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