Protein Protocols Protein Protocols

700 Jensen and Wilm
the same as used for nanoelectrospray emitters but they are not sputter-coated with metal.
The chromatographic material is visible against a dark background but nearly invisible in
front of white paper.
3. When sufficient chromatographic resin has been loaded into the capillary (1–2 mm resin
height) the glass tip is widened/broken by gently touching it against the tabletop. The
opening should allow liquid but not column resin to exit the capillary during centrifugation.
Do not centrifuge the resin too fast; otherwise it becomes compressed and may block
the flow of liquid. The centrifugal capillary column is only used once to avoid sample-tosample
contamination.
4. Rinse the capillary column by injecting 5 µL of 50% MeOH followed by gentle centrifugation.
5. Equilibrate the column resin by injecting 5 µL of 5% formic acid into the capillary
followed by centrifugation until all liquid has passed through the column.
6. Dissolve the sample in 10–20 µL of 5% formic acid and inject it onto the capillary
column in aliquots of 5 µL followed by centrifugation. For best recovery, dissolve the
dried protein or peptide sample in 80% formic acid and then immediately dilute it to 5%
by addition of water.
7. Wash the column resin twice by centrifugation with 5 µl of 5% formic acid solution. This
desalting step is very efficient as the column is washed with 50–100× its resin volume.
Before beginning the elution step the washing solution must be completely removed by
gentle centrifugation.
8. Elution of sample into a nanoelectrospray needle.
Mount the capillary column in-line with a premade nanoelectrospray needle in a custommade
capillary holder that fits into a microcentrifuge (Fig. 3A). Elute the peptide mixture
into the nanoelectrospray capillary by centrifugating twice with 0.5 µL of 60% methanol–
5% formic acid. Elute proteins with 60–70 % acetonitrile–5 % formic acid. This procedure
allows handling of elution volumes between 10 µL and 0.2 µL. Elution, however,
should be performed twice because the first elution does not completely deplete the
column. Keep in mind that signal intensity in an electrospray spectrum is concentration
dependent, so keep the elution volume as small as possible.
9. Mount the loaded nanoelectrospray needle onto the ion source and begin the experiment.
2.5. Protocol 3. Sample Desalting and Concentration Employing
Microcolumns Packed in GELoader Tips
This sample desalting/concentration method is very simple and can be used prior to
MALDI-MS and nanoelctrospray MS. The resin is held in place by making a constriction
at the end of a GELoader pipettor tip. Sample loading, washing and elution is
performed by loading liquid on top of the resin and applying air pressure to generate a
low flow through the column. No frits are necessary.
2.5.1. Materials
1-mL plastic syringe; GELoader tips (Eppendorf brand). The syringe is adapted to
the GELoader tips by using the top part of a “yellow” plastic tip.
1 Prepare a slurry of 100–200 µL of chromatographic resin, for example, Poros R1, R2, or
OligoR3, in 1 mL of methanol.
2 Make a partially constricted GELoader pipet tip by gently squeezing or twisting the end of
the tip (Fig. 3B). This allows liquid to flow through the tip while retaining the chromatographic
resin.
3 Use another GELoader tip to load 5 µL of slurry of resin into the GELoader tip from the
top and pack it by applying air pressure with the 1-µL syringe adapted to fit the GELoader
tip microcolumn. The column height should be 2–4 mm.