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Advances in the Mass Spectrometry of Membrane Proteins: From Individual Proteins to Intact Complexes Nelson P. Barrera and Carol V. Robinson Annu. Rev.

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Presentation on theme: "Advances in the Mass Spectrometry of Membrane Proteins: From Individual Proteins to Intact Complexes Nelson P. Barrera and Carol V. Robinson Annu. Rev."— Presentation transcript:

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 : Bi/Ch 132 Adam Boynton Fall 2012

2 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 Micelles protect membrane complexes from solution to vacuum. Science 321:243–46)

3 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 Promising Development: Using ES- MS with Micelles A86E51EC6D

4 Study: ATP-binding cassette (ABC) transporter BtuC 2 D 2 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 Science 321:243–46)

5 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 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 :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): Experimental ATD calibrated against ATDs of ions of known structure Can determine collision cross section (CCS) for a given ion Compare CCSs to elucidate 3D structures of protein complexes mobility/index.shtml Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Science 2005, 310, 1658–1661

8 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 BtuC 2 D 2 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 J. Am. Chem. Soc. 132:15468– V accel. voltage 240 V accel. voltage

9 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 phase 5)Ions accelerated and analyzed via TOF reflectron MS N. Morgner, H.D. Barth, B. Brutschy, Austral. J. Chem. 59 (2006) 109–114.

10 LILBID-MS: Study of P. furiosus ATP synthase Low laser intensity: ions gently desorbed - detect intact complexes - subunit stoichiometry: A 3 B 3 CDE 2 FH 2 ac 10 High laser intensity: non-covalent interactions broken - detect complex subunits Vonck J, Pisa KY, Morgner N, Brutschy B, Muller V J. Biol. Chem. 284:10110–19

11 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 :247-71

12 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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