title: A multidimensional liquid chromatography-tandem mass spectrometry platform to improve protein identification in high-throughput shotgun proteomics.
abstract: A new on-line multidimensional system for sequential trapping and individual elution and separation of peptides based on their molecular weight is described. By sequentially using two chemically different trapping columns, a polymethacrylate monolith and a packed C18 one, peptides from complex samples can be on-line trapped and divided into two fractions, containing respectively mainly medium-large peptides and smaller peptides. Then, by means of two switching valves working in parallel, the two fractions were individually separated by reversed phase chromatography. The whole gradient consisted of two subgradients, with the first one dedicated to the separation of smaller peptides and the second one to the separation of larger peptides. Such configuration allowed to identify up to 1476 proteins in a standard E. coli tryptic digest, with improved performance, increased average sequence coverage and reduced single unique peptide identifications compared to a conventional shotgun proteomics configuration comprising only the C18 trapping column and the analytical column.
authors.affiliation: Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.
authors.forename: Anna Laura
authors.initials: AL
authors.lastname: Capriotti
authors.affiliation: Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy. Electronic address: chiara.cavaliere@uniroma1.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Cavaliere
authors.affiliation: Department of Chemistry and Pharmaceutical Sciences, University of Ferrara, via L. Borsari 46, Ferrara, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Cavazzini
authors.affiliation: Department of Drug Chemistry and Technology, Sapienza University of Rome, P.le Aldo Moro 5, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Gasparrini
authors.affiliation: Department of Drug Chemistry and Technology, Sapienza University of Rome, P.le Aldo Moro 5, Rome, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pierri
authors.affiliation: Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.
authors.forename: Susy
authors.initials: S
authors.lastname: Piovesana
authors.affiliation: Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.
authors.forename: Aldo
authors.initials: A
authors.lastname: Laganà
mesh_terms: D002851:Chromatography, High Pressure Liquid; D030562:Databases, Protein; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D036103:Nanotechnology; D010455:Peptides / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; D014357:Trypsin
keywords: Monolithic trapping column; Multidimensional chromatography; NanoHPLC–MS/MS; Peptide fractionation; Shotgun proteomics