TY - JOUR
T1 - From protein complexes to subunit backbone fragments
T2 - A multi-stage approach to native mass spectrometry
AU - Belov, Mikhail E.
AU - Damoc, Eugen
AU - Denisov, Eduard
AU - Compton, Philip D.
AU - Horning, Stevan
AU - Makarov, Alexander A.
AU - Kelleher, Neil L.
PY - 2013/12/3
Y1 - 2013/12/3
N2 - 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)-MS3, 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.
AB - 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)-MS3, 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.
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U2 - 10.1021/ac4029328
DO - 10.1021/ac4029328
M3 - Article
C2 - 24237199
AN - SCOPUS:84889048480
VL - 85
SP - 11163
EP - 11173
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 23
ER -