C18 desalting protocol This protocol describes how to make and use a C18 desalting tip for desalting and purifying in-gel and in-solution digests prior to mass spectrometry. The C18 tip will bind peptides and allow salt and other contaminants to be washed off the sample. The clean, desalted peptides can then be eluted into a clean tube and run on the mass spectrometer. A C18 tip can be manufactured by punching a small diameter plug of C18 material from a disk of Empore Octadecyl C18 47mm diameter Extraction disk (SIGMA part number 66883U) using punches available in the proteomics facility and packing the small disk into a P10 pipette tip. Staff in the Central Proteomics Facility can show you how to do this and provide the necessary materials. How to Use You will need the following buffers: 0.1% formic acid in water (sample buffer, loading buffer and wash buffer), 0.1% formic acid in acetonitrile (wetting buffer), and 0.1% formic acid in 70% acetonitrile/30% water (elution buffer). These buffers and chemical are available in the Central Proteomics Facility. 1) Your samples containing peptides should be acidified by the addition of a small amount of formic acid or acetic acid. Aim to make the sample < 1% formic/acetic acid. 2) Prepare the C18 tip by loading 30ul of wetting buffer into the top of the C18 tip. Wetting buffer can be pushed through the C18 material by using a large plastic syringe (eg 10mL plastic disposable syringe) OR a vacuum manifold (one is located in the Central Proteomics Facility). 3) Equilibrate the C18 column by loading 30 ul of equilibration buffer onto the C18 tip and pushing/sucking the buffer through. The device is now ready to bind your peptides. THE PEPTIDE CONTAINING SAMPLE SHOULD BE LOADED IMMEDIATELY ONOT THE C18 TIP 4) The peptide sample in acidified aqueous buffer (typically 0.1% formic acid in water) can now be loaded onto the C18 tip. Load the sample solution onto the C18 tip and SLOWLY push the sample over the C18 phase or suck it through SLOWLY on a vacuum manifold. Collect the flowthrough in a clean Eppendorf tube. If the peptides fail to bind to the C18 tip this will contain your peptides! Capture efficiency can be improved by passing the flowthrough over the C18 tip again – this will lead to more efficient binding of the peptides to the C18 tip. 5) Desalt the peptides by loading 30 ul of wash buffer onto the C18 tip. Collect the washes in a clean eppendorf tube (this might contain your peptides if something has gone wrong). 6) Elute the desalted peptides by loading 30 ul of elution buffer onto the C18 tip and SLOWLY passing it over the C18 tip. Collect the sample in a clean eppendorf tube. 7) The sample should now be concentrated to dryness/near dryness in a SpeedVac (available in the Central Proteomics Facility). Samples can now be flash frozen and stored at -20C or -80 C prior to mass spec analysis.