Protein Isoform Determination
Protein isoforms
Posttranslational modification (PTM), like glycosylation, results in a huge number of protein variants (isoforms) showing micro heterogeneity of different kinds. The dominating heterogeneity is found in the carbohydrate moieties of glycoproteins. Other examples are genetic polymorphism, modified functional groups in amino acids by e.g. phosphorylation, acetylation, or limited proteolysis. No protein seems to be homogenous and many proteins show a huge number of different isoforms. The distribution of the isoforms might change due to physiological regulation and pathological processes and the distribution pattern seems to have high clinical significance. Production of a typical pattern from one cell-type may affect the whole isoform distribution in the circulation. The glycosylation of proteins seems to be an important regulator for the fine-tuning of biological activity. Isoforms show various interactions with receptors which also can affect their survival time. In cancer, inflammation or liver diseases, the protein glycosylation pattern is typically changed at an early stage of the disease. The need for protein isoform detection ranges from high abundance proteins like haemoglobin (10^-3 M) down to proteins like erythropoietin (10^-15 M).Protein isoform determination
The distribution of isoforms seems to have high impact on the biological function as well as being an indicator of pathological conditions, but the lack of suitable methods hampers the clinical use of isoform diagnostics.Few quantitative methods are available for determination of isoforms, especially for measurement of low concentrations in biological specimens. A combination of discrete steps of separation (electrophoresis or chromatography) and detection (immunological technique) is used. The quality of the test depends on the type of specific isoforms that can be distinguished, how efficiently these isoforms are separated (resolution), and how well they can be quantified (specificity and sensitivity). The electrophoretic or chromatographic separations in columns are gentle techniques which do not require extensive pre-preparation of the biological sample. The advantage with chromatographic separation is the possibility to use different ligands for isoform separation. Analysis of the fractions using immunoassay is quantitative, sensitive and specific, although quite expensive. Slab-gel and capillary electrophoresis separate isoforms due to charge differences but lack suitable means to quantify and specify correctly. Mass spectrometry detection does not solve these specificity and quantification problems, especially not when analysing low abundance glycoproteins. An affinity based pre-treatment step that specifically captures the protein of interest and concentrates it in relation to other proteins will enhance the possibilities to use these types of methods. None of the available techniques is suitable for high-throughput testing or for point-of-care applications. The chromatographic approach, utilizing selected ligands for separation, in combination with immunoassay seems to be the best way for quantitative isoform detection but a miniaturisation is needed.