Authors: Yanbao Yu, Madeline Smith & Rembert Pieper ### Abstract The stop-and-go-extraction tips (StageTips) have been widely used in shotgun proteomics to clean/desalt peptide samples prior to LC-MS/MS analysis. Here, an extremely simple and high throughput StageTip protocol is described. In this protocol, an adaptor is introduced to the StageTip, and makes it readily available for bench-top centrifugation. Each spin step (with 200 μL buffer loaded) takes around 2 min at 4,000 rpm. Compared with previous syringe-based manual pressure device, the spinnable StageTip is completely labor-free, automatable, and can be easily scaled up. Considered with the recently developed 96FASP method, the spinnable StageTips provide another component to the high throughput workflow for clinical proteomics and biomarker discoveries. ### Introduction 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 (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 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. ### References 1. Konvalinka, A.; Scholey, J. W.& Diamandis, E. P. Searching for New Biomarkers of Renal Diseases through Proteomics. *Clin. Chem.* 58, 353-65 (2012). - Wood, S. L., et al. Proteomic studies of urinary biomarkers for prostate, bladder and kidney cancers. *Nat. Rev. Urol*. 10, 206-18 (2013). - Gerszten, R. E.; Asnani, A.& Carr, S. A. Status and Prospects for Discovery and Verification of New Biomarkers of Cardiovascular Disease by Proteomics. *Circ.Res*. 109, 463-74 (2011). - Ahrens, C. H., et al. Generating and navigating proteome maps using mass spectrometry. *Nat. Rev. Mol. Cell Biol*. 11, 789-801 (2010). - Zhang, Y., et al. Protein Analysis by Shotgun/Bottom-up Proteomics. *Chem. Rev*. 113, 2343-94 (2013). - Rappsilber, J.; Ishihama, Y.& Mann, M. Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption/Ionization, Nanoelectrospray, and LC/MS Sample Pretreatment in Proteomics. *Anal. Chem*. 75, 663-70 (2002). - Rappsilber, J.; Mann, M.& Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. *Nat. Protoc*. 2, 1896-906 (2007). - Wiśniewski, J. R.; Zougman, A.& Mann, M. Combination of FASP and StageTip-Based Fractionation Allows In-Depth Analysis of the Hippocampal Membrane Proteome. *J. Proteome Res*. 8, 5674-8 (2009). - Kleifeld, O., et al. Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates. *Nat. Protocols* 6, 1578-611 (2011). - Han, D., et al. Characterization of the membrane proteome and N-glycoproteome in BV-2 mouse microglia by liquid chromatography-tandem mass spectrometry. *BMC Genomics* 15, 95 (2014). - Han, D., et al. In-depth proteomic analysis of mouse microglia using a combination of FASP and StageTip-based, high pH, reversed-phase fractionation. *Proteomics* 13, 2984-8 (2013). - Nagaraj, N., et al. Deep proteome and transcriptome mapping of a human cancer cell line. *Mol. Syst. Biol*. 7 (2011). - Nakagami, H. 2014. StageTip-Based HAMMOC, an Efficient and Inexpensive Phosphopeptide Enrichment Method for Plant Shotgun Phosphoproteomics. In Plant Proteomics, ed. JV Jorrin-Novo, S Komatsu, W Weckwerth, S Wienkoop, pp. 595-607: Humana Press - Pozniak, Y.& Geiger, T. 2014. Design and Application of Super-SILAC for Proteome Quantification. In Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC), ed. B Warscheid, pp. 281-91: Springer New York - Schoof, E. M.& Linding, R. 2014. Experimental and Computational Tools for Analysis of Signaling Networks in Primary Cells. In Current Protocols in Immunology, pp. 1–23: John Wiley & Sons, Inc. - Yu, Y., et al. Urine Sample Preparation in 96-Well Filter Plates for Quantitative Clinical Proteomics. *Anal. Chem*. 86, 5470–7 (2014). ### Figures  **Figure 1: A six-port valve detached from a common HPLC system**. The arrows point to two ports that are damaged. Clear erosions (when comparing with the other four normal ports) can be seen on both of them.  **Figure 2: Three types of tip adaptors**. Type one and two adaptors are commercially available, and can fit in the 1.5-mL or 2.0-mL microtubes well (as shown in the lower panel). Type three is self-made in the lab, and serves as a simple adaptor or StageTips.  **Figure 3: Four different types of 200 μL tips**. The white arrows indicate the varied lengths of the support ribs of different tips. The red arrows on the tips point to the position the adaptor stops when sliding them in (as also seen on the adaptors in the lower panel). The right panel shows 24 StageTips with adaptors are loaded into the centrifuge with the safely closed.  **Figure 4: Two different sizes of collection tubes (1.5-mL and 2.0-mL)**. The red arrows point to the 0.5-mL level of each tube. This is to show the approximate level of liquids when doing StageTips with different volume of solvents (for example, eluting peptides with 20 μL or 200 μL each time). *Source: [Protocol Exchange (2014)](http://www.nature.com/protocolexchange/protocols/3421). Originally published online 8 September 2014 *