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. 2023 Oct 6;22(10):3178-3189.
doi: 10.1021/acs.jproteome.3c00207. Epub 2023 Sep 20.

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

Wenrong Chen  1 , Zhengming Ding  2 , Yong Zang  3   4 , Xiaowen Liu  5   6
Affiliations

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

Wenrong Chen et al. J Proteome Res. .

Abstract

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 top-down MS, PTM-TBA verified 435 mass shifts using the bottom-up MS data and UniProt annotations.

Keywords: bottom-up mass spectrometry; post-translational modification; top-down mass spectrometry.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Overall scheme of the PTM-TBA pipeline for PTM identification, localization, and verification using top-down MS, bottom-up MS, and UniProt annotations. (a) Top-down MS data are first used for identifying mass shifts/PTMs in proteoforms, and then mass shifts/PTMs identified from bottom-up MS data and PTM annotations are employed to verify the mass shifts/PTMs identified from top-down MS data. MSFragger, MetaMorpheus, and MaxQuant are three options for peptide identification by bottom-up MS. TopPIC and MSPathFinder are two tools in the pipeline for proteoform identification by top-down MS. PTM annotations in UniProt and dbPTM are used for verifying PTM identifications. (b) The numbers of mass shift/PTM identifications are shown for the PTM-TBA analysis of the first replicate of the SW480 top-down and bottom-up MS data using MSFragger, TopPIC, and UniProt annotations. Mass shifts identified from bottom-up and top-down MS data are divided into three levels: level 1: mass shifts with identified PTM types and localized sites; level 2A: mass shifts with identified PTM types but without localized sites; level 3: mass shifts without PTM identification and localized sites.
Figure 2
Figure 2
Illustration of duplication removal of mass shifts in proteoforms. (a) Proteoform identifications A and B are from the same protein. Proteoform A contains the whole protein sequence with two mass shifts (green and orange), and proteoform B is a truncated one with one mass shift (yellow). The colored parts show protein regions containing possible modification sites of the mass shift. (b) Three single mass shifts are extracted from the two proteoform identifications. (c) The orange and yellow mass shifts have similar shifts and their possible modification site regions overlap, so they are treated as duplicated ones and only one is kept.
Figure 3
Figure 3
Histogram of mass shifts reported by MSFragger (round 1) from the first replicate of the SW480 bottom-up MS data in the range of [0, 200] Da.
Figure 4
Figure 4
Histogram of mass shifts reported by TopPIC from the first replicate of the SW480 top-down MS data in the range of [0, 200] Da.
Figure 5
Figure 5
Comparison of mass shift identifications across the three replicates of SW480 top-down and bottom-up MS data. (a) NTA, (b) level 1 excluding NTA, (c) level 2A, and (d) level 3 mass shifts identified from the top-down MS data. (e) NTA, (f) level 1 excluding NTA, (g) level 2A, and (h) level 3 mass shifts identified from the top-down MS data and verified by the bottom-up MS data.
Figure 6
Figure 6
Comparison of PTMs identified by TopPIC and MSPathFinder from the first replicate of the SW480 top-down MS data.
Figure 7
Figure 7
Comparison of mass shifts identified from the first replicate of the SW480 top-down MS data and verified by the first replicate of the SW480 bottom-up MS data and UniProt annotations. (a) NTA, (b) level 1 excluding NTA, (c) level 2A, (d) level 3.
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