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.