7 research outputs found
Enhanced Global Post-translational Modification Discovery with MetaMorpheus
Correct identification of protein
post-translational modifications
(PTMs) is crucial to understanding many aspects of protein function
in biological processes. G-PTM-D is a recently developed technique
for global identification and localization of PTMs. Spectral file
calibration prior to applying G-PTM-D, and algorithmic enhancements
in the peptide database search significantly increase the accuracy,
speed, and scope of PTM identification. We enhance G-PTM-D by using
multinotch searches and demonstrate its effectiveness in identification
of numerous types of PTMs including high-mass modifications such as
glycosylations. The changes described in this work lead to a 20% increase
in the number of identified modifications and an order of magnitude
decrease in search time. The complete workflow is implemented in MetaMorpheus,
a software tool that integrates the database search procedure, identification
of coisolated peptides, spectral calibration, and the enhanced G-PTM-D
workflow. Multinotch searches are also shown to be useful in contexts
other than G-PTM-D by producing superior results when used instead
of standard narrow-window and open database searches
Ultrafast Peptide Label-Free Quantification with FlashLFQ
The
rapid and accurate quantification of peptides is a critical
element of modern proteomics that has become increasingly challenging
as proteomic data sets grow in size and complexity. We present here
FlashLFQ, a computer program for high-speed label-free quantification
of peptides following a search of bottom-up mass spectrometry data.
FlashLFQ is approximately an order of magnitude faster than established
label-free quantification methods. The increased speed makes it practical
to base quantification upon all of the charge states for a given peptide
rather than solely upon the charge state that was selected for MS2
fragmentation. This increases the number of quantified peptides, improves
replicate-to-replicate reproducibility, and increases quantitative
accuracy. We integrated FlashLFQ into the graphical user interface
of the MetaMorpheus search software, allowing it to work together
with the global post-translational modification discovery (G-PTM-D)
engine to accurately quantify modified peptides. FlashLFQ is also
available as a NuGet package, facilitating its integration into other
software, and as a standalone command line software program for the
quantification of search results from other programs (e.g., MaxQuant)
Enhanced Global Post-translational Modification Discovery with MetaMorpheus
Correct identification of protein
post-translational modifications
(PTMs) is crucial to understanding many aspects of protein function
in biological processes. G-PTM-D is a recently developed technique
for global identification and localization of PTMs. Spectral file
calibration prior to applying G-PTM-D, and algorithmic enhancements
in the peptide database search significantly increase the accuracy,
speed, and scope of PTM identification. We enhance G-PTM-D by using
multinotch searches and demonstrate its effectiveness in identification
of numerous types of PTMs including high-mass modifications such as
glycosylations. The changes described in this work lead to a 20% increase
in the number of identified modifications and an order of magnitude
decrease in search time. The complete workflow is implemented in MetaMorpheus,
a software tool that integrates the database search procedure, identification
of coisolated peptides, spectral calibration, and the enhanced G-PTM-D
workflow. Multinotch searches are also shown to be useful in contexts
other than G-PTM-D by producing superior results when used instead
of standard narrow-window and open database searches
Expanding Proteoform Identifications in Top-Down Proteomic Analyses by Constructing Proteoform Families
In top-down proteomics,
intact proteins are analyzed by tandem
mass spectrometry and proteoforms, which are defined forms of a protein
with specific sequences of amino acids and localized post-translational
modifications, are identified using precursor mass and fragmentation
data. Many proteoforms that are detected in the precursor scan (MS1)
are not selected for fragmentation by the instrument and therefore
remain unidentified in typical top-down proteomic workflows. Our laboratory
has developed the open source software program Proteoform Suite to
analyze MS1-only intact proteoform data. Here, we have adapted it
to provide identifications of proteoform masses in precursor MS1 spectra
of top-down data, supplementing the top-down identifications obtained
using the MS2 fragmentation data. Proteoform Suite performs mass calibration
using high-scoring top-down identifications and identifies additional
proteoforms using calibrated, accurate intact masses. Proteoform families,
the set of proteoforms from a given gene, are constructed and visualized
from proteoforms identified by both top-down and intact-mass analyses.
Using this strategy, we constructed proteoform families and identified
1861 proteoforms in yeast lysate, yielding an approximately 40% increase
over the original 1291 proteoform identifications observed using traditional
top-down analysis alone
Proteoform Suite: Software for Constructing, Quantifying, and Visualizing Proteoform Families
We present an open-source, interactive
program named Proteoform
Suite that uses proteoform mass and intensity measurements from complex
biological samples to identify and quantify proteoforms. It constructs
families of proteoforms derived from the same gene, assesses proteoform
function using gene ontology (GO) analysis, and enables visualization
of quantified proteoform families and their changes. It is applied
here to reveal systemic proteoform variations in the yeast response
to salt stress
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ProForma: A Standard Proteoform Notation
The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard