6 research outputs found
Active Site Modifications of the Brain Isoform of Creatine Kinase by 4-Hydroxy-2-nonenal Correlate with Reduced Enzyme Activity: Mapping of Modified Sites by Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry
High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function
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
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