Advances in HDX Techniques

Transcription

1 Waters HDX 24 meeting Advances in HDX Techniques Thomas J. D. Jørgensen Associate professor, Ph.D. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense

2 Part I Structural dynamics of PAI-

3 RNA aptamers as PAI- conformational probes Paionap 5 Binds both active and latent PAI- with low nm affinity Delays latency transition 4-fold No effect on the inhibitory activity Madsen et al., Biochemistry, 2 Paionap 4 Binds active PAI- with low nm affinity Delays latency transition 8-fold No effect on the inhibitory activity PAI- Paionap 5

4 From mass spectra to deuterium uptake plots PAI- PAI- aptamer (paionap 4) QS378MBT (::) 9.8E2 QS378MBT (::) 9.8E2 Full Deu. min min min. min Undeu. QS2334MBT (:4:) QS2332MBT (::) QS233MBT (::) QS2328MBT (::6) QS2322MBT (::) m/z 7.95E E2 9.E2.8E3.22E3 QS2377MBT (:4:) m/z QS2375MBT (::) QS2373MBT (::) QS2372MBT (::6) 5.6E E2 7.5E2 7.5E2 QS2322MBT (::).22E m/z Deuterium content FRLFRSTVKQVDFSE Full deuteration control Paionap 5. minutes Paionap 4 no aptamer m/z m/z 627. Trelle, Dupont, Madsen, Andreasen, Jørgensen, ACS Chemical Biology, 23

5 Effect of aptamer binding 7 26 FRSTVKQVDF Deuterium Content Full deuteration control Paionap 5 no aptamer Paionap 4 K8 R76 R8. Min F4 K22 Trelle, Dupont, Madsen, Andreasen, Jørgensen, ACS Chemical Biology, 23

6 Effect of aptamer binding, Helix D FKIDDKGMAPALRHLYKELMGPWNKDEIS 2 8 Full deuteration control 6 Deuterium Content no aptamer Paionap 4 Paionap 5 K8 R8 R Min K22 F4 Trelle, Dupont, Madsen, Andreasen, Jørgensen, ACS Chemical Biology, 23

7 Helix C is strongly protected upon aptamer binding LQLTTGGETQQQIQAAMG 6 4 Full deuteration control 2 no aptamer Deuterium Content Paionap 5 Paionap 4. Min Trelle, Dupont, Madsen, Andreasen, Jørgensen, ACS Chemical Biology, 23

8 Transient local unfolding in PAI (without aptamer) a c aa d aa 6-4 e aa b 8 Relative intensity 4 sec. at 5 C 2 sec. sec. F U F U F U 5 sec. 3 sec. 5 sec. No D 6 min. at 37 C 4 min. 3 min. 2 min. min. 2min. No D 6 min. at 37 C 4 min. 3 min. 2 min. min. 2min. No D m/z.5 Da 7 Da 5 Da 8-9 Da 8-9 Da -2 Da 8.5 Da 7 Da m max m max m max Trelle, Madsen, Andreasen, Jørgensen, Angewandte Chemie (in press), 24

9 Local unfolding for the residues 6 4 follows first order kinetics (helix A/β6B/Helix B ) a Active PAI b Latent PAI c Relative intensity m Full D = 9.8 Da Full D m max = 8 9 Da No D min. 8 min. 4 min. 3 min. 2 min. min. 5min. 2 min m/z Full D 4 min. active PAI 6 min. 8 min. 4 min. 2 min. 2min. No D d Folded / (Folded Unfolded) Latent PAI Active PAI 5 5 Exchange time [min] Rate constant (k U ) Half life (t /2 ) Active.289 min 23.9 min Latent.5 min 4664 min Trelle, Madsen, Andreasen, Jørgensen, Angewandte Chemie (in press), 24

10 Energy landscape: unfolding > latency transition Energy Trelle, Madsen, Andreasen, Jørgensen, Angewandte Chemie (in press), 24 M** Helix B/C and A β strand 6B and 5A M* Helix B/C? t /2 ~ 2 min? Active t /2 ~ 45 min Latent Reaction coordinate

11 D 2 O is not H 2 O Trelle, Dupont, Madsen, Andreasen, Jørgensen, ACS Chemical Biology, 23

12 The binding interface is protected against exchange PAI- Deuterium content SMB SMB Exchange time (min) 4 FRLFRSTVKQVDF 26 Covers part of helix E & strand A SMB domain of vitronectin Trelle, Hirschberg, Jansson, Ploug, Roepstorff, Andreasen, Jørgensen Biochemistry, 22

13 Protection remote from binding site, Helix C (& B) PAI- Deuterium Content SMB SMB Time (min) Trelle, Hirschberg, Jansson, Ploug, Roepstorff, Andreasen, Jørgensen Biochemistry, 22

14 Part II Electrochemical reduction of disulfide bonds for HDX-MS analyses

15 Reduction at quench conditions using TCEP ph 2.5 TCEP efficiency is severely reduced at ph 2.5 High concentrations Accumulation in RP columns Extensive washing Cline, D. J.; Thorpe, C. Biochemistry 24, 43, 595

16 On-column accumulation of TCEP Three consecutive injections with 4 mm TCEP pmol upar wt (A) s exchange-in rep 2 - Q PBS and 4 mm TCEP for 2 min (2 ul) - Pepsin column QS4855SM : TOF MS ES BPI 29 Injection QS4857SM : TOF MS ES BPI QS4859SM : TOF MS ES BPI 45 % % % Injection 2 Injection Time

17 On-column accumulation of TCEP Three consecutive injections with 4 mm TCEP pmol upar wt (A) s exchange-in rep2 - Q PBS an QS4859SM 955 (8.695) Cm (955:84) : TOF MS ES e4 pmol upar wt (A) s exchange-in rep 2 - Q PBS and 4 mm TCEP for 2 min ( ul) - Pepsin column QS4855SM : TOF MS ES Injection BPI 29 % m/z pmol upar wt (A) s exchange-in rep2 - Q PBS an QS4859SM (8.695) 4. Cm (955:84) 6. : TOF MS ES QS4857SM : TOF MS ES.97e4 Injection BPI 2 38 % % % m/z QS4859SM : 675 TOF MS 68 ES Injection BPI 3 pmol upar wt (A) s exchange-in rep Q PBS an QS4859SM 955 (8.695) Cm (955:84) : TOF MS ES e3 % % Time m/z

18 Reduction Chain A H S H S H S H S H S H S Chain B

19 Reduction of using TCEP Quench conditions - C and ph 2.5 MH 6 MH 5 MH 4 4 mm TCEP 2 min. incubation Chain B MH 4 MH 6 MH 5 MH 4 4 mm TCEP min. incubation Chain B MH 5 Chain B MH 4 MH 6 MH 5 MH 4 4 mm TCEP 5 min. incubation min. incubation less than 5% reduction 5 min. Incubation less than 2% reduction Mysling, Salbo, Ploug, Jørgensen Anal. Chem. 24

20 Electrochemical reduction cell (Antec) Reference electrode Able to reduce insulin efficiently ul internal volume Solvent flow Running conditions: 5 bar (725 PSI) pressure limit with first generation model Working Electrode % FA in solvent

21 Injection Digestion chamber C Reduction cell Trap and analytical column.2 C From loop To desalting trap Pepsin column Mysling, Salbo, Ploug, Jørgensen Anal. Chem. 24

22 Electrochemical reduction of insulin MH 6 MH 5 Cell off μl/min. Reduction efficiency is dependent on residence time (Flow rate) Relative intensity [AU] Chain B MH 5 Chain B MH 5 Chain B MH 4 Chain B MH 4 Chain A MH 3 MH 6 MH 5 Cell on μl/min. Residence time: 7.2 s. Cell on 5 μl/min. Residence time: 4.4 s. Tweak reduction using the desalting flow rate m/z [Th] Mysling, Salbo, Ploug, Jørgensen Anal. Chem. 24

23 Impact on deuterium back-exchange Labeled insulin B-chain Deuterons Theoretical maximum Observed Deuterons Back-exchange 28.% 36.% 42.2% Cell active - - YO Cell present - - YO Buffer.23% FA % FA % FA Desalting.5 min. 3 ul/min 3 min. 5 ul/min 3 min. 5 ul/min Main contributor to back-exchange Increased desalting time Mysling, Salbo, Ploug, Jørgensen Anal. Chem. 24

24 Other observations PBS and ammonium acetate had a negative impact on the reduction - Alleviated by diluting samples x when quenching exchange - Other buffers could have less dramatic effects Electrochemical reduction was not found to alter deuteration patterns Mysling, Salbo, Ploug, Jørgensen Anal. Chem. 24

25 Acknowledgement Peter A. Andreasen Jeppe B. Madsen Daniel M. Dupont Jan K. Jensen Michael Ploug Antec, NL, Agnieszka Kraj Biolab, DK Morten Beck Trelle Simon Mysling Daniel Hirschberg Anna Jansson Peter Roepstorff Sabine Amon Rune Salbo Kim Stjerne Britta Gribsholt Funding Lundbeck Foundation The Danish Council for Independent Research Technology and Production Sciences The Danish Council for Independent Research Natural Sciences