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