pI-based fractionation of serum proteomes versus anion exchange after enhancement of low-abundance proteins by means of peptide libraries |
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Authors: | Umberto Restuccia Egisto Boschetti Elisa Fasoli Frederic Fortis Luc Guerrier Angela Bachi Alexander V. Kravchuk Pier Giorgio Righetti |
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Affiliation: | aSan Raffaele Scientific Institute, 20132 Milano, Italy;bBio-Rad Laboratories, 92430 Marnes-la-Coquette, France;cDepartment of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, 20131 Milano, Italy |
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Abstract: | The pre-treatment of biological extracts with the aim of detecting very low-abundance proteins generates complexity requiring a proper fractionation. Therefore the success of identifying all newly detectable species depends on the selected fractionation methods.In this context and starting from a human serum, where the dynamic concentration range was reduced by means of a preliminary treatment with a combinatorial hexapeptide ligand library, we fractionated the sample using a novel method based on the differences in isoelectric points of proteins by means of Solid-State Buffers (SSB) associated with cation exchangers. The number of fractions was limited to four and was compared to a classical anion exchange method generating the same number of fractions.What was observed is that when using SSB technology the protein redundancy between fractions was significantly reduced compared to ion exchange fractionation allowing thus a better detection of novel species. The analysis of trypsinized protein fractions by nanoLC-MS/MS confirmed that the SSB technology used is more discriminant than anion exchange chromatography fractionation.A sample fractionation by SSB after the reduction of dynamic concentration range can be accomplished without either adjustment of pH and ionic strength or protein concentration and cleanup. Both advantages over either classical chromatography or isoelectric fractionations allow approaching the discovery of markers of interest under easier conditions applicable in a variety of fields of investigation. |
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Keywords: | Proteomics Sample treatment Fractionation Solid-State Buffers Dynamic range |
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