pI-based fractionation of serum proteomes versus anion exchange after enhancement of low-abundance proteins by means of peptide libraries.
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.
Restuccia
Umberto
U
San Raffaele Scientific Institute, Milano, Italy.
Boschetti
Egisto
E
Fasoli
Elisa
E
Fortis
Frederic
F
Guerrier
Luc
L
Bachi
Angela
A
Kravchuk
Alexander V
AV
Righetti
Pier Giorgio
PG
D001798:Blood Proteins / Q000737:chemistry*; D002021:Buffers; D002412:Cations; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000379:methods*
D001798:Blood Proteins; D002021:Buffers; D002412:Cations; D008024:Ligands; D019151:Peptide Library; D010455:Peptides