2 3 Weeks 1-4 Days data processing time 211 Waters Corporation 48

49 Leap HDX Automation Manager Independent temperature zones for automated sampling processing HDX manager 211 Waters Corporation 49

50 Automated Waters HDX-MS system 211 Waters Corporation 5

51 Enzymate Online Digestion Column Pepsin immobilized BEH column, Waters P/N mm X 3 mm 15, psi compatibility 211 Waters Corporation 51

52 HDX Phos B Check Standard Intact phosphorylase b protein that can be used to evaluate HDX system performance, measure back exchange, and optimize methods P/N Waters Corporation 52

53 DynamX 3. Software Expanding beyond peptide HDX-MS workflow support 211 Waters Corporation 53

54 HDX/ETD Workflow ETD/HDX can pinpoint conformational changes to a single AA residue ETD presents a practical alternative to fragmentation techniques for high-spatial-resolution HDX studies 211 Waters Corporation 54

55 ETD with the SYNAPT G2-Si System Front-end ETD using glow discharge source Compatible with LC separations Permanently mounted Easy operation & maintenance 211 Waters Corporation 55

56 ETD with the SYNAPT G2-Si System The peptide(s) of interest are selected from peptide-level HDX studies Quick MS system test can show instrument is set up for minimal D scrambling Targeted DDA experiment used to acquire peptide ETD MS/MS data DynamX 3. will be able to automate processing of the ETD fragmentation data 211 Waters Corporation 56

57 ETD/HDX Data can Provide Near AA Residue Spatial Resolution of D Uptake 211 Waters Corporation 57

58 New HDX-MS online resource Waters Corporation 58

59 New team member Susan Fang, Senior Scientist HDX-MS Jing (Susan) Fang Sept, 212 Jan, 214 Postdoc fellowship Amgen, CA Sept, 27 July, 212 PhD candidate NEU Prof. John Engen Dr. Zhongqi Zhang Since Jan, 214 Senior scientist 211 Waters Corporation 59

60 Overview HDX-MS theory Example applications Structural biology Epitope mapping Protein-ligand binding Reducing failure rates in crystallography and protein NMR studies Biopharma comparability HDX-MS at Waters System ACQUITY UPLC M-Class with HDX Technology Ion mobility and HDMS DynamX Automation Enzymate online pepsin digestion columns HDX Check Standard ETD, DynamX 3. and Synapt G2-Si Waters.com HDX toolkit 211 Waters Corporation 6