- The pipette tip adaptors:
Figure 2 shows three types of adaptors for ordinary pipette tips. Both type one and two can fit perfectly in the 1.5-mL or 2.0-mL microtubes (as shown in the lower panel of Figure 2). The adaptor one uses economically less plastics and works as good as adaptor two. So we use type 1 adaptors in all of our experiments. Some publications suggested puncturing holes on the lid of microtubes, and making them as simple spin adaptors (type 3 in Figure 2) 7,8. Apparently, the first two types are much more convenient and simple to use, and even more durable than the lid-based adaptors.
- The pipette tips:
There are many different types of pipette tips, but even for the tips with the same volume size, different manufactures provide slightly different lengths and shapes Figure 3 shows four different types of 200 μL tips commonly found in the lab. Regarding the support ribs in the upper portion of the tips (indicated by the white arrows), each one has a different length. The width near the top is also different from each other, which will determine if the adaptors can fit the tips (indicated by the red arrows on the tips and on the adaptors). Ideally, the tips should be narrow enough so the adaptors can fit in smoothly (all the way up to the support rib), and the support ribs should be wide enough to block the adaptors and short enough so the lid of the centrifuge can be closed tightly. If not, the lid may pop off during centrifugation and throw out the tip. Before packing, it is wise to test the centrifuge with empty tips and confirm the lid can be closed completely (as shown in Figure 3, right panel).
The first two tips (Figure 3) are too wide to fit in the adaptors, and the fourth tip has too long of a support rib with which the lid of centrifuge cannot be closed. The third type of tip was used in all our experiments. Some labs use gel loading tips as StageTips 7. Because their capillary tails are too long to fit in the microtubes, they are not suitable for spinnable StageTips.
- The collection tubes:
Because of their low bindings, we prefer the maximum recovery tubes for all the mass spec sample preparations. In contrast to other reports that used small volumes (10 or 20 μL) to do StageTipping 7, we prefer working with large volumes of processing buffers (100 or 200 μL) in order to effectively load samples onto the StageTips, and efficiently wash and then elute peptides out. Figure 4 shows two types of microtubes that StageTip can work with. The red marks on the tubes (also indicated by red arrows) show the 0.5-mL level. The packed StageTip in the 2.0-mL tube (right side) seems to stay above that level, and we used this type of tube in all our experiments. When one wants to process samples with small volumes, the 1.5-mL tube (left side) works well too.
- The centrifugation speed:
We tested centrifuging StageTips with four different speed, 2000 rpm, 3000 rpm, 4000 rpm and 5000 rpm. Spin with 2000 rpm usually takes a significantly long time (around 10 min for preparing tips, and >40 min for binding, washing and elution), while spin with 3000 rpm or above decrease the processing time dramatically. For example, to active the tips with 200 μL Buffer A and Buffer D only takes <2 min; to load 100 μL samples (resuspended with Buffer B) takes 1 to 2 min as well. Elution with 200 μL Buffer C or D takes 2 to 3 min with 4000 or 5000 rpm, whereas takes >5 min with 3000 rpm. We analyzed the samples (with three replicates under each condition) with nanoLC-Q Exactive MS/MS. The number of protein identifications did not show significant variations. We used 4000 rpm to process StageTips in all our experiments.
- Peptide desalting using adaptors and StageTips:
This procedure is adapted from the published protocol 7. Several changes have been made in order to better fit the sample processing in our lab.
1) Follow the instructions on the published protocol, pack single or multiple layers of C18 into the tips. Pack as many as you need.
2) Place packed tips with the adaptor into the 2.0 mL microtubes (as shown in Figure 4).
3) Conditioning I: load 200 µL buffer A (methanol) into the tips, spin at 4000 rpm for ~1 min;
Conditioning II: load 200 µL buffer D (0.5% acetic acid, 80% acetonitrile and 20% H2O) into the tips, spin at 4000 rpm for ~1 min.
4) Equilibration: load 200 µL buffer B (0.5% acetic acid in H2O) into the tips, spin at 4000 rpm for ~1 min.
5) Resuspend the dried peptide samples into 100 µL of buffer B, and vortex for around 10 min. The peptides may come from in-gel digestion, in-solution digestion, filter aided sample preparation (FASP) or 96FASP 16.
6) Binding: load 100 µL peptide solutions in the tips and spin at 4000 rpm for about 1.5 min. Re-load the flow-through into the tips and spin again. Repeat this binding step 2~3 times.
7) Wash: load 200 µL buffer B and spin at 4000 rpm for 2~3 min. Discard the flow-through.
8) Elution: place the StageTips into new collection tubes; load 200 µL buffer C, spin at 4000 rpm for 2 min; load 200 µL buffer D, spin at 4000 rpm for 2 min, repeat elution with buffer D one more time. The total volume of the elution is ~600 μL.
9) Dry the peptide elutes in Speed-Vac, re-suspend with HPLC buffer for immediate LC-MS/MS analysis, or store at -80°C until further use.