Shotgun (or bottom-up)-based proteomic technologies in clinical researches have enabled the identification of many potential biomarkers for various diseases 1-3. A typical workflow of such technologies usually involves protein extraction from tissues or body fluids followed by enzymatic digestion and then chromatographic fractionation and mass spectrometric identification (LC-MS/MS) 4,5. To prepare high quality peptide samples for LC-MS, it is very important to ensure the overall quality of shotgun proteomics experiments. Peptide samples collected after digestion usually need to be cleaned to remove salts, possible gel pieces (for in-gel digested samples) or particles (for in-solution digested samples), which otherwise will damage the LC switching valves or clog the columns. As shown in Figure 1, the arrows indicate two ports of the six-port valve are damaged probably by salt crystals or other type of particles. While running under nano flow (usually ≤ 300 nL/min), these damages may cause inconsistent backpressure or sample loss which are not easily seen. This is one of the reasons our lab prefers cleaning the samples prior to LCMS analysis instead of doing on-line desalting. The protocol to use stop-and-go-extraction tips (StageTips) for desalting is simple, flexible and has been applied widely in proteomics labs 6,7. In this protocol, an ordinary pipette tip is packed with single or multiple layers of C18 materials that are pre-embedded into the Teflon support 7. This tip can then serve as a desalting tip, and works in a way similar to commercial ZipTips (Millipore). The packing can also be customized to include different types of materials or in combination such as SCX and SAX to perform fractionation 7-9, or to serve as a barrier to support other types of chromatographic beads loaded on top 10. This tip-based, column-free and pump-free chromatographic separation provides a simple alternative to traditional HPLC-based separation 8,10-12.
To process samples using StageTips, one has to find a pressure device to load samples in and elute samples out of the tips. As originally suggested, a plastic syringe is commonly used to manually force buffers through the tips. In our lab, depending on the amount of samples and salt conditions inside, finishing one sample with a syringe may take up to ten minutes. This sounds relatively easy when dealing with only a few samples (<10). However, when tens of samples have to be processed, manual push with syringe pump seems to be impractical and lack throughput. Previously, pipette tip boxes were suggested as StageTip adaptors to simultaneously process multiple samples by centrifugation 7. Yet, this protocol seems complicated regarding assembling the tip boxes with centrifuge. Another type of adaptor which seems a bit more violent was described to make a hole on the lid of a 1.5-mL tube, insert the StageTip and make the whole unit spinnable 7. However, puncturing holes using scissors on the tube lids cannot be well controlled and is less reproducible. Sometime it is hard to make holes with the right size so the tips can sit in the middle of the tube. Also, in our experiments, this type of hole is not strong enough to hold tips during centrifugation, and the tips may be bent and damaged in some cases. In addition, a mini-centrifuge specially designed for StageTips was also developed and commercially available (Sonation, Germany). However, this centrifuge seems good for waste collection only; the elution has to be collected elsewhere.
We recently noticed a commercially available adaptor for pipette tips, and applied it to StageTips in our lab. This unit can perfectly fit in the 1.5- or 2.0-mL microtubes. Then the whole module is completely spinnable on bench top centrifuge, thus making the entire procedures totally automatable and labor-free. From our experience, this method can significantly speed up the desalting steps without compromising any binding or elution efficiencies. The adaptor is not a new product to the market; to our own knowledge, a couple of labs have been using it for a while 13-15. However, this adaptor unit has not been fully realized by most proteomics labs. We described herein a simple protocol for using the adaptors and StageTips to clean peptide samples for proteomics. With most adaptions from the published protocol 7, the current method serves as a note to the StageTips protocol 7.