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View additional product information for SMART Digest™ Trypsin Kits - FAQs (60109-101-B, 60109-102-B, 60113-101, 60113-101-10PK, 60109-103-B, 60109-103-MB, 60109-102, 60109-101-LPH, 60109-103, 60109-102-MB, 60109-101, 60109-101-MB)
16 product FAQs found
Yes, we recommend the following protocol, which can be optimized as needed:
1. Rinse a Fisher Scientific Vivacon 500, 30kDa MWCO filter (Cat. No. 14558347) to remove trace amounts of glycerin using a fill volume of water. Decant the filtrate and collection vessel. The filter is now ready for use. If you do not want to use the Vivacon filter immediately, then store the pre-rinsed device in the refrigerator with water or buffer covering the membrane surface. Do not allow the membrane to dry out.
2. Dilute sample water to a total volume of 150 µL on the filter.
3. Centrifuge 14,000 RPM for 15 minutes and discard the flow-through.
4. Add 150 µL water.
5. Centrifuge 14,000 RPM for 15 minutes and discard the flow-through.
6. Add 200 µL of SMART Digest buffer.
7. Change the collection vial and add 5 µL of the SMART Digest soluble enzyme to the filter.
8. Parafilm the filters to prevent evaporation of sample during digestion. Incubate the units in a heated bath at 70°C for 60 - 90 minutes.
9. Optional: As needed, add DTT (3 µL of 50 mM DTT), heat at 37°C and incubate for 30 minutes. Alkylate as needed (using 3 µL of 150 mM alkylating reagent).
10. After incubation, add 2 µL of 1% trifluoroacetic acid to the filter. Mix for 1 minute.
11. Centrifuge the filter units at 14,000 RPM for 10 min.
12. As needed, measure protein concentration using a Thermo Scientific NanoDrop Spectrophotometer, or similar procedure.
To automate the SMART Digest Soluble Trypsin Kit workflow, we recommend using deepwell plates with Sepra Seals, or PCR plates with iron-on seals in combination with an automated liquid handling platform. Any heating unit with a heated lid, capable of providing uniform heating at 70°C may be used.
The kit is compatible with up to 1M urea, but incompatible with most concentrations of guanidine.
The kit is compatible with many surfactants including OGS, CHAPS, Nonidet, and NP-40.
Thus far, studies have shown that by accelerating the reaction and minimizing the reaction time, fewer chemical modifications are observed in comparison to protocols utilizing elongated digestion times at lower temperatures. For more details, refer to the application note titled 'SMART Digest Compared to Classic In-Solution Digestion of Rituximab for In-Depth Peptide Mapping Characterization'.
See the publication, 'Thiol-Disulfide Exchange in Human Growth Hormone' for details (Pharm Res (2016) 33:1370–1382 DOI 10.1007/s11095-016-1879-3). No disulfide bond rearrangement was observed. However, free cysteines could potentially lead to scrambling. Alkylate free cysteines to prevent disulfide bond scrambling, as needed.
The SMART Digest Soluble Trypsin Kit was engineered to be thermally stable. When operated at high temperatures (e.g. 70°C), denaturation and digestion happen simultaneously. Therefore, for many quantitative workflows there is no need to perform the additional steps of denaturation, reduction and alkylation. However, during this process many disulfide bonds will remain intact. As such, for many identification workflows it is recommended that you perform reduction and alkylation after digestion. In our experience, denaturants and reducing reagents negatively impact digestion when using the SMART Digest Soluble Trypsin Kits.
The pH of the digestion buffer as received in the SMART Digest Soluble Trypsin Kit is approximately 8. When used at 70°C, the pH of the buffer is approximately 7. Operation at this lower pH helps to prevent the formation of artificial modifications.
The SMART Digest buffer in the SMART Digest Soluble Trypsin Kit contains ~ 0.5 M salts. The salts greatly aid in achieving rapid digestion at high temperatures. Desalting through the use of valve switching is highly advised, although the use of SPE cleanup has also been successful.
The SMART Digest buffer was optimized for maximum trypsin activity at elevated temperatures. Other buffers can be used, but their use may negatively impact trypsin activity. If your application requires the use of an alternative buffer, digestion time and temperature should be optimized accordingly.
All proteins vary with regards to digestion so you will need to adjust the temperature and incubation time accordingly. We recommend the following strategy for screening digestion time:
1. Create a method in your heating unit setting the desired operating temperature (we recommend 70°C). Start this method and allow the temperature to reach equilibrium for at least 5 minutes before adding samples.
2. Prepare eight identical samples using a relatively high known concentration of native analyte in the matrix of operation, diluting them to 50 µL each with ultrapure water, if necessary.
3. Add each sample along with 150 µL of SMART Digest buffer and 5 µL of SMART Digest soluble enzyme to each of the eight wells.
4. Place all samples firmly into the preheated unit.
5. Periodically (e.g. every 15 minutes) remove a sample and quench the reaction using an equivalent volume of 1% trifluoroacetic acid, or 1% formic acid.
6. Following incubation analyze the samples to determine the extent of digestion.
7. Once the intact protein peak has disappeared, digestion is complete and the corresponding digestion time can be used for subsequent analyses.
Do not reduce and alkylate sample prior to digestion. Our findings indicate that the chemicals used for denaturation, reduction and alkylation may negatively impact the activity of the enzyme and the solubility of the protein sample. If the peptides of interest require reduction prior to analysis it is recommended that these steps be performed post digestion.
Generally, there is no requirement to vary the amount of trypsin used for any given sample. Studies have shown that when operating at elevated temperatures with a stable enzyme, the reactions are significantly less concentration dependent than traditional protocols. For samples containing less than 5 µg of total protein, titering the enzyme amount may prevent an excess of enzyme from interfering with the analysis.
For every 5 µL of the SMART Digest Soluble Trypsin Kit up to 50 µL of plasma (approx. 3.5 mg of protein) is readily digested.
The trypsin enzyme concentration is 1 µg/µL so there would be 5 µg of trypsin in 5µL.
As long as the substrate is soluble, shaking is not required.
We recommend using any heating unit with a heated lid that is capable of providing uniform heating at 70°C.