Isoelectric point region pI≈7.4 as a treasure island of abnormal proteoforms in blood

Theoretical distribution of isoelectric points (pI values) of human blood proteins exhibits multi-modality with a deep minimum in the range between pI 7.30 and 7.50. Considering that the pH of human blood is 7.4±0.1, normal forms of human proteins tend to eschew this specific pI region, thus avoiding charge neutrality that can result in enhanced precipitation. However, abnormal protein isoforms (proteoforms), which are the hallmarks and potential biomarkers of certain diseases, are likely to be found everywhere in the pI distribution, including this “forbidden” region. Therefore, we hypothesized that damaging proteoforms characteristic for neurodegenerative diseases are best detected around pI≈7.4. Blood serum samples from 14 Alzheimer's disease patients were isolated by capillary isoelectric focusing and analyzed by liquid chromatography hyphenated with tandem mass spectrometry. Consistent with the pI≈7.4 hypothesis, the 8 patients with fast memory decline had a significantly (p<0.003) higher concentration of proteoforms in the pI=7.4±0.1 region than the 6 patients with a slow memory decline. Moreover, protein compositions differed more from each other than for any other investigated pI region, providing absolute separation of the fast and slow decliner samples. The discovery of the “treasure island” of abnormal proteoforms in form of the pI≈7.4 region promises to boost biomarker development for a range of diseases.