From protein complexes to subunit backbone fragments: a multi-stage approach to native mass spectrometry.
Native mass spectrometry (MS) is becoming an important integral part of structural proteomics and system biology research. The approach holds great promise for elucidating higher levels of protein structure: from primary to quaternary. This requires the most efficient use of tandem MS, which is the cornerstone of MS-based approaches. In this work, we advance a two-step fragmentation approach, or (pseudo)-MS(3), from native protein complexes to a set of constituent fragment ions. Using an efficient desolvation approach and quadrupole selection in the extended mass-to-charge (m/z) range, we have accomplished sequential dissociation of large protein complexes, such as phosporylase B (194 kDa), pyruvate kinase (232 kDa), and GroEL (801 kDa), to highly charged monomers which were then dissociated to a set of multiply charged fragmentation products. Fragment ion signals were acquired with a high resolution, high mass accuracy Orbitrap instrument that enabled highly confident identifications of the precursor monomer subunits. The developed approach is expected to enable characterization of stoichiometry and composition of endogenous native protein complexes at an unprecedented level of detail.
Thermo Fisher Scientific , 28199 Bremen, Germany.
Mikhail E
ME
Belov
Eugen
E
Damoc
Eduard
E
Denisov
Philip D
PD
Compton
Stevan
S
Horning
Alexander A
AA
Makarov
Neil L
NL
Kelleher
D013058:Mass Spectrometry / Q000379:methods* / Q000639:trends; D010763:Phosphorylase b / Q000032:analysis / Q000737:chemistry; D021122:Protein Subunits / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods / Q000639:trends; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000639:trends; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000639:trends
D021122:Protein Subunits; D010763:Phosphorylase b