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

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