Human plasma contains far more forms of proteins than standard protein measurements may capture, according to a bioRxiv preprint describing a draft atlas of the plasma proteome.
Many proteins circulate in several versions, known as proteoforms. These can arise through processes such as alternative splicing, protein cleavage and chemical modifications after a protein is produced. Different proteoforms may have different biological functions, but most proteomic tests report a single value for each protein.
Researchers developed an analytical workflow to map this protein diversity. The approach combined established protein-separation methods with data-independent acquisition mass spectrometry and computational analysis.
The researchers from the Institute of Biochemistry, Charité - University Medical Center Berlin, Germany, first reduced the concentration of 14 highly abundant plasma proteins, which can obscure less abundant molecules. They then separated intact proteins by their size and electrical charge using denaturing size-exclusion chromatography, isoelectric focusing, and two-dimensional gel electrophoresis.
This process produced 3,114 protein fractions. The proteins were digested into peptides and analyzed by liquid chromatography-mass spectrometry using data-independent parallel accumulation-serial fragmentation, or diaPASEF. Software then reconstructed the positions of the peptide signals across the separated fractions to identify different forms of each protein.
Across the complete dataset, the researchers detected 179,049 potential proteoforms representing 5,077 protein groups. They classified 44,083 proteoforms from 2,089 protein groups as high confidence because they were found in at least three of four replicate gel experiments. The median was 11 high-confidence proteoforms per protein.
The greatest diversity was observed among abundant proteins and those involved in nutrient transport, blood clotting and the complement immune system. The most abundant form of a protein accounted for less than half of its total measured peptide signal, suggesting that less abundant forms make up a substantial proportion of the plasma proteome.
The researchers also examined previously reported differences between the Olink and SomaScan affinity-based proteomic platforms. Measurements generally agreed more closely for relatively abundant proteins with low or moderate proteoform diversity. Poor agreement was most often associated with proteins present at low concentrations, although high proteoform diversity may explain some discrepancies involving more abundant proteins.
The findings are relevant to biomarker research because two assays designed to measure the same protein may recognize different proteoforms. This could affect comparisons between technologies and the interpretation of results, particularly when individual forms have different biological functions or associations with disease.
The atlas is an initial reference rather than a complete account of the plasma proteome. Most proteoforms were separated according to their physical properties but were not fully identified at the molecular level. Low-abundance and disease-associated forms may be underrepresented, and some findings may represent groups of closely related proteoforms rather than individual molecules. The analysis may also include proteins introduced through cellular contamination.
Further studies using top-down mass spectrometry, clinical cohorts, and samples from different populations will be needed to determine which proteoforms are associated with normal biology and which may provide disease-specific information.
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