Hydrogen/Deuterium Exchange Mass Spectrometry:A Mini-Tutorial

George Bou-Assaf56th ASMS Conference

June 2nd, 2008

Florida State UniversityNational High Magnetic Field Laboratory

Tallahassee-Florida

Hydrogen Exchange : A Brief HistoryLinderstrøm-Lang (1950’s): “Amide hydrogen exchange rates

should reflect the presence of hydrogen bonded structure”

Rosa & Richards (1979) : Spacial resolution enhancement by combination of proteolysis with HPLC

Englander (1960’s) : studied H/T exchange by liquid scintillation

(1980) improved the protocol by lowering the temperature of the separation step to reduce back-

exchange.

Zhang and Smith (1993) : combined HDX to MS, used FAB for ionization

Johnson and Walsh (1994) : first to use ESIL.S. Busenlehner, R.N. Armstrong / Archives of Biochemistry and Biophysics – 2005, 433, 34-46

A.N. Hoofnagle, K.A.Resing, N.G.Ahn / Annu. Rev. Biophys. Biomol. Struct. – 2003, 32:1-25

Advantages over NMR & X-Ray

• Crystallization problems

• Rigid systems

http://fig.cox.miami.edu/~cmallery/255/255tech/mcb3.38.xray.jpg http://nmr.magnet.fsu.edu/facilities/900_105mm_TLH.htm

• Low sensitivity

• High concentration required

• Protein size < 50 kDa

X-Ray Crystallography

Nuclear Magnetic Resonance

HDX at the molecular level

H3NN

N COOH

R

O R

ROH

H

D

D

Low exchange rates : α-helices, β-sheets and core of the protein.

High exchange rates:Loops, turns and solvent accessible regions.

HDX at the macromolecular level

Akashi, Satoko. Medicinal Research Reviews, 2006, 26(3), 339-368.

H/D exchange

H/D exchange

Quench

Proteolysis

Quench

Proteolysis

Formation of a complex

Comparison of deuteration level

Types of HDX

Thomas E. Wales, John R. Engen, Mass Spectrometry Reviews, 2006, 25, 158– 170

Types of HDX

Thomas E. Wales, John R. Engen, Mass Spectrometry Reviews, 2006, 25, 158– 170

Types of HDX

E.A. Komives / International Journal of Mass Spectrometry 240, 2005, 285–290

• Off-exchange: it is hard to fully deuterate a protein, takes very long time…

• On exchange is preferred

Limitations of HDX

• Exchange in D2O buffer while digestion, HPLC, MS in hydrogenated solvents and denaturing conditions.

• Use SFC1, fast RPLC2…

• Good sequence coverage

• Spatial resolution: multiple overlapping fragments.

H3NN

N COOH

R

O R

ROD

D

H

H

1. M.R. Emmett, S. Kazazic, A.G. Marshall, W. Chen, S.D.H. Shi, B. Belaños and M.J. Greig, Anal. Chem. 2006, 78:7058-7060

2. G.M. Bou-Assaf, M.R. Emmett, A.G. Marshall, 56th Amer. Soc. Mass Spectrom. Conf. on Mass Specrometry and applied topics 2008

Experimental Setup at NHMFL

protein or complex

D

D

D

D

D

D

D

H

H

dilute 10 foldw/ D2O buffer

H/D exchange

Quench pD 2.3 ~ 2.5

digest with active enzyme at ~1.0 oC

Peptide separationESI - MS

Temp ~0 oCJasco HPLC/SFC System

Time (min)

0.0

0.5

2.0

60

240

4080

Desalt

The exchange rate constant

8

[ ] [ ]

10

ex H OH

OH H

k k H k OH

and k k

+ −= +

=

We need enzymes that work in these

conditions!

PEPSIN –PROTEASES TYPE

XIII & TYPE XVIII

As temperature decreases, the exchange also decreases.

David L. Smith, JOURNAL OF MASS SPECTROMETRY, VOL. 32, 1997, 135-146

GSPEFGTGTRFGTDLAKEAKKVHQTT

RTVPAKRGTIYDRNGVPIAEDATSYNV

YAVIDENYKSATGKILYVEKTQFNKVA…

Peptide Mapping

Sequence coverage:Pepsin : 93% Type XVIII : 84% Type XIII : 40%

99.7% Laetitia Cravello, Rapid Commun. Mass Spectrom. 2003; 17: 2387–2393

Spatial resolution

Apomyo 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 SLOW MEDIUM FASTSequence 1 L F T G H P E T L E K F D K F K H L K T E A E MProtease 2 F D K F K H L K T E A E 6 0 5

XIII 3 F K H L K T E 4 0 2Fragments 4 P E T L E K 4 1 0

5 T L E K F D K F K H 5 0 4Pepsin 6 E K F D K F K H L K T E A E 8 0 5

Fragments 7 E K F D K F K H L K T E A E M 8 1 58 L E K F 3 0 0

Rate 9 S S S S 4F + 1S M1F + 5S1M+1S

10

8

6

4

2

0

Zero H between0.4 h-1 and 10 h-1

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0-2.3

5 H Faster than 10 h-1

Apomyoglobin SegmentF43DKFKHLKTEAE54

from protease type XIII Cleavage

Log k (h-1)

6 H Slowerthan 0.4 h-1

Number ofDeuteriumsin StatedRate ConstantRange

Zhang H., Kazazic S., Schaub T.M., Tipton J.D., Emmett M.R., and Marshall, A.G. (manuscript submitted)

ApplicationsFolding and unfolding of proteins

Thomas E. Wales, John R. Engen, Mass Spectrometry Reviews, 2006, 25, 158– 170

Influence of mutation on the 3D structures of proteins

L.S. Busenlehner, R.N. Armstrong / Archives of Biochemistry and Biophysics 433 (2005) 34–46

Applications

L.S. Busenlehner, R.N. Armstrong / Archives of Biochemistry and Biophysics 433 (2005) 34–46

Conformational change induced by ligand binding

Ligand : Ion, Metabolite or other Protein

Applications

J. Lisal, T.T. Lam, D.E. Kainov, M.R. Emmett, A.G. Marshall and R. Tuma / Nat. Struc. Mol. Biol. (2005) 12 (5): 34–46

Molecular Mechanisms