S-Trap™ General FAQ
SDS is necessary for efficient capture in the protein-trapping S-Trap™ matrix. Five percent (5%) is standard and recommended; the absolute minimum SDS concentration for effective use is 2%. Even high concentrations of SDS (up to 20%) do not affect S-Trap™ performance.
S-Trap™ Spin Columns have an expiration of 2 years post-manufacturing date.
S-Trap™ is compatible with essentially all proteins, with the single possible exception of alcohol-soluble prolamin storage proteins found in cereal grains and seeds of the grass family Poaceae. Prolamins include gliadins (wheat), avenins (oats), zeins (corn/maize), hordeins (barley), secalins (rye), kafirins (sorghum), oryzin (rice prolamins), coixins (Job’s tears/Coix), eragrostins (teff), caneins (sugarcane), pennisetins (pearl millet), setarins (foxtail/Italian millet), and panicin (proso millet); other species have homologous unnamed prolamins. To verify the absence of methanol (MeOH)-soluble proteins, combine and SpeedVac the flow-through and wash fractions, and analyze by SDS-PAGE.
S-Traps™ capture low molecular weight proteins, especially if they are in a complex mixture with many other proteins which tend to carry them into the protein trap; binding depends on the sequence and structure of both the low molecular weight protein and co-bound proteins.
No. The protein-trapping S‑Trap™ matrix retains proteins but not peptides. Peptides will typically appear in the SDS-containing flow–through.
Typically < 10% CVs for replicate digestions. Due to stochastic sampling, high abundance proteins have lower CVs and low abundance higher CVs.
S-Trap™ has been shown to remove urea (up to 8M), NaCl, KCl, PBS, tris, glycerol, NP-40, CHAPS, phenol red, PEG and other detergents, Ficoll, tween, triton X-100, Lameli loading buffer, phosphate and histidine buffers, NaHCO₃, NaOH, glucose, sucrose, trehalose, HEPES, EDTA, imidazole, guanidine-HCL, DTT, TCEP, Brij-35, DMSO, and triethanolamine. Avoid the use of GuHCl with S-Traps™: SDS and GuHCl make an insoluble ionic salt that precipitates. We recommend substituting SDS in protocols requiring GuHCl for sample solubilization. In the case of a buffer component that seems to not be fully removed, simply wash more. As always, we recommend testing your specific conditions in particular for important samples.
Add SDS to your buffer to at least a 2% w/v final concentration. Then proceed as normal: add phosphoric acid, then binding buffer and everything will be great! Like with other buffer components, if you run into a buffer component which is “sticky” and appears to not be fully removed by three washes, simply wash more.
S-Trap™ is compatible with Rapigest™ or ProteaseMAX™. However, ProteaseMAX™ should not be used at elevated digestion temperatures due to accelerated autolysis.
Yes. Lyse tissue in 20x volume of HYPERsol Lysis Solution (10% SDS, 100 mM Tris, pH 8.5) and follow the HYPERsol protocol available on the Protocols page.
Protein Concentration FAQ
Use a BCA assay and not Bradford: BCA is not sensitive to detergents and Bradford is. Note that the original BCA assay is not compatible with reducing agents. There exist, however, reducing agent compatible BCA assays, e.g. BCA-RAC assay from Pierce.
| Size | Protein Sample |
|---|---|
| Micro | 1 µg ‑ 100 µg |
| Mini | 100 µg ‑ 300 µg |
| Midi | ≤ 1 mg |
Extraction / Lysis / Solubilization FAQ
This is highly dependent on the sample type being processed. Please see Appendix A for specific recommendations.
If your sample is viscous, there are a few methods that might help. These include ultrasonication, probe/horn sonication, and the addition of Benzonase. Refer to Appendix D for more information.
Buffers and pH FAQ
Tris-HCl can be used in place of TEAB at the same concentrations and pH value for S-Trap™ Solution 5, Binding/Wash Solution and S-Trap™ Solution 6, Digestion Solution. Note that Tris is not compatible with iTRAQ, TMT, or other amine labeling chemistries. Ammonium bicarbonate cannot be used in the S-Trap™ Solution 5, Binding/Wash Solution due to insufficient solubility in 90% MeOH; however, it can be used in place of TEAB in the S-Trap™ Solution 6, Digestion Solution.
For the S-Trap™ Solution 1, 2x Lysis Solution and S-Trap™ Solution 5, Binding/Wash Solution, we recommend using phosphoric acid for pH adjustment. Note that TEAB is volatile and can change pH if left to sit or stored with a large air headspace. We thus recommend adjusting the pH of a 1 M TEAB solution with 85% phosphoric acid, aliquoting this solution and keeping it frozen.
S-Traps™ are compatible with most buffer systems; you should match your digestion solution to the protease being used. For trypsin, we recommend 50 mM TEAB at pH 7.5 – 8. Note that typical digestion buffers of ammonium bicarbonate and TEAB do not require pH adjustment for trypsin. However, buffers made with Tris or HEPES free base (or acid) require pH adjustment. The recommended error range when adjusting the pH is 0.05. If phosphoproteomics is of interest, use HCl for pH adjustment and perform additional washes: the bicarbonate anion will displace any free phosphate ionically bound to trapped proteins.
Note that extended incubation in basic conditions (pH > 8.5) can partially solubilize the silica matrix; this is insoluble at low pH, does not harm a sample, and will not bind to reverse phase columns. To prevent formation, ensure the pH is ≤ 8.0 for 2 – 4 hour digests and 7.5 – 8.0 for overnight digests. If precipitation is present, remove it by centrifugation, filtration, or high-pH C18 desalting.
Yes, lactic acid at 3% or citric acid at 4% final concentration can be used.
S-Trap™ Solution 1, 2x Lysis Solution is 10% (w/v) SDS, 100 mM TEAB in LC-MS grade Water, pH 7.55.
The S-Trap™ High Recovery Lysis Solution is 5% (w/v) SDS, 8 M urea, 100 mM glycine in LC-MS grade Water, pH 7.55. TEAB can also be used in place of glycine. The S-Trap™ High Recovery Acidifier is 55% (v/v) Phosphoric Acid in LC-MS grade Water. The S-Trap™ High Recovery protocol also calls for protease addition into the acidified sample prior to binding. Follow the S-Trap™ High Recovery protocol available on the Protocols page.
Enzymatic Digestion FAQ
Yes. Many proteases can be used for protein digestion. Adjusted protease concentration, as well as digestion time and temperature, may need to be optimized depending on sample and protease. A matrix of different w/w concentrations of protease, digestion temperatures and/or times is useful to determine the parameters best suited to your study; additionally, a MS-compatible detergent like Rapigest™ or ProteaseMAX™ can be included (ProteaseMAX™ should not be used at elevated digestion temperatures due to accelerated autolysis). To prevent drying out, increase digestion volume with extended digestion times or temperatures (³ 37 °C and/or 4 hours). Do not apply < 1 µg of trypsin per S-Trap™ for efficient digestion or significantly increase incubation time; other proteases may require different minimum amounts. Trypsin/Lys-C mixes work better than trypsin alone. See Appendix C.
It depends heavily on the analytical instrument and acquisition parameters: new, fast DIA techniques generally pick up low level missed cleavages more efficiently; these are typically only a few percent of overall ion current. In general, trypsin at 47 °C for 1 hour is empirically optimal. However, this is highly dependent on the sample type being processed, as well as your workflow. Please see Appendix A for specific sample type recommendations and Appendix C for different protease recommendations. Other enzymes might require different temperatures and times.
If the digest has dried on the spin column (for example, if the cap was not applied during incubation, samples were forgotten, or the digestion took place outside a water-saturated atmosphere), the protein-trapping S-Trap™ matrix will need to be rehydrated to solubilize the peptides. Add S-Trap™ Solution 6, Digestion Solution and let sit for 30 minutes, then centrifuge out. Repeat these steps again and combine. Additional elutions may assist in peptide recovery. Concentrate by lyophilization. The S-Trap™ is designed to have no affinity for digested peptides; they just need to be solubilized.
The easiest way to remove bubbles is to “flick” the tubes. Alternatively, you can pipette the S-Trap™ Solution 6, Digestion Solution up and down, making sure that all the solution is on the trap and not on the side of the spin column/well where it will dry out.
The S-Trap™ Spin Columns/plates cannot be air-tight during incubation with protease: when you cap/seal the spin column/plate tightly and heat, pressure on the top of the spin column will rise, forcing the S-Trap™ Solution 6, Digestion Solution out of the bottom. Thus, you must allow the pressure to equilibrate. Leave S-Trap™ Micro and Mini Spin Columns slightly open and use the included 96-well cover for plates. S-Trap™ Mini Spin Columns have a built-in vent and should be completely closed.
There are two ways to limit evaporative loss. Preload dry incubators with beakers of water to saturate the air with water vapor and limit evaporation; let the beakers come to temperature before using the incubator or fill with hot water. If using a thermomixer, fill unused wells with water and cap. If samples appear to be drying out despite a water-saturated atmosphere, add additional S-Trap™ Solution 6, Digestion Solution to compensate for evaporative loss as described above.
Peptide Elution and Cleanup FAQ
Generally, ~85% of peptides come off with TEAB, ~5% with formic acid, and remainder with acetonitrile. This is highly sample-dependent and hydrophobic peptides tend to come out with organic (acetonitrile) elutions.
TMT labeling is sensitive to pH and water content. Therefore, it is important to dry down the eluted peptides and resuspend to control for both of these factors.
We generally recommend concentrating the peptides using desalting columns, however, C18 columns and tips have much lower recoveries than most people are aware of: around 50% in typical use. If you are working with low-level and/or hydrophobic samples, the serial loss over desalting, a LC trap column. and then an analytical column can result in seeing nothing or almost nothing. We and customers have observed this in particular with exosome samples. If you are doing a large-scale digestion for PTM enrichment, you will want to do desalting so that you can dry your peptides down and solubilize them in the binding buffer for your PTM enrichment. In particular, unless trapped proteins are sufficiently washed with TEAB to exchange the phosphate anion with bicarbonate, phosphate anions from the denaturation/binding process can remain due to ionic interactions with basic groups (RKH); desalting replaces them, if present, with (typically) formate or TFA anions.
Both of these are signs that the protein must be washed more to further remove SDS and/or other contaminants/components: perform further washes. Note that SDS and analogs (Rapigest™) are moderately hydrophobic and will come off C18 columns. Before it does however, it alters the surface to make it more of a cation exchange.
S-Trap™ Applications
We refer to Appendix D for various troubleshooting help.
As indicated in Appendix A; post-IP, wash beads three times with an appropriate wash solution (ex. 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM MgCl2, 0.2% Triton, and 1 mM DTT). Monitor sample purity via SDS-PAGE: there should be visible differences (bands) between the control and experimental lanes. Elute from beads with S-Trap™ Solution 1, 2x Lysis Solution (100 mM Tris-HCl pH 7.5, 10% SDS). Follow the S-Trap™ protocol, wash six times with S-Trap™ Solution 5, Binding/Wash Solution.
S-Traps™ are designed for use with isobaric amine labels (i.e. iTRAQ and TMT).
Yes. S-Traps™ can be used to clean up click-chemistry reactions of either CuACC or copper-free strain-promoted click-chemistry reactions.
See Appendix A: aliquot 0.5 mL urine and add 2 mL of cold acetone. Precipitate the proteins overnight at −20 °C. Pellet, then dissolve in S-Trap™ 1x Lysis Solution. Proceed with S-Trap™ digestion.
If your CSF samples are already aliquoted and frozen, lyophilize them. Bring them up in 8 M urea, 5% SDS, 50 mM TEAB, pH 7.4 to approximately 1 – 2 mg/mL. Sonicate the samples, reduce, alkylate, and proceed with S-Trap™ digestion. Avoid SpeedVacing: proteins in solution are subject to chemical or enzymatic changes. If you have to aliquot CSF, minimize freeze-thaw.
See Appendix A: denature sample by adding S-Trap™ Solution 1, 2x Lysis Solution to 1x and proceed with S-Trap™ digestion.
Peptidoglycans (PG) are challenging samples because the intact, crosslinked sacculus is a robust, covalently connected matrix. SDS can strip membranes and liberate intracellular proteins, but sacculi remain insoluble unless enzymatically depolymerized. PG analysis with S-Traps™, yields three fractions:
Fraction 1: Intracellular and other detergent-solubilized proteins (i.e. a “whole-cell lysate”);
Fraction 2: Trypsin-accessible PG-associated proteins (digested to peptides);
Fraction 3a and 3b: Proteins covalently attached to, embedded or physically trapped within the cell wall (also digested to peptides).
To obtain Fraction 1, harvest bacteria, wash and resuspend thoroughly in S-Trap™ 1× Lysis Solution. Lyse mechanically (e.g. bead beating and/or sonication; monitor lysis by microscopy) then heat at 95 °C for 1 hour, vortexing 1 minute every 10 minutes during the incubation; this will break open and remove proteins from within the sacculi. Resuspend as thoroughly as possible, perform a protein assay, and reduce and alkylate at this step. Centrifuge ~15,000 × g for 10 minutes; the supernatant is Fraction 1. Keep the pellet. If desired, the pellet can be extracted more than once.
To obtain Fraction 2, wash the Fraction 1 pellet as thoroughly as possible in 90% water, 10% MeOH: resuspend thoroughly, centrifuge, remove supernatant, repeat at least 8x with maximum volume, vigorously vortexing between washes. (Note that wash supernatants can be dried down and combined with Fraction 1; they will contain some additional protein released during SDS removal.) After resuspending for the last wash, transfer the homogenate to an S-Trap™. Spin out the last wash and resuspend the washed pellet in S-Trap™ Solution 6, Digestion Solution containing trypsin at 200 µg/mL (final). Incubate 6 hours to overnight at 37 C. Spin out the supernatant and wash anything remaining three times with S-Trap™ Solution 6, Digestion Solution. These are peptides from all trypsin-accessible peptidoglycan associated proteins; concentrate.
To obtain Fraction 3, depolymerize the sacculus to release proteins inaccessible in Fraction 2. For Gram+ Firmicutes (e.g., Enterococcus, Streptococcus, Listeria-like) and SDS extracted Gram− bacteria, carefully resuspend the pellet, being sure not to harm the matrix, in 25 mM ammonium formate (pH 6.0) + 1 mM MgCl₂ + 0.5 mM diminazene containing mutanolysin (200 µg/mL final) and lysozyme (200 µg/mL final). Incubate 12 hours to overnight at 37 °C. If Staphylococcus is suspected, apply lysostaphin before the mutanolysin/lysozyme digestion: resuspend the pellet in 250 mM ammonium acetate (pH 7.5) + 20 µM ZnCl₂ + 0.5 mM diminazene containing lysostaphin at 1 µg/mL and incubate 1 hour at 37 °C. Spin out to remove lysostaphin and proceed with mutanolysin/lysozyme as above. If acid-fast bacteria or Mycobacteria are suspected, in place of mutanolysin/lysozyme, lyse PG with LysB and LysA: resuspend the pellet in 100 mM ammonium acetate (pH 7.3) containing 5 mM CaCl₂ and 0.5 mM diminazene containing LysA at 200 µg/mL (final) and LysB at 200 µg/mL (final). Incubate 12 hours to overnight at 37 °C.
In all cases, spin out the flow through after enzymatic PG digestion and collect the soluble material; this is Fraction 3a and contains wall-embedded/covalently tethered proteins released from sacculi plus PG fragments and added enzymes. Assay protein concentration (include a sample with lysis buffers alone), add S-Trap™ Lysis Solution to 1x and process per standard protocol on a separate S-Trap™. Resuspend any insoluble material in S-Trap™ 1x Lysis Solution, perform a protein assay, and proceed with all steps of standard S-Trap™ sample processing. This is Fraction 3b and contains “hard leftovers”: residual wall fragments not fully solubilized, highly hydrophobic or aggregated proteins, membrane remnants, and other insoluble complexes. The exact composition will vary by organism and how thorough the PG digestion was.
Yes. As indicated in Appendix A; pellet cells and wash three times with PBS solution. To facilitate lysis and protein extraction, resuspend samples in PBS containing 100 µg/mL lysostaphin, 100 U/mL DNAsel, 25 U/mL RNAsel, and 2x EDTA free protease inhibitor cocktail; ideally cryopulverize the sample (e.g. Retsch CryoMill) or alternatively bead beat using silica beads; five rounds of bead beating for 45 seconds each, with 1 minute rest intervals in between on ice. Warm/melt (if applicable) and incubate for 30 minutes at 37 °C; sonication will improve results. Centrifuge 17,000 x g for 10 minutes. Collect soluble fraction and insoluble fraction. Wash the insoluble fraction with PBS and centrifuge at 17,000 x g for 5 minutes. Proceed with S-Trap™ digestion for the different fractions, solubilizing in S-Trap™ Solution 1, 2x Lysis Solution.
Yes. S-Trap™ can be used for protein extraction of secreted proteins. Take the solution containing your analyte molecules such as serum-free cell culture supernatant, add SDS such that the final concentration will be 5% after resuspension and concentrate (if needed) by lyophilization. After concentration, resuspend in 50 mM TEAB, perform a protein assay and proceed with S-Trap™ digestion.
As indicated in Appendix A; dissolve stool with S-Trap™ 1x Lysis Solution. Incubate at 96 °C for 5 minutes, followed by six cycles of 30 second sonication. Proceed with a protein assay and standard S-Trap™ digestion.
Yes, it is completely possible to use S-Traps™ to clean up sample that have high salt or detergent, etc. Concentrate and clean your proteins in exactly the same way on S-Trap™ Spin Columns but do not add trypsin. Rather, for gels, add 1x SDS-PAGE buffer to the trap (the volume depends on the size of the wells in your gel; at least 20 µL however), heat the trap to solubilize the proteins (5 minutes at 95 °C) and spin out your eluted proteins. For ³ 50 µg protein, this single elution is fine, and recovery is typically around 90%. If you have only a small amount of protein, perform three elutions with 0.5x Laemmli loading buffer, concentrate by SpeedVac (the SDS will become solid) and resuspend back to necessary volume. It is a good idea to both reduce and alkylate proteins before purifying them with an S-Trap™: this will prevent disulfide bond formation that can lead to a multimeric ladder in the gel. Additionally, you can then go straight to Gel-LC if MS analysis is desired.
Protein precipitation can be combined with S-Trap™ sample processing by performing precipitation and resuspending the pellet in SDS. Perform a protein assay, reduce, alkylate, precipitate, resolubilize in S-Trap™ 1x Lysis Solution, and proceed with S-Trap™ digestion.
As indicated in Appendix A; Dissect out ~1 mg tissue and freeze immediately with a dry ice bath or liquid nitrogen. Transfer frozen tissue to a 1.0 mL homogenizer. Add 100 µL (or desired volume; aim for 3- 10x w/v tissue weight: S-Trap™ 1x Lysis Solution) of S-Trap™ 1x Lysis Solution. Homogenize at 4 °C. Transfer homogenized tissue in S-Trap™ 1x Lysis Solution to a sample tube and boil for 2 minutes. Centrifuge the sample for 10 minutes at 14,000 x g at room temperature, then collect the supernatant and proceed with S-Trap™ digestion. Bone will require demineralization with EDTA; larger pieces may require days. Length and power of sonication must be optimized for each tissue. Pellets can be analyzed separately and note that they are NOT necessarily uninteresting, simply less soluble. Analyze pellets separately by making a suspension, performing all standard S-Trap™ steps and loading the suspension onto an S-Trap™.
The S-Trap™ Solution 1, 2x Lysis Solution plus methanolic S-Trap™ washes are usually sufficient to remove lipids from common samples like cell culture. However, not all lipids are fully soluble in MeOH. For particularly lipid-rich tissues like brain or adipose, perform additional on-column lipid cleanup as follows: perform the S-Trap™ protocol up to the binding. Then, rinse the proteins with 50% chloroform/50% MeOH three times filling to the max volume of the S-Trap™ Spin Column or plate. Next perform the normal washes with the 90% MeOH S-Trap™ Solution 5, Binding/Wash Solution and proceed with S-Trap™ digestion.
Alternatively, it is possible to remove the lipids before sample processing. The most effective approach is to use a cryogenic bead beater at liquid nitrogen temperature where the sample and an organic such as DCM, chloroform, or ether are pulverized together. (Note at –196 ºC, some of these solvents can be solid.) Typically, use a 5 – 10-fold volume excess of organic solvent over tissue. Warm the sample to 4 ºC, vortex, and remove the organic (filtration or centrifugation; be cautious with the density of chloroform and DCM as proteins float; add MeOH to decrease solvent density and pellet the proteins). Dissolve the sample in S-Trap™ Solution 1, 2x Lysis Solution with harsh sonication. Proteins tend to be denatured by organic lipid extractions and require extra encouragement to go into solution.
The S-Trap™ Solution 1, 2x Lysis Solution plus methanolic S-Trap™ washes are usually sufficient to remove lipids from common samples like cell culture. However, not all lipids are fully soluble in MeOH. For particularly lipid-rich tissues like brain or adipose, perform additional on-column lipid cleanup as follows: perform the S-Trap™ protocol up to the binding. Then, rinse the proteins with 50% chloroform/50% MeOH three times filling to the max volume of the S-Trap™ Spin Column or plate. Next perform the normal washes with the 90% MeOH S-Trap™ Solution 5, Binding/Wash Solution and proceed with S-Trap™ digestion.
Alternatively, it is possible to remove the lipids before sample processing. The most effective approach is to use a cryogenic bead beater at liquid nitrogen temperature where the sample and an organic such as DCM, chloroform, or ether are pulverized together. (Note at –196 ºC, some of these solvents can be solid.) Typically, use a 5 – 10-fold volume excess of organic solvent over tissue. Warm the sample to 4 ºC, vortex, and remove the organic (filtration or centrifugation; be cautious with the density of chloroform and DCM as proteins float; add MeOH to decrease solvent density and pellet the proteins). Dissolve the sample in S-Trap™ Solution 1, 2x Lysis Solution with harsh sonication. Proteins tend to be denatured by organic lipid extractions and require extra encouragement to go into solution.
| Solvent mixture(s); all v/v | Citation |
|---|---|
| 10:3:2.5 MTBE/MeOH/water; 60:30:4.5 chloroform/MeOH/water | Cai, T., et al. Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling. Journal of Lipid Research, 57(3): 388-397 (2016). |
| 30:25:41.5:3.5 chloroform/IPA/ MeOH/water | Shiva, S., et al. An efficient modified method for plant leaf lipid extraction results in improved recovery of phosphatidic acid. Plant methods, 14(1), 14 (2018). |
| 10:3:2.5 MTBE/MeOH/water | Matyash V., et al. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lipid Res. 49: 1137-1146 (2008). |
| 2:1 chloroform/MeOH |
Folch J., et al. A simple method for the isolation and purification of total lipides from animal tissues. The Journal of Biological Chemistry, 226: 497-509 (1957). Knittelfelder, O. L., et al. A versatile ultra-high-performance LC-MS method for lipid profiling. Journal of Chromatography B, 951: 119-128 (2014). |
| 4:1 MeOH/chloroform | Dawson, G. Measuring brain lipids. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 1851(8): 1026-1039 (2015). |
| 1:2 chloroform/MeOH | Bligh E. G. and Dyer W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 37: 911-917 (1959). |
| 1:1 chloroform/MeOH; others | Reis, A., et al. A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL. Journal of Lipid Research, jlr-M034330 (2013). |
| 3:1 butanol/MeOH | Löfgren, L., et al. The BUME method: a new rapid and simple chloroform-free method for total lipid extraction of animal tissue. Scientific Reports, 6, 27688 (2016). |
| Many | Christie, W. W. and Han, X. Lipid Analysis-Isolation, Separation, Identification and Lipidomic Analysis, 446 pages (2010). |
High-Throughput Proteomics
We recommend Deep 96 Square Well Plates; a 1 mL V-bottom plate for elution and 2 mL for washes.
Absolutely! Unused wells can be used later if they are covered, especially during the protein digestion step in a humidified incubator. A particularly effective approach is to cover the plate with tape folded over at the bottom of the plate. Cut the tape for the needed number of wells and remove the tape by pulling up.
S-Trap™ plates work with a variety of automation devices including KingFisher.
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