Proteoforms, the different forms of a protein with sequence variations including post-translational modifications (PTMs), execute vital functions in biological systems, such as cell signaling and epigenetic regulation. Advances in top-down mass spectrometry (MS) technology have permitted the direct characterization of intact proteoforms and their exact number of modification sites, allowing for the relative quantification of positional isomers (PI). Protein positional isomers refer to a set of proteoforms with identical total mass and set of modifications, but varying PTM site combinations. The relative abundance of PI can be estimated by matching proteoform-specific fragment ions to top-down tandem MS (MS2) data to localize and quantify modifications. However, the current approaches heavily rely on manual annotation. Here, we present IsoForma, an open-source R package for the relative quantification of PI within a single tool. Benchmarking IsoForma's performance against two existing workflows produced comparable results and improvements in speed. Overall, IsoForma provides a streamlined process for quantifying PI, reduces the analysis time, and offers an essential framework for developing customized proteoform analysis workflows. The software is open source and available at https://github.com/EMSL-Computing/isoforma-lib.
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Separation, identification, and confirmation of cyclic and tadpole macromolecules via UPLC-MS/MS
Macrocyclic poly(glycidyl phenyl ether) (pGPE) synthesized via zwitterionic ring opening polymerization is typically contaminated by chains with linear and tadpole architecture. Although mass spectrometry (MS) analysis can readily confirm the presence of the linear byproduct, due to its unique mass, it is unable to differentiate between the cyclic and tadpole structures, which are constitutional isomers produced by backbiting reactions in monomeric or dimeric chains, respectively. To overcome this problem, ultraperformance reversed-phase liquid chromatography interfaced with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) was employed. The separation achieved by UPLC revealed that the tadpole isomer elutes before the cyclic structure because of the increased polarity afforded by its distinctive substituents. The ratio of tadpole to cyclic species increased with the degree of polymerization, in agreement with the synthetic method used, as the potential for forming tadpole structures by backbiting is entropically favored in longer polymer chains. Once separated, the two isomers could be independently characterized by tandem mass spectrometry. The macrocyclic and tadpole species exhibit unique fragmentation patterns, including structurally diagnostic fragments for each structure.
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- Award ID(s):
- 1808115
- NSF-PAR ID:
- 10335027
- Date Published:
- Journal Name:
- The Analyst
- Volume:
- 147
- Issue:
- 10
- ISSN:
- 0003-2654
- Page Range / eLocation ID:
- 2089 to 2096
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Introduction Condensed tannins (CTs) are proanthocyanidin heteropolymers that are widely distributed among plants. Their biochemical properties are determined by molecular structure (e.g. polymer size, hydroxylation, stereochemistry). In
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Populus tremuloides Methodology CTs were extracted from dried foliage of six
P. tremuloides P. tremuloides Results Populus tremuloides Conclusion This thiolysis‐UPLC‐PDA‐(−)esiMS method was optimised for analysis of CT polymer size, hydroxylation, stereochemistry, and total concentration in
Populus foliage. It revealed significant variation in each of these properties amongP. tremuloides
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/d2an00208f
