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Nelson P. Barrera and Carol V. Robinson Annu . Rev. Biochem . 2011. 80:247-71 Bi/ Ch 132

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.

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Nelson P. Barrera and Carol V. Robinson Annu . Rev. Biochem . 2011. 80:247-71 Bi/ Ch 132

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  1. 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

  2. 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 proteincomplexeshas 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)

  3. Promising Development: Using ES-MS with Micelles http://www.piercenet.com/browse.cfm?fldID=9AB987DA-C4D4-4713-8312-08A86E51EC6D • 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 complex

  4. Using ES-MS with Micelles • Study: ATP-binding cassette (ABC) transporter BtuC2D2 • TwotransmembraneBtuC subunits • Twosoluble 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 (Barrera NP, Di Bartolo N, Booth PJ, Robinson CV. 2008. Science 321:243–46)

  5. ES with Micelles: Role of Activation Energy • 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)

  6. 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)

  7. 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 complexes http://bowers.chem.ucsb.edu/theory_analysis/ion-mobility/index.shtml

  8. IM–MS: Studying 3D Structure of Protein Complexes in the Gas Phase • KirBac3.1 potassium ion channel • Homotetramer with 4 transmembranesubunits • CCS suggests compact structure • Native quaternary structure maintained in gas phase 240 V accel. voltage 180 V accel. voltage • BtuC2D2 transporter protein • Tetramer with 2 transmembrane & 2 soluble subunits • 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

  9. Laser-Induced Liquid Bead Ion Desorption (LILBID)-MS N. Morgner, H.D. Barth, B. Brutschy, Austral. J. Chem. 59 (2006) 109–114. 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) Introduced into vacuum and irradiated one by one with nanosecond mid-IR pulses (pulse energies of 1-15 mJ) Pulses tuned to 3 μm wavelength (water absorption maximum) Liquid reaches “supercritical state”, droplets explode, release charged biomolecules into gas phase Ions accelerated and analyzed via TOF reflectron MS

  10. LILBID-MS: Study of P. furiosusATP 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

  11. Comparing “Micelle ES-MS” and LILBID-MS • Study of EmrE dimer • * = +N-formyl Met PTM • + = unmodified wild type • Three dimers formed • (++, +*,**) • 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 Nelson P. Barrera and Carol V. Robinson. Annu. Rev. Biochem. 2011. 80:247-71

  12. Future Direction • 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

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