Infrared Laser Activation of Soluble and Membrane Protein Assemblies in the Gas Phase.
Collision-induced dissociation (CID) is the dominant method for probing intact macromolecular complexes in the gas phase by means of mass spectrometry (MS). The energy obtained from collisional activation is dependent on the charge state of the ion and the pressures and potentials within the instrument: these factors limit CID capability. Activation by infrared (IR) laser radiation offers an attractive alternative as the radiation energy absorbed by the ions is charge-state-independent and the intensity and time scale of activation is controlled by a laser source external to the mass spectrometer. Here we implement and apply IR activation, in different irradiation regimes, to study both soluble and membrane protein assemblies. We show that IR activation using high-intensity pulsed lasers is faster than collisional and radiative cooling and requires much lower energy than continuous IR irradiation. We demonstrate that IR activation is an effective means for studying membrane protein assemblies, and liberate an intact V-type ATPase complex from detergent micelles, a result that cannot be achieved by means of CID using standard collision energies. Notably, we find that IR activation can be sufficiently soft to retain specific lipids bound to the complex. We further demonstrate that, by applying a combination of collisional activation, mass selection, and IR activation of the liberated complex, we can elucidate subunit stoichiometry and the masses of specifically bound lipids in a single MS experiment.
Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford , Oxford, OX1 3QZ, United Kingdom.
Victor A
VA
Mikhailov
Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford , Oxford, OX1 3QZ, United Kingdom.
Idlir
I
Liko
Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford , Oxford, OX1 3QZ, United Kingdom.
Todd H
TH
Mize
Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford , Oxford, OX1 3QZ, United Kingdom.
Matthew F
MF
Bush
Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford , Oxford, OX1 3QZ, United Kingdom.
Justin L P
JL
Benesch
Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford , Oxford, OX1 3QZ, United Kingdom.
Carol V
CV
Robinson
D041541:Acidianus / Q000201:enzymology; D001360:Avidin / Q000737:chemistry / Q000528:radiation effects; D018834:Chaperonin 60 / Q000737:chemistry / Q000528:radiation effects; D005740:Gases / Q000737:chemistry / Q000528:radiation effects*; D007259:Infrared Rays; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry / Q000528:radiation effects*; D008823:Micelles; D046912:Multiprotein Complexes / Q000737:chemistry / Q000528:radiation effects*; D010715:Phosphatidylglycerols / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000528:radiation effects; D016963:Thermus thermophilus / Q000201:enzymology; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000528:radiation effects
D018834:Chaperonin 60; D005740:Gases; D008565:Membrane Proteins; D008823:Micelles; D046912:Multiprotein Complexes; D010715:Phosphatidylglycerols; D021122:Protein Subunits; D001360:Avidin; D025262:Vacuolar Proton-Translocating ATPases