Ricerc@Sapienza

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. The whole gradient consisted of twosubgradients, with the first one dedicated to the separation of smaller peptides and the second one to theseparation of larger peptides. Such configuration allowed to identify up to 1476 proteins in a standardE. coli tryptic digest, with improved performance, increased average sequence coverage and reducedsingle unique peptide identifications compared to a conventional shotgun proteomics configurationcomprising only the C18 trapping column and the analytical column