Ricerc@Sapienza

The work describes the development of an enrichment method for the analysis of endogenous phosphopeptides in serum. Endogenous peptides can play significant biological roles, and some of them could be exploited as future biomarkers. In this context, blood is one of the most useful biofluids for screening, but a systematic investigation of the endogenous peptides, especially phosphorylated ones, is still lacking, mainly due to the lack of suitable analytical methods.

Magnetic materials in sample preparation for shotgun phosphoproteomics offer several advantages over conventional systems, as the enrichment can be achieved directly in solution, but they still suffer from some drawbacks, due to limited stability and selectivity, which is supposed to be affected by the hydrophilicity of the polymeric supports used for cation immobilization. The paper describes the development of an improved magnetic material with increased stability, thanks to a two-step covering of the magnetic core, for the enrichment of phosphopeptides in biological samples.

A new on-line multidimensional system for sequential trapping and individual elution and separationof peptides based on their molecular weight is described. By sequentially using two chemically differenttrapping columns, a polymethacrylate monolith and a packed C18 one, peptides from complex samplescan be on-line trapped and divided into two fractions, containing respectively mainly medium-large pep-tides and smaller peptides. Then, by means of two switching valves working in parallel, the two fractionswere individually separated by reversed phase chromatography.