Deciphering Drift Time Measurements from Travelling Wave Ion Mobility Spectrometry-Mass Spectrometry Studies

Abstract

Detailed knowledge of the tertiary and quaternary structure of proteins and protein complexes is of immense importance in understanding their functionality. Similarly, variations in the conformational states of proteins form the underlying mechanisms behind many biomolecular processes, numerous of which are disease-related. Thus, the availability of reliable and accurate biophysical techniques that can provide detailed information concerning these issues is of paramount importance. Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) offers a unique opportunity to separate multi-component biomolecular entities and to measure the molecular mass and collision cross-section of individual components in a single, rapid (≤ 2 min) experiment, providing 3D-architectural information directly. Here we report a method of calibrating a commercially available electrospray ionisation (ESI)-travelling wave ion mobility spectrometry (TWIMS)–mass spectrometer using known cross-sectional areas determined for a range of biomolecules by conventional IMS-MS. Using this method of calibration, we have analysed a range of proteins of differing mass and 3D architecture in their native conformations by ESI-TWIMS-MS and found that the cross-sectional areas measured in this way compare extremely favourably with cross-sectional areas calculated using an in-house computing method based on Protein Data Bank NMR-derived co-ordinates. This not only provides a high degree of confidence in the calibration method, but also suggests that the gas phase ESI-TWIMS-MS measurements relate well to solution-based measurements derived from other biophysical techniques. In order to determine which instrumental parameters affect the ESI-TWIMS-MS cross-sectional area calibration, a systematic study of the parameters used to optimise TWIMS drift time separations has been carried out, observing the effect each parameter has on drift times and IMS resolution. Finally, the ESI-TWIMS-MS cross-sectional area calibration has been applied to the analysis of the amyloidogenic protein β₂-microglobulin and measurements for three co-populated conformational families, present under denaturing conditions, have been made: the folded, partially unfolded and unfolded states.

Keywords

ion mobility spectrometry mass spectrometry cross-sectional areas travelling wave ion guide electrospray ionisation proteins peptides

Get full access to this article

View all access options for this article.

References

Verbeck

G.F.

Ruotolo

B.T.

Sawyer

H.A.

Gillig

K.J.

and Russell

D.H.

, “A fundamental introduction to ion mobility mass spectrometry applied to the analysis of biomolecules”, J. Biomol. Tech. 13, 56–61 (2002).

Creaser

C.S.

Griffiths

J.R.

Bramwell

C.J.

Noreen

Hill

C.A.

and Thomas

C.L.P.

, “Ion mobility spectrometry: A review. Part 1. Structural analysis by mobility measurement”, Analyst 129, 1658–1661 (2004). doi: 10.1039/b404531a

Hoaglund-Hyzer

C.S.

Counterman

A.E.

and Clemmer

D.E.

, “Anhydrous protein ions”, Chem. Rev. 99, 3037–3079 (1999). doi: 10.1039/b404531a

Borsdorf

Nazarova

E.G.

and Miller

R.A.

, “Atmospheric-pressure ionization studies and field dependence of ion mobilities of isomeric hydrocarbons using a miniature differential mobility spectrometer”, Anal. Chim. Acta 575, 76–88 (2006). doi: 10.1016/j.aca.2006.05.066

McCooeye

Ding

Gardner

G.J.

Fraser

C.A.

Lam

Sturgeon

R.E.

and Mester

, “Separation and quantitation of the stereoisomers of Ephedra alkaloids in natural health products using flow injection-electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry”, Anal. Chem. 75, 2538–2542 (2003). doi: 10.1021/ac0342020

Dwivedi

Matz

L.M.

Clowers

B.H.

Siems

W.F.

and Hill

H.H.

, “Gas-phase chiral separations by ion mobility spectrometry”, Anal. Chem. 78, 8200–8206 (2006). doi: 10.1021/ac0608772

Smith

D.P.

Giles

Bateman

R.H.

Radford

S.E.

and Ashcroft

A.E.

, “Monitoring co-populated conformational states during protein folding events using electrospray ionisation-ion mobility spectrometry-mass spectrometry”, J. Am. Soc. Mass Spectrom. 18, 2180–2190 (2007). doi:10.1016/j.jasms.2007.09.017

Valentine

S.J.

and Clemmer

D.E.

, “H/D exchange levels of shape-resolved cytochrome c conformers in the gas phase”, J. Am. Chem. Soc. 119, 3558–3566 (1997). doi: 10.1021/ja9626751

Kanu

A.B.

Dwivedi

Tam

Matz

L.M.

and Hill

H.H.

, “Ion mobility-mass spectrometry”, J. Mass Spectrom. 43, 1–22 (2008). doi: 10.1002/jms.1383

10.

Kindy

J.M.

Taraszka

J.A.

Regnier

F.E.

and Clemmer

D.E.

, “Quantifying peptides in isotopically labeled protease digests by ion mobility/time-of-flight mass spectrometry”, Anal. Chem. 74, 950–958 (2002). doi: 10.1021/ac010807p

11.

Hoaglund-Hyzer

C.S.

Valentine

S.J.

and Clemmer

D.E.

, “An ion trap Interface for ESI-ion mobility experiments”, Anal. Chem. 69, 4156–4161 (1997). doi: 10.1021/ac970526a

12.

Mosier

P.D.

Counterman

A.E.

Jurs

P.C.

and Clemmer

D.E.

, “Prediction of peptide ion collision cross sections from topological molecular structure and amino acid parameters”, Anal. Chem. 74, 1360–1370 (2002). doi: 10.1021/ac0112059

13.

Revercomb

H.E.

and Mason

E.A.

, “Theory of plasma chromatogra-phy/gaseous electrophoresis: A review”, Anal. Chem. 47, 970–983 (1975). doi: 10.1021/ac60357a043

14.

Clemmer

D.E.

and Jarrold

M.F.

, “Ion mobility measurements and their applications to clusters and biomolecules”, J. Mass Spectrom. 32, 577–592 (1997). doi: 10.1002/(SICI)1096-9888(199706)

15.

Mason

E.A.

and McDaniel

E.W.

, Transport properties of ions in gases. John Wiley & Sons Inc., New York, USA (1988).

16.

Szumlas

A.W.

and Hieftje

G.M.

, “Phase-resolved detection in ion-mobility spectrometry”, Anal. Chim. Acta 566, 45–54 (2006). doi: 10.1016/j.aca.2006.02.036

17.

Purves

R.W.

and Guevremont

, “Electrospray ionisation high-field asymmetric waveform ion mobility spectrometry-mass spectrometry”, Anal. Chem. 71, 2346–2357 (1999). doi: 10.1021/ac981380y

18.

Guevremont

Siu

K.W.M.

Wang

and Ding

, “Combined ion mobility/time-of-flight mass spectrometry study of electrospray-generated ions”, Anal. Chem. 69, 3959–3965 (1997). doi: 10.1021/ac970359e

19.

Barnett

D.A.

Ells

Guevremont

and Purves

R.W.

, “Separation of leucine and isoleucine by electrospray ionization – high-field asymmetric waveform ion mobility spectrometry-mass spectrometry”, J. Am. Soc. Mass Spectrom. 10, 1279–1284 (1999). doi: 10.1016/S1044-0305(99)00098-7

20.

McCooeye

M.A.

Mester

Ells

Barnett

D.A.

Purves

R.W.

and Guevremont

, “Quantitation of amphetamine, methamphetamine and their methylenedioxy derivatives in urine by solid-phase microextraction coupled with electrospray ionization-high-field asymmetric waveform ion mobility spectrometry-mass spectrometry”, Anal. Chem. 74, 3071–3075 (2002). doi: 10.1021/ac011296+

21.

Purves

R.W.

Barnett

D.A.

Ells

and Guevremont

, “Elongated conformers of charge states +11 to +15 of bovine ubiquitin studied using ESI-FAIMS-MS”, J. Am. Soc. Mass Spectrom. 12, 894–901 (2001). doi: 10.1016/S1044-0305(01)00272-0

22.

Purves

R.W.

Barnett

D.A.

Ells

and Guevremont

, “Investigation of bovine ubiquitin conformers separated by high-field asymmetric waveform ion mobility spectrometry: Cross section measurements using energy loss experiments with a triple quadrupole mass spectrometer”, J. Am. Soc. Mass Spectrom. 11, 738–745 (2000). doi: 10.1016/S1044-0305(00)00136-7

23.

Borysik

A.J.H.

Radford

S.E.

and Ashcroft

A.E.

, “Co-populated conformational ensembles of beta2-microglobulin uncovered quantitatively by ESI-MS”, J. Biol. Chem. 279, 27069–27077 (2004). doi: 10.1074/jbc.M401472200

24.

Giles

Pringle

S.D.

Worthington

K.R.

Little

D.R.

Wildgoose

J.L.

and Bateman

R.H.

, “Applications of a travelling wave-based radio-frequency-only stacked ring ion guide”, Rapid Commun. Mass Spectrom. 18, 2401–2414 (2004). doi: 10.1002/rcm.1641

25.

Pringle

S.D.

Giles

Wildgoose

J.L.

Williams

J.P.

Slade

S.E.

Thalassinos

Bateman

R.H.

Bowers

M.T.

and Scrivens

J.H.

, “An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument”, Int. J. Mass Spectrom. 261, 1–12 (2007). doi: 10.1016/j.ijms.2006.07.021

26.

Thalassinos

Slade

S.E.

Jennings

K.R.

Scrivens

J.H.

Giles

Wildgoose

J.L.

Hoyes

Bateman

R.H.

and Bowers

M.T.

, “Ion mobility spectrometry of proteins in a modified commercial mass spectrometer”, Int. J. Mass Spectrom. 236, 55–63 (2004). doi: 10.1016/j.ijms.2004.05.008

27.

Eckers

Laures

A.M.

Giles

Major

and Pringle

S.D.

, “Evaluating the utility of ion mobility separation in combination with high-pressure liquid chromatography/mass spectrometry to facilitate detection of trace impurities in formulated drug products.” Rapid Commun. Mass Spectrom. 21, 1255–1263 (2007). doi: 10.1002/rcm.2938

28.

Ruotolo

Giles

Campuzano

Sandercock

A.M.

Bateman

R.H.

and Robinson

C.V.

, “Evidence for macromolecular protein rings in the absence of bulk water”, Science 310, 1658–1661 (2005). doi: 10.1126/science.1120177

29.

Ruotolo

B.T.

Hyung

S.-J.

Robinson

P.M.

Giles

Bateman

R.H.

and Robinson

C.V.

, “Ion mobility-mass spectrometry reveals long-lived, unfolded intermediates in the dissociation of protein complexes”, Angew. Chemie Int. Ed. 46, 8001–8004 (2007). doi: 10.1002/anie.200702161

30.

Lorenzen

Olia

A.S.

Uetrecht

Cingolani

and Heck

A.J.

, “Determination of stoichiometry and conformational changes in the first step of the P22 tail assembly”, J. Mol. Biol. 379, 385–396 (2008). doi: 10.1016/j.jmb.2008.02.017

31.

Clemmer

D.E.

, http://www.indiana.edu/∼clemmer/Research/research.htm

32.

Valentine

S.J.

Counterman

A.E.

and Clemmer

D.E.

, “A database of 660 peptide ion cross sections: Use of intrinsic size parameters for bona fide prediciton of cross sections”, J. Am. Soc. Mass Spectrom. 10, 1188–1211 (1999). doi: 10.1016/S1044-0305(99)00079-3

33.

Williams

J.P.

and Scrivens

J.H.

, “Coupling desorption electrospray ionisation and neutral desorption/extractive electrospray ionisation with a travelling-wave based ion mobility mass spectrometer for the analysis of drugs”, Rapid Commun. Mass Spectrom. 22, 187–196 (2008). doi: 10.1002/rcm.3346

34.

Lerme

Dugourd

Hudgins

R.R.

and Jarrold

M.F.

, “High-resolution ion mobility measurements of indium clusters: Electron spill-out in metal cluster anions and cations”, Chem. Phys. Lett. 304, 19–22 (1999). doi: 10.1016/S0009-2614(99)00281-X

35.

Wildgoose

J.L.

Giles

Pringle

S.D.

Koeniger

Valentine

S.J.

Bateman

R.H.

and Clemmer

D.E.

, “A comparsion of travelling wave and drift tube ion mobility separations”, Proc. 54th ASMS Conference on Mass Spectrometry & Allied Topics May 28–June 1st 2006, Seattle, WA, USA, (2006).

36.

Ruotolo

Benesch

J.L.

Sandercock

A.M.

Hyung

S.-J.

and Robinson

C.V.

, “Ion mobility – mass spectrometry analysis of large protein complexes”, Nature Protocols 3, 1139–1152 (2008). doi: 10.1038/nprot.2008.78

37.

Kad

N.M.

Thomson

N.H.

Smith

D.P.

Smith

D.A.

and Radford

S.E.

, “Beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro”, J. Mol. Biol. 313, 559–571 (2001). doi: 10.1006/jmbi.2001.5071

38.

von Helden

Hsu

M.-T.

Gotts

and Bowers

M.T.

, “Carbon cluster cations with up to 84 atoms: Structures, formation mechanism and reactivity”, J. Phys. Chem. 97, 8182–8192 (1993). doi: 10.1021/j100133a011

39.

Cornell

W.D.

Cleplak

Bayly

C.I.

Gould

I.R.

Merz

K.M.

Ferguson

D.M.

Spellmeyer

D.C.

Fox

Caldwell

J.W.

and Kollman

P.A.

, “A second generation force field for the simulation of proteins, nucleic acids, and organic molecules”, J. Am. Chem. Soc. 117, 5179–5197 (1995). doi: 10.1021/ja00124a002

40.

Shelimov

K.B.

Clemmer

D.E.

Hudgins

R.R.

and Jarrold

M.F.

, “Protein structure in vacuo: Gas-phase conformations of BPTI and cytochrome c”, J. Am. Chem. Soc. 119, 2240–2248 (1997). doi:10.1021/ja9619059

41.

Jarrold

M.F.

, “Helices and sheets in vacuo”, Phys. Chem. Chem. Phys. 9, 1659–1671 (2007). doi: 10.1039/b612615d

42.

Shvartsburg

A.A.

and Jarrold

M.F.

, “An exact hard-spheres scattering model for the mobilities of polyatomic ions”, Chem. Phys. Lett. 261, 86–91 (1996). doi: 10.1016/0009-2614(96)00941-4

43.

Mesleh

M.F.

Hunter

J.M.

Shvartsburg

A.A.

Schatz

G.C.

and Jarrold

M.F.

, “Structural information from ion mobility measurements: Effects of the long-range potential.” J. Phys. Chem. 100, 16082–16086 (1996). doi: 10.1016/0009-2614(96)00941-4

44.

Jarrold

M. F.

, http://www.indiana.edu/∼nano/Software.html

45.

Stockley

P.G.

Rolfsson

Thompson

G.S.

Basnak

Francese

Stonehouse

N.J.

Homans

S.W.

and Ashcroft

A.E.

, “A simple, RNA-mediated allosteric switch controls the pathway to formation of a T=3 capsid”, J. Mol. Biol. 369, 541–522 (2007). doi: 10.1016/j.jmb.2007.03.020

46.

Smith

A.M.

Jahn

T.J.

Ashcroft

A.E.

and Radford

S.E.

, “Direct observation of oligomeric species formed in the early stages of amyloid fibril formation using electrospray ionisation mass spectrometry”, J. Mol. Biol. 364, 9–19 (2006). doi: 10.1016/j.jmb.2006.08.081

47.

Thomas

J.J.

Bothner

Traina

Benner

W.H.

and Siuzdak

, “Electrospray ion mobility spectrometry of intact viruses”, Spectroscopy 18, 31–36 (2004).

48.

Clemmer

D.E.

Hudgins

R.R.

and Jarrold

M.F.

, “Naked protein conformation: Cytochrome c in the gas phase”, J. Am. Chem. Soc. 117, 10141–10142 (1995). doi: 10.1021/ja00145a037

49.

Badman

E.R.

Hoaglund-Hyzer

C.S.

and Clemmer

D.E.

, “Monitoring structural changes of proteins in an ion trap over ∼100–200 ms: Unfolding transitions in cytochrome c ions”, Anal. Chem. 73, 6000–6007 (2001). doi:10.1021/ac010744a

50.

Sobott

Benesch

J.L.

Vierling

and Robinson

C.V.

, “Subunit exchange of multimeric protein complexes. Real-time monitoring of subunit exchange between small heat shock proteins by using electrospray mass spectrometry”, J. Biol. Chem. 277, 38921–38929 (2002). doi: 10.1074/jbc.M206060200

51.

Sobott

Hernandez

McCammon

M.G.

Tito

M.A.

and Robinson

C.V.

, “A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies”, Anal. Chem. 74, 1402–1407 (2002). doi: 10.1021/ac0110552

52.

van Berkel

W.J.

van den Heuval

R.H.

Versluis

and Heck

A.J.

, “Detection of intact megaDalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry”, Proteome Sci. 9, 435–439 (2000).

53.

Tahallah

Pinkse

Maier

C.S.

and Heck

A.J.

, “The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time-of-flight instrument”, Rapid Commun. Mass Spectrom. 15, 596–601 (2001). doi: 10.1002/rcm.275

54.

Verdone

Corazza

Viglino

Pettirossi

Giorgetti

Mangione

Andreola

Stoppini

Bellotti

and Esposito

, “The solution structure of human beta2-microglobulin reveals the prodomes of its amyloid transition”, Proteome Sci. 11, 487–499 (2002). doi: 10.1110/ps.29002