3 research outputs found
Top-Down Characterization of Proteins with Intact Disulfide Bonds Using Activated-Ion Electron Transfer Dissociation
Here we report the
fragmentation of disulfide linked intact proteins
using activated-ion electron transfer dissociation (AI-ETD) for top-down
protein characterization. This fragmentation method is then compared
to the alternative methods of beam-type collisional activation (HCD),
electron transfer dissociation (ETD), and electron transfer and higher-energy
collision dissociation (EThcD). We analyzed multiple precursor charge
states of the protein standards bovine insulin, α-lactalbumin,
lysozyme, β-lactoglobulin, and trypsin inhibitor. In all cases,
we found that AI-ETD provides a boost in protein sequence coverage
information and the generation of fragment ions from within regions
enclosed by disulfide bonds. AI-ETD shows the largest improvement
over the other techniques when analyzing highly disulfide linked and
low charge density precursor ions. This substantial improvement is
attributed to the concurrent irradiation of the gas phase ions while
the electron-transfer reaction is taking place, mitigating nondissociative
electron transfer, helping unfold the gas phase protein during the
electron transfer event, and preventing disulfide bond reformation.
We also show that AI-ETD is able to yield comparable sequence coverage
information when disulfide bonds are left intact relative to proteins
that have been reduced and alkylated. This work demonstrates that
AI-ETD is an effective fragmentation method for the analysis of proteins
with intact disulfide bonds, dramatically enhancing sequence ion generation
and total sequence coverage compared to HCD and ETD
Improved Precursor Characterization for Data-Dependent Mass Spectrometry
Modern
ion trap mass spectrometers are capable of collecting up
to 60 tandem MS (MS/MS) scans per second, in theory providing acquisition
speeds that can sample every eluting peptide precursor presented to
the MS system. In practice, however, the precursor sampling capacity
enabled by these ultrafast acquisition rates is often underutilized
due to a host of reasons (e.g., long injection times and wide analyzer
mass ranges). One often overlooked reason for this underutilization
is that the instrument exhausts all the peptide features it identifies
as suitable for MS/MS fragmentation. Highly abundant features can
prevent annotation of lower abundance precursor ions that occupy similar
mass-to-charge (<i>m</i>/<i>z</i>) space, which
ultimately inhibits the acquisition of an MS/MS event. Here, we present
an advanced peak determination (APD) algorithm that uses an iterative
approach to annotate densely populated <i>m</i>/<i>z</i> regions to increase the number of peptides sampled during
data-dependent LC-MS/MS analyses. The APD algorithm enables nearly
full utilization of the sampling capacity of a quadrupole-Orbitrap-linear
ion trap MS system, which yields up to a 40% increase in unique peptide
identifications from whole cell HeLa lysates (approximately 53 000
in a 90 min LC-MS/MS analysis). The APD algorithm maintains improved
peptide and protein identifications across several modes of proteomic
data acquisition, including varying gradient lengths, different degrees
of prefractionation, peptides derived from multiple proteases, and
phosphoproteomic analyses. Additionally, the use of APD increases
the number of peptides characterized per protein, providing improved
protein quantification. In all, the APD algorithm increases the number
of detectable peptide features, which maximizes utilization of the
high MS/MS capacities and significantly improves sampling depth and
identifications in proteomic experiments
Coupling Capillary Zone Electrophoresis with Electron Transfer Dissociation and Activated Ion Electron Transfer Dissociation for Top-Down Proteomics
Top-down proteomics offers the potential
for full protein characterization,
but many challenges remain for this approach, including efficient
protein separations and effective fragmentation of intact proteins.
Capillary zone electrophoresis (CZE) has shown great potential for
separation of intact proteins, especially for differentially modified
proteoforms of the same gene product. To date, however, CZE has been
used only with collision-based fragmentation methods. Here we report
the first implementation of electron transfer dissociation (ETD) with
online CZE separations for top-down proteomics, analyzing a mixture
of four standard proteins and a complex protein mixture from the Mycobacterium marinum bacterial secretome. Using
a multipurpose dissociation cell on an Orbitrap Elite system, we demonstrate
that CZE is fully compatible with ETD as well as higher energy collisional
dissociation (HCD), and that the two complementary fragmentation methods
can be used in tandem on the electrophoretic time scale for improved
protein characterization. Furthermore, we show that activated ion
electron transfer dissociation (AI-ETD), a recently introduced method
for enhanced ETD fragmentation, provides useful performance with CZE
separations to greatly increase protein characterization. When combined
with HCD, AI-ETD improved the protein sequence coverage by more than
200% for proteins from both standard and complex mixtures, highlighting
the benefits electron-driven dissociation methods can add to CZE separations