3 research outputs found
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<sup>3</sup>, 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 (<i>m</i>/<i>z</i>) 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
Development of a GC/Quadrupole-Orbitrap Mass Spectrometer, Part I: Design and Characterization
Identification of unknown compounds
is of critical importance in
GC/MS applications (metabolomics, environmental toxin identification,
sports doping, petroleomics, and biofuel analysis, among many others)
and remains a technological challenge. Derivation of elemental composition
is the first step to determining the identity of an unknown compound
by MS, for which high accuracy mass and isotopomer distribution measurements
are critical. Here, we report on the development of a dedicated, applications-grade
GC/MS employing an Orbitrap mass analyzer, the GC/Quadrupole-Orbitrap.
Built from the basis of the benchtop Orbitrap LC/MS, the GC/Quadrupole-Orbitrap
maintains the performance characteristics of the Orbitrap, enables
quadrupole-based isolation for sensitive analyte detection, and includes
numerous analysis modalities to facilitate structural elucidation.
We detail the design and construction of the instrument, discuss its
key figures-of-merit, and demonstrate its performance for the characterization
of unknown compounds and environmental toxins
Novel Parallelized Quadrupole/Linear Ion Trap/Orbitrap Tribrid Mass Spectrometer Improving Proteome Coverage and Peptide Identification Rates
Proteome coverage and peptide identification
rates have historically
advanced in line with improvements to the detection limits and acquisition
rate of the mass spectrometer. For a linear ion trap/Orbitrap hybrid,
the acquisition rate has been limited primarily by the duration of
the ion accumulation and analysis steps. It is shown here that the
spectral acquisition rate can be significantly improved through extensive
parallelization of the acquisition process using a novel mass spectrometer
incorporating quadrupole, Orbitrap, and linear trap analyzers. Further,
these improvements to the acquisition rate continue to enhance proteome
coverage and general experimental throughput