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. 2022 Mar 17;29:19. doi: 10.1186/s12929-022-00802-5

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Mass-spectrometry based approaches to isolate microproteins. Sample preparation prior to LC–MS/MS analysis to isolate microprotein species < 30 kDa in size includes size exclusion approaches. Molecular weight cut off filters (MWCOs) can sieve for microproteins depending on the type of filter used i.e., 10 kDa or 30 kDa. Acid precipitation is a common enrichment step for to precipitate larger proteins. Solid phase extraction (SPE) enrichment occurs via reverse-phase C8 cartridges and elutes microproteins of interest. Further methods in reducing sample complexities include electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and high-resolution isoelectric focusing (Hi-RIEF). ERLIC separates based on charged analytes and utilises SAX resin for strong anion exchange, whereas Hi-RIEF seperates peptides based on their isoelectric points (pI) on a pH gradient gel. Post-fractionation accuracy is dependent on high sequence coverage and low background noise in mass spectra. This can be achieved with using High-energy Collision Induced Dissociation (HCD) on Fusion Tribrid MS or Q-Exactive MS