Advances in the Mass
Spectrometry of Membrane
Proteins: From Individual Proteins
to Intact Complexes
Nelson P. Barrera and Carol V. Robinson
Annu. Rev. Biochem. 2011. 80:247-71
Bi/Ch 132
Adam Boynton
Fall 2012
Membrane Protein Complex Challenge
 Mass spectrometry has been become a powerful method for
studying soluble protein complexes
 Structural determinations
 Subunit stoichiometries
 Topology
 Application to studying intact membrane protein complexes
has remained a challenge
 Insolubility in ES buffers
 Noncovalent interactions between transmembrane and
cytoplasmic subunits easily disrupted
(Barrera NP, Di Bartolo N, Booth PJ, Robinson CV. 2008. Micelles protect membrane complexes from solution to vacuum. Science 321:243–46)
Promising Development: Using ESMS with Micelles
 Idea: encapsulate protein complex
within a non-ionic detergent micelle
 e.g. n-dodecyl-b-D-maltoside (DDM)
Both hydrophobic and
hydrophilic properties
Provides lipid-like environment
for membrane protein
Preserve membrane protein
structure and activity
 Use nanoelectrospray-MS to disrupt
micelle and release intact protein
Using ES-MS with Micelles
 Study: ATP-binding cassette (ABC)
transporter BtuC2D2
 Two transmembrane BtuC subunits
 Two soluble BtuD subunits
 Instrumentation: quadrupole-TOF
(tandem MS)
 Maximum acceleration voltages
applied in both ESI source & collision
cell (≈ 200 V)
 Changing pressure in collision cell
yields different dissociation pathways
 Bottom: lower pressure, micelle still
 Middle: higher pressure, intact
 Top: highest pressure, BtuC subunit
dissociates, form trimer
 Charge states/splitting patterns can
be analyzed to detect PTMs and
ligand binding
(Barrera NP, Di Bartolo N, Booth PJ, Robinson CV. 2008. Science 321:243–46)
ES with Micelles: Role of Activation
 Study: ABC transporter dimer
protein MacB
Highest activation energy: micelle
completely evaporated, sharp signals
observed; two lipid molecules remain
bound; dimer still intact!
Increase activation energy: micelle
undergoes evaporation, can start to
see protein dimer charge states
Low activation energy: micelles still
bound to complex = broad peak
(Barrera NP, Isaacson SC, Zhou M, Bavro VN, Welch A, et al. 2009. Mass spectrometry
of membrane transporters reveals subunit stoichiometry and interactions. Nat.
Methods 6:585–87)
ES in Micelles: Role of Activation
Activation coefficient
 Indicator of energy
required to release protein
complex from micelle
 Larger for greater
molecular mass
 Higher for membrane
complexes than soluble
 Micelle protective
(Nelson P. Barrera and Carol V. Robinson Annu. Rev. Biochem. 2011. 80:247-71)
Ion-mobility (IM)–MS
 Ions separated based
on ability to move
through a neutral gas in
drift region, in presence
of electric field
 Time taken for ion to
travel through drift
region recorded
(“arrival time
distribution” or ATD):
Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson,
C. V. Science 2005, 310, 1658–1661
 Experimental ATD calibrated against ATD’s of ions of
known structure
 Can determine collision cross section (CCS) for a given ion
 Compare CCS’s to elucidate 3D structures of protein
IM–MS: Studying 3D Structure of Protein
Complexes in the Gas Phase
KirBac3.1 potassium ion channel
 Homotetramer with 4 transmembrane subunits
 CCS suggests compact structure
 Native quaternary structure maintained in gas
240 V accel.
180 V accel.
BtuC2D2 transporter protein
 Tetramer with 2 transmembrane & 2 soluble
 More readily dissociates than KirBac3.1
• KirBac3.1 better protected by micelle
Wang SC, Politis A, Di Bartolo N, Bavro VN, Tucker SJ, et al. 2010. J. Am. Chem. Soc. 132:15468–70
Laser-Induced Liquid Bead Ion
Desorption (LILBID)-MS
N. Morgner, H.D. Barth, B. Brutschy, Austral. J. Chem. 59 (2006) 109–114.
1) Microdroplets of solution (diameter 50 μm, volume 65 pl) produced by 10 Hz droplet
generator (e.g. 3 μm protein complex in 10 mm ammonium acetate with 0.05% DDM)
2) Introduced into vacuum and irradiated one by one with nanosecond mid-IR pulses (pulse
energies of 1-15 mJ)
3) Pulses tuned to 3 μm wavelength (water absorption maximum)
4) Liquid reaches “supercritical state”, droplets explode, release charged biomolecules into
gas phase
5) Ions accelerated and analyzed via TOF reflectron MS
LILBID-MS: Study of P. furiosus ATP synthase
Low laser intensity: ions “gently” desorbed
- detect intact complexes
- subunit stoichiometry: A3B3CDE2FH2ac10
High laser intensity: non-covalent interactions broken
- detect complex subunits
Vonck J, Pisa KY, Morgner N, Brutschy B, Muller V. 2009. J. Biol. Chem. 284:10110–19
Comparing “Micelle ES-MS” and
 Both provide a means to study intact membrane protein complexes
 LILBID-MS more tolerable to wider range of buffers
 Better resolution achievable with ES
 Easier to study post-translational modifications (below)
 Easier to study small-molecule binding to complex
Study of EmrE dimer
* = +N-formyl Met PTM
+ = unmodified wild type
Three dimers formed
(++, +*,**)
Nelson P. Barrera and Carol V. Robinson. Annu. Rev. Biochem. 2011. 80:247-71
Future Direction
 Combining IM-MS with imaging techniques such as EM
and AFM
 IM-MS is very powerful for studying protein complex
 Locate subunit interactions in EM density maps/AFM images

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