title: Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.
abstract: Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding <0.1 cent in material costs to the price of each tip. The system allows fast loading with low backpressure (>300 micro/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diameter, 0.5 mm length) is 2-4 microg of protein digest, which can be increased by using larger diameter or stacked disks. Five femtomole of tryptic BSA digest could be recovered quantitatively. We have found that the Stage system is well-suited as a universal sample preparation system for proteomics.
authors.affiliation: Center of Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D012710:Serum Albumin, Bovine