Advances in the Mass Spectrometry of Membrane Proteins: From Individual
Proteins to Intact ComplexesNelson P. Barrera and Carol V. Robinson Annu. Rev. Biochem. 2011. 80:247-71
Bi/Ch 132 Adam Boynton
Fall 2012
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
Membrane Protein Complex Challenge
(Barrera NP, Di Bartolo N, Booth PJ, Robinson CV. 2008. Micelles protect membrane complexes from solution to vacuum. Science 321:243–46)
Idea: encapsulate protein complex within a non-ionic detergent micellee.g. n-dodecyl-b-D-maltoside
(DDM)Both hydrophobic and
hydrophilic propertiesProvides lipid-like environment
for membrane protein Preserve membrane protein
structure and activity Use nanoelectrospray-MS to disrupt
micelle and release intact protein complex
Promising Development: Using ES-MS with Micelles
http://www.piercenet.com/browse.cfm?fldID=9AB987DA-C4D4-4713-8312-08A86E51EC6D
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
intact Middle: higher pressure, intact
tetramer Top: highest pressure, BtuC subunit
dissociates, form trimer
Charge states/splitting patterns can be analyzed to detect PTMs and ligand binding
Using ES-MS with Micelles
(Barrera NP, Di Bartolo N, Booth PJ, Robinson CV. 2008. Science 321:243–46)
ES with Micelles: Role of Activation Energy
Low activation energy: micelles still bound to complex = broad peak
Increase activation energy: micelle undergoes evaporation, can start to see protein dimer charge states
Highest activation energy: micelle completely evaporated, sharp signals observed; two lipid molecules remain bound; dimer still intact!
Study: ABC transporter dimer protein MacB
(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 Energy
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):
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 complexes
http://bowers.chem.ucsb.edu/theory_analysis/ion-mobility/index.shtml
Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Science 2005, 310, 1658–1661
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 phase
BtuC2D2 transporter protein Tetramer with 2 transmembrane & 2
soluble subunits More readily dissociates than
KirBac3.1 • KirBac3.1 better protected by
micelleWang SC, Politis A, Di Bartolo N, Bavro VN, Tucker SJ, et al. 2010. J. Am. Chem. Soc. 132:15468–70
180 V accel. voltage
240 V accel. voltage
Laser-Induced Liquid Bead Ion Desorption (LILBID)-MS
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 phase5) Ions accelerated and analyzed via TOF reflectron MS
N. Morgner, H.D. Barth, B. Brutschy, Austral. J. Chem. 59 (2006) 109–114.
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
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
Comparing “Micelle ES-MS” and LILBID-MS
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
Combining IM-MS with imaging techniques such as EM and AFM IM-MS is very powerful for studying protein
complex subunits Locate subunit interactions in EM density
maps/AFM images
Future Direction
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