Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
Mass spectrometry (MS)-based proteomics measures peptides derived from proteins by proteolytic cleavage. Before performing the analysis by matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS), nanoelectrospray-MS/MS (NanoES-MS/MS) or liquid chromatography-MS/MS (LC-MS/MS), the peptide mixtures need to be cleaned, concentrated and often selectively enriched or pre-fractionated, for which we employ simple, self-made and extremely economical stop-and-go-extraction tips (StageTips). StageTips are ordinary pipette tips containing very small disks made of beads with reversed phase, cation-exchange or anion-exchange surfaces embedded in a Teflon mesh. The fixed nature of the beads allows flexible combination of disks with different surfaces to obtain multi-functional tips. Disks containing different surface functionalities and loose beads such as titania and zirconia for phosphopeptide enrichment can be combined. Incorporation into an automated workflow has also been demonstrated. Desalting and concentration takes approximately 5 min while fractionation or enrichment takes approximately 30 min.
Rappsilber
Juri
J
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK. juri.rappsilber@ed.ac.uk
Mann
Matthias
M
Ishihama
Yasushi
Y
D005591:Chemical Fractionation / Q000295:instrumentation / Q000379:methods*; D002853:Chromatography, Liquid; D004867:Equipment Design; D013058:Mass Spectrometry; D010455:Peptides / Q000302:isolation & purification*; D011138:Polytetrafluoroethylene; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D013499:Surface Properties
D010455:Peptides; D011138:Polytetrafluoroethylene