Abstract
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.
Highlights
Abnormal forms of human proteins[1] are often associated with human disease, such as Alzheimer's disease (AD), Amyotrophic lateral sclerosis, prion disease, Creutzfeldt–Jakob disease, Parkinson's disease (PD), amyloidosis, and a wide range of other disorders
Analysis of all liquid chromatography (LC)-mass spectrometry (MS)/MS datasets resulted in quantification of 650 protein groups that passed 1% false discovery rate (FDR) threshold at both peptide and protein levels
Top ten proteins with significant abundance changes (p
Summary
Abnormal forms of human proteins (abnormal proteoforms)[1] are often associated with human disease, such as Alzheimer's disease (AD), Amyotrophic lateral sclerosis, prion disease, Creutzfeldt–Jakob disease, Parkinson's disease (PD), amyloidosis, and a wide range of other disorders. Abnormalities in the protein primary or higherorder structure as well as in the PTM status result in changes in protein’s physico-chemical properties, such as molecular weight (MW) and isoelectric point pI. The latter is of particular analytical interest, as even a small change in amino acid sequence or 3D structure can result in a pI shift[5]. Some researchers believe that this trough is due to a combination of physico-chemical properties of amino acids, protein MW and length distribution[9], while others find it to be consistent with the tendency of proteins to avoid pI equal to the media’s pH, as at such conditions the proteins acquire neutral overall charge and become prone to aggregation[10]. As experimentally-determined pH values for subcellular compartments differ significantly (lysosome 4.8; vacuole 5.3; Golgi 6.6; endoplasmic reticulum 7.1; cytoplasm 7.3; mitochondrion 7.5; nucleus 7.7; peroxisome 8.2)[11], it was found that the average predicted pI for a subcellular compartment tends to deviate from the subcellular pH, consistent with mitigating against neutral-charge aggregation[10]
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