Protein Protocols Protein Protocols

Nanoelectrospray MS/MS for Peptide Sequencing 699
Fig. 3. Microcolumns for peptide and protein desalting/concentration prior to nanoelectrospray
mass spectrometry. (A) Capillary column made from a pulled glass capillary and
filled with a small volume of Poros resin. The sample is rinsed and then eluted from the column
capillary directly into the nanoelectrospray needle by centrifugal force using a tabletop
centrifuge. (B) Microcolumn made from a GELoader tip. The sample is loaded and eluted by
air pressure from a syringe adapted to the GELoader tip.
ing and transfer of sample from capillary column to nanoelectrospray needle (available
from MDS Proteomics, Odense, Denmark).
1. Mount a pulled glass capillary into a custom-made capillary holder (Fig. 3A) or into a
pierced lid of a 1.5-mL microcentrifuge tube. Use a micropipet with a gel loader tip to
transfer 5 µL of resin slurry into the capillary.
2. Load the chromatographic resin into the tip of the glass capillary by centrifugal force
using a manually operated tabletop minicentrifuge (e.g. PicoFuge, Stratagene, Palo Alto,
CA) at low speed (500–2000 rpm). The pulled glass capillaries used for the columns are