advances in the mass spectrometry of membrane proteins from individual proteins to intact complexes n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Nelson P. Barrera and Carol V. Robinson Annu . Rev. Biochem . 2011. 80:247-71 Bi/ Ch 132 PowerPoint Presentation
Download Presentation
Nelson P. Barrera and Carol V. Robinson Annu . Rev. Biochem . 2011. 80:247-71 Bi/ Ch 132

Loading in 2 Seconds...

play fullscreen
1 / 12

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


  • 106 Views
  • Uploaded on

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.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

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


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
advances in the mass spectrometry of membrane proteins from individual proteins to intact complexes

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
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)

promising development using es ms with micelles
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
using es ms with micelles
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)

es with micelles role of activation energy
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)

es in micelles role of activation energy
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
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

im ms studying 3d structure of protein complexes in the gas phase
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

laser induced liquid bead ion desorption lilbid ms
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

lilbid ms study of p furiosus atp synthase
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

comparing micelle es ms and lilbid ms
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

future direction
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