HDX-MS at Waters Waters Corporation 1

Transcription

1 HDX-MS at Waters 2011 Waters Corporation 1

2 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 2011 Waters Corporation 2

3 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 0 C Automates HDX experiments Accurate measurement ETD capability Ion mobility Reliable peptic peptide ID by MS E Automated HDX data Processing for deuteration determination 2011 Waters Corporation 3

4 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 7.40 at room temp Add 20-fold excess D 2 O Protein labeling occurs For various times Quench ph to 2.50, temp to 0 C OPTIONAL AUTOMATION Inside HDX Manager at 0 C Quenched protein is injected into HDX manager at ph 2.5 Local Analysis at Peptide Level Online Pepsin Digestion ph 2.50 nanoacquity UPLC using 1.7 µm BEH130 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 2011 Waters Corporation 4

5 Measurement of average mass change over time Labeling time 240 m 60 m 100 % % Uptake of deuterium from 0 solution for different regions 100 vary according to time % % 0 10 m 1 m 100 % % % % 0 Changes in mass (distribution) correlated to conformational changes s % % 0 0 s 100 % 0 Time % 0 m/z Waters Corporation 5

6 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 0 degrees C up to 15K psi 2011 Waters Corporation 6

7 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. 2008; 22: Waters Corporation 7

8 IMS separates co-eluting labeled peptides UCA064_100901_077_bsa_2m 287 (3.504) Cm (278:306) 100 1: TOF MS ES+ 9.08e4 2 m Labeling No IMS separation % 0 m/z Ion Mobility Enabled, p,,, p ( p) UCA064_100901_077_bsa_2m_rt_04_JA2 291 (3.567) Cm (278:294) 1: TOF MS ES e4 ASIQKFGERALKA 2+ % with IMS-enabled separation 0 m/z UCA064_100901_077_bsa_2m_rt_04_JA 284 (3.472) Cm (278:294) 1: TOF MS ES e3 % AVEGPKL 1+ 0 m/z UCA b (3 504) C ( ) 1 TOF MS ES 2011 Waters Corporation 8

9 Ion Mobility Separation No Deuterium Exposure 2011 Waters Corporation 9

10 Ion Mobility Separation 100 Minute Exposure 2011 Waters Corporation 10

11 Waters HDX software: DynamX TM Automated uptake calculation Easy view Convenient interpretation 2011 Waters Corporation 11

12 DynamX: IMS Support 100 min Time Course Changes in Uptake 10 min Peptide Identified Interfering Peptide separated by Mobility 1 min 10 sec Ref 2011 Waters Corporation 12

13 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 10 sec 1 min 12 min 60 min 240 min Blue dotted line at y-axis values +0.5 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;100(6): Waters Corporation 13

14 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 2011 Waters Corporation 15

15 Leap HDX Automation Manager Independent temperature zones for automated sampling processing HDX manager 2011 Waters Corporation 16

16 Automated Waters HDX-MS system 2011 Waters Corporation 17

17 Enzymate Online Digestion Column Pepsin immobilized BEH column, Waters P/N mm X 30 mm 15,000 psi compatibility 2011 Waters Corporation 18

18 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 19

19 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 2011 Waters Corporation 20

20 ETD with the SYNAPT G2-Si System Front-end ETD using glow discharge source Compatible with LC separations Permanently mounted Easy operation & maintenance 2011 Waters Corporation 21

21 ETD/HDX Data can Provide Near AA Residue Spatial Resolution of D Uptake 2011 Waters Corporation 22

22 Inlet Editor 2011 Waters Corporation 23

23 Example Inlet Method 2011 Waters Corporation 24

24 Example Inlet Method Binary Solvent Manager Example gradient is for a 1.0 x 50 mm HSS T3 column. If using a 1.0 x 100 mm BEH column, typical flow rates are 40 to 45 µl/min Fluidic Configuration is set for Single Pump Trapping 2011 Waters Corporation 25

25 Example Inlet Method Binary Solvent Manager Trapping flow for the BSM should be set to be identical as the initial gradient conditions. Sample loading time (trapping and digestion) is set on this tab Waters Corporation 26

26 Example Inlet Method Auxiliary Solvent Manager The ASM flow rates have been increased to 1000 µl/min. Once software has been released, the ASM will be capable of different flow rates for both analytical and trapping conditions Waters Corporation 27

27 Example Inlet Method HDX Manager The HDX Manager has independent settings for the digestion and chamber temperature Waters Corporation 28

28 Example Inlet Method HDX Manager Data channels must be activated. Errors will occur if they are not Waters Corporation 29

29 Source Conditions 2011 Waters Corporation 30

30 Instrument 2011 Waters Corporation 31

31 TriWave 2011 Waters Corporation 32

32 TriWave DC 2011 Waters Corporation 33

33 MSe and HDMSe HDMS E Training April Waters Corporation 34

34 UPLC/HDMS E deconvoluting chimericy Co-Eluting Peptides Ionized Precursors Precursors IMS Separated Precursors Transferred to TOF MS 2011 Waters Corporation 35

35 UPLC/HDMS E deconvoluting chimericy Co-Eluting Peptides Ionized Precursors Precursors IMS Separated Precursors & Products Time Aligned 2011 Waters Corporation 36

36 UPLC/HDMS E Ion mobility 2011 Waters Corporation 37

37 IMS MS E MS Method Settings Waters Corporation 38

38 IMS MS E MS Method Settings Waters Corporation 39

39 IMS MS E MS Method Settings 30 Lockspray sampling set to every 30 seconds Waters Corporation 40