TopFD: A Proteoform Feature Detection Tool for Top–Down Proteomics

Basharat, Abdul Rehman; Zang, Yong; Sun, Liangliang; Liu, Xiaowen

doi:10.1021/acs.analchem.2c05244

Cited by 16 publications

(12 citation statements)

References 36 publications

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“…In the first step, RAW files were converted into mzML files using the Msconvert tool . The spectral deconvolution which converted precursor and fragment isotope clusters into the monoisotopic masses and proteoform features were then performed using TopFD (Top-down mass spectrometry Feature Detection, version 1.5.6) . The resulting mass spectra and proteoform feature information were stored in msalign and text files, respectively.…”

Section: Discussionmentioning

confidence: 99%

Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis–Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample

Sadeghi,

Chen,

Wang

et al. 2024

J. Proteome Res.

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Mass spectrometry (MS)-based top-down proteomics (TDP) has revolutionized biological research by measuring intact proteoforms in cells, tissues, and biofluids. Capillary zone electrophoresis−tandem MS (CZE-MS/MS) is a valuable technique for TDP, offering a high peak capacity and sensitivity for proteoform separation and detection. However, the long-term reproducibility of CZE-MS/MS in TDP remains unstudied, which is a crucial aspect for large-scale studies. This work investigated the long-term qualitative and quantitative reproducibility of CZE-MS/MS for TDP for the first time, focusing on a yeast cell lysate. Over 1000 proteoforms were identified per run across 62 runs using one linear polyacrylamide (LPA)-coated separation capillary, highlighting the robustness of the CZE-MS/MS technique. However, substantial decreases in proteoform intensity and identification were observed after some initial runs due to proteoform adsorption onto the capillary inner wall. To address this issue, we developed an efficient capillary cleanup procedure using diluted ammonium hydroxide, achieving high qualitative and quantitative reproducibility for the yeast sample across at least 23 runs. The data underscore the capability of CZE-MS/MS for large-scale quantitative TDP of complex samples, signaling its readiness for deployment in broad biological applications. The MS RAW files were deposited in ProteomeXchange Consortium with the data set identifier of PXD046651.

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Section: Discussionmentioning

confidence: 99%

Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis–Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample

Sadeghi,

Chen,

Wang

et al. 2024

J. Proteome Res.

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“…Proteoform features were extracted from the LC-MS map of each run using a modified version of TopFD 47 (Supplementary Table S1). A proteoform feature may have isotopic envelopes with different charge states in different scans.…”

Section: Methodsmentioning

confidence: 99%

“…TopFD 47 was used to analyze the E. coli TD-DDA-MS data for proteoform feature detection and spectral deconvolution (Supplementary Table S3), and the pseudo spectrum generation method was employed to produce pseudo MS/MS spectra from the E. coli TD-DIA-MS data. The deconvoluted MS/MS spectra or pseudo MS/MS spectra were searched against the UniProt E. coli proteome sequence database (4530 entries, version September 06, 2023) using TopPIC 49 (version 1.7.2).…”

Section: Methodsmentioning

confidence: 99%

TopDIA: A Software Tool for Top-Down Data-Independent Acquisition Proteomics

Basharat,

Xiong,

et al. 2024

Preprint

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Top-down mass spectrometry is widely used for proteoform identification, characterization, and quantification owing to its ability to analyze intact proteoforms. In the last decade, top-down proteomics has been dominated by top-down data-dependent acquisition mass spectrometry (TD-DDA-MS), and top-down data-independent acquisition mass spectrometry (TD-DIA-MS) has not been well studied. While TD-DIA-MS produces complex multiplexed tandem mass spectrometry (MS/MS) spectra, which are challenging to confidently identify, it selects more precursor ions for MS/MS analysis and has the potential to increase proteoform identifications compared with TD-DDA-MS. Here we present TopDIA, the first software tool for proteoform identification by TD-DIA-MS. It generates demultiplexed pseudo MS/MS spectra from TD-DIA-MS data and then searches the pseudo MS/MS spectra against a protein sequence database for proteoform identification. We compared the performance of TD-DDA-MS and TD-DIA-MS usingEscherichia coliK-12 MG1655 cells and demonstrated that TD-DIA-MS with TopDIA increased proteoform and protein identifications compared with TD-DDA-MS.

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“…All raw files were converted to centroided mzML files using msconvert in ProteoWizard . In the TopPIC pipeline, TopFD (version 1.6.0) was used to deconvolute the centroided top-down mass spectra to neutral monoisotopic masses of precursor and fragment ions. The deconvoluted MS/MS spectra were searched against the GENCODE-SWISS database concatenated with a decoy database with the same size using TopPIC (version 1.6.0) .…”

Section: Methodsmentioning

confidence: 99%

Characterization of Proteoform Post-Translational Modifications by Top-Down and Bottom-Up Mass Spectrometry in Conjunction with Annotations

Chen,

Ding,

Zang

et al. 2023

J. Proteome Res.

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Many proteoforms can be produced from a gene due to genetic mutations, alternative splicing, post-translational modifications (PTMs), and other variations. PTMs in proteoforms play critical roles in cell signaling, protein degradation, and other biological processes. Mass spectrometry (MS) is the primary technique for investigating PTMs in proteoforms, and two alternative MS approaches, top-down and bottom-up, have complementary strengths. The combination of the two approaches has the potential to increase the sensitivity and accuracy in PTM identification and characterization. In addition, protein and PTM knowledge bases, such as UniProt, provide valuable information for PTM characterization and verification. Here, we present a software pipeline PTM-TBA (PTM characterization by Top-down and Bottom-up MS and Annotations) for identifying and localizing PTMs in proteoforms by integrating top-down and bottom-up MS as well as PTM annotations. We assessed PTM-TBA using a technical triplicate of bottom-up and top-down MS data of SW480 cells. On average, database search of the top-down MS data identified 2000 mass shifts, 814.5 (40.7%) of which were matched to 11 common PTMs and 423 of which were localized. Of the mass shifts identified by topdown MS, PTM-TBA verified 435 mass shifts using the bottom-up MS data and UniProt annotations.

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TopFD: A Proteoform Feature Detection Tool for Top–Down Proteomics

Cited by 16 publications

References 36 publications

Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis–Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample

Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis–Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample

TopDIA: A Software Tool for Top-Down Data-Independent Acquisition Proteomics

Characterization of Proteoform Post-Translational Modifications by Top-Down and Bottom-Up Mass Spectrometry in Conjunction with Annotations

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