Comparing Top-Down Proteoform Identification: Deconvolution, PrSM Overlap, and PTM Detection

Generating top-down tandem mass spectra (MS/MS) from complex mixtures of proteoforms benefits from improvements in fractionation, separation, fragmentation, and mass analysis. The algorithms to match MS/MS to sequences have undergone a parallel evolution, with both spectral alignment and match-count...

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Vydáno v:Journal of proteome research Ročník 22; číslo 7; s. 2199
Hlavní autoři: Tabb, David L, Jeong, Kyowon, Druart, Karen, Gant, Megan S, Brown, Kyle A, Nicora, Carrie, Zhou, Mowei, Couvillion, Sneha, Nakayasu, Ernesto, Williams, Janet E, Peterson, Haley K, McGuire, Michelle K, McGuire, Mark A, Metz, Thomas O, Chamot-Rooke, Julia
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States 07.07.2023
Témata:
Humans
Protein Processing, Post-Translational
Proteome - genetics
Proteomics
Software
Tandem Mass Spectrometry
identification algorithms
deconvolution
proteoforms
post-translational modifications
top-down proteomics
bioinformatics
ISSN:1535-3907, 1535-3907
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Popis
Shrnutí:Generating top-down tandem mass spectra (MS/MS) from complex mixtures of proteoforms benefits from improvements in fractionation, separation, fragmentation, and mass analysis. The algorithms to match MS/MS to sequences have undergone a parallel evolution, with both spectral alignment and match-counting approaches producing high-quality proteoform-spectrum matches (PrSMs). This study assesses state-of-the-art algorithms for top-down identification (ProSight PD, TopPIC, MSPathFinderT, and pTop) in their yield of PrSMs while controlling false discovery rate. We evaluated deconvolution engines (ThermoFisher Xtract, Bruker AutoMSn, Matrix Science Mascot Distiller, TopFD, and FLASHDeconv) in both ThermoFisher Orbitrap-class and Bruker maXis Q-TOF data (PXD033208) to produce consistent precursor charges and mass determinations. Finally, we sought post-translational modifications (PTMs) in proteoforms from bovine milk (PXD031744) and human ovarian tissue. Contemporary identification workflows produce excellent PrSM yields, although approximately half of all identified proteoforms from these four pipelines were specific to only one workflow. Deconvolution algorithms disagree on precursor masses and charges, contributing to identification variability. Detection of PTMs is inconsistent among algorithms. In bovine milk, 18% of PrSMs produced by pTop and TopMG were singly phosphorylated, but this percentage fell to 1% for one algorithm. Applying multiple search engines produces more comprehensive assessments of experiments. Top-down algorithms would benefit from greater interoperability.
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ISSN:1535-3907
1535-3907
DOI:10.1021/acs.jproteome.2c00673