title: Microcolumns with self-assembled particle frits for proteomics.
abstract: LC-MS-MS experiments in proteomics are usually performed with packed microcolumns employing frits or outlets smaller than the particle diameter to retain the packing material. We have developed packed microcolumns using self-assembled particles (SAPs) as frits that are smaller than the size of the outlet. A five to one ratio of outlet size to particle diameter appears to be the upper maximum. In these situations the particles assembled into an arch over the outlet like the stones in a stone bridge. When 3 microm particles were packed into a tapered column with an 8 microm outlet, two particles bridged the outlet with 0.3 pl dead volume and perfect success rate. In peptide analysis by LC-MS, the peak width at half height was normally less than 6 s, compared to 12 s without SAPs. The LC-MS-MS system provided 37% sequence coverage (21 matched peptides) for a tryptically-digested sample of 10 fmol bovine serum albumin. We also describe application of the SAP principle to make disposable pipette tip columns with short pieces of fused-silica capillary as the outlet.
authors.affiliation: Protein Interaction Laboratory in the Center of Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
authors.forename: Jens S
authors.initials: JS
authors.lastname: Andersen
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D002853:Chromatography, Liquid / Q000295:instrumentation; D013058:Mass Spectrometry / Q000295:instrumentation; D040901:Proteomics