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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)
27 product FAQs found
• CHAPS – results in ~ 30% reduction in digest efficiency.
• OGS – no reduction in digestion efficiency.
• TWEEN – no reduction in digestion efficiency.
• RIPA – results in ~ 20% reduction in digestion efficiency. The addition of RIPA, for ribonuclease A digestion, results in a concentration-dependent effect, where initial enzyme inhibition is overcome by improved substrate solubilization at higher concentrations only.
The SMART Digest Trypsin Kit is not affected by concentrations of up to 0.5 M of urea. If the concentration is higher than this, we recommend that the sample is diluted to less than 0.5 M of urea prior to beginning digestion using the SMART Digest Trypsin Kit.
The composition of the SMART Digest Trypsin Kit buffer is as follows:
Chemical Name (CAS No., EINECS No.): Kit Component Quantity, Weight %
• Water (7732-18-5, 231-791-2): 2, 50-95%
• Glycerol (56-81-5, 200-289-5): 2, < 20%
• Tris Base (77-86-1, 201-064-4): 2, < 10%
• Tris-HCI (1185-53-1, 214-684-5): 2, < 10%
• Calcium Chloride (10043-52-4, 233-140-8): 2, < 10%
• Sodium Azide (26628-22-8, 247-852-1): 2, < 0.1%
The SMART Digest Trypsin Kit contains an excess of enzyme capable of digesting between 200 pg and 3.5 mg of protein. As most samples will fall in this range, it is not necessary to routinely quantitate protein concentration prior to digestion.
The reason urea is needed as a first step in an in-solution protocol is to disrupt the sample and partially unfold the proteins. The proteins need to be partially unfolded so that the trypsin enzyme can have better access to the internal amino acid chain, not just the surface of the protein of interest. The reason the SMART Digest Trypsin kit does not need urea is that it uses heat to unfold the protein.
Yes, the SMART Digest Trypsin Kit can be used with Lys-C and other enzymes. We recommend to perform the additional steps post-digestion.
No the SMART Digest Trypsin Kit is not compatible with gels, as the beads are unable to penetrate gels and start digestion.
Yes, the SMART Digest Trypsin Kit is compatible with isobaric tagging. We recommend to perform the tagging post-digestion.
The reducing agent lowers digestion efficiency and adds extra steps unless you are specifically looking for disulphides.
The resin is made of 10 µm PS-DVB core made hydrophilic with a two-tailed coating.
Yes, extensive studies have shown that complete sample digestion is achieved in as little as 5 minutes for simple mono-protein samples, to 3.5 hours for complex matrices such as plasma.
The immobilization of trypsin prevents it from attacking and digesting itself, contrary to what happens in an in-solution digestion.
During the immobilization process the trypsin is chemically modified in such a way that it is chemically stabilized while maintaining its specificity. The selectivity of the cleavage site is not affected.
Many surfactants negatively impact not only digestion, but LC-MS performance as well. Of the surfactants we have screened, octylglucoside is the only surfactant that does not negatively impact trypsin activity. It is not charged so it does not impact MS ionization, and it exists as one molecular weight so it does not result in multiple background peaks.
In comparison to in-solution digests, a comparable number of post-translational modifications (PTMs) have been observed when screening for deamidation, amidation, methylation and oxidation. No modifications to existing PTMs, such as phosphorylated sites, have been observed.
If there are free cysteines, it is possible for disulfides to scramble before, during or after digestion. We would therefore recommend performing alkylation prior to digestion.
The SMART Digest Trypsin kits were engineered to be thermally stable. When operated at high temperatures (e.g. 70°C), denaturation and digestion occurs 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 a result, for characterization workflows where maximum sequence coverage is required, it is recommended that you perform reduction and alkylation after digestion. Denaturants and reducing reagents can negatively impact digestion using the SMART Digest Trypsin kits.
The pH of the SMART Digest buffer is approximately 7.2.
The SMART Digest buffer that comes with the SMART Digest Trypsin Kit contains about 0.5M salts. These salts greatly assist in achieving rapid digestion at high temperatures. Desalting through the use of valve switching, or the use of Thermo Scientific SOLAµ SPE cleanup is advised.
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 and you will need to adjust temperature and incubation time accordingly. A recommended strategy for screening digestion time is outlined below:
1. Create a method in your heater/shaker to set temperature to 70°C and RPM to1400.
2. Allow the temperature to reach equilibrium for at least 5 minutes.
3. Prepare 8 identical samples using a relatively high known concentration of native analyte in the matrix of operation (dilute them to 50 µL with ultrapure water, if necessary) and add to individual SMART Digest wells.
4. Add 150 µL of SMART Digest buffer to each well and cap.
5. Place all wells firmly into the preheated Heater Shaker.
6. Periodically (e.g. every 5 to 15 minutes) remove a sample from the strip.
7. Centrifuge, filter or perform an SPE process with a SOLAµ HRP plate (cat. No. 60209-001) then analyze the samples to determine the extent of digestion.
8. Once the intact protein peak has disappeared, digestion of the protein is complete and the corresponding digestion time can be used for subsequent analyses.
Refer to the following recommended digestion starting conditions (200µL protein solution at 100µg/µL concentration, 70°C):
• Insulin - 4 minutes
• BSA - < 5 minutes
• Carbonic anhydrase - < 5 minutes
• Lysozyme - < 5 minutes
• Apo-B - 30 minutes
• IgG - 45 minutes
• IgG in 50 µL plasma - 75 minutes (17.5 µg/mL)
• Ribonuclease A - 150 minutes
• Thyroglobulin - 240 minutes
• C-reactive protein - 240 minutes
One of the benefits of using immobilized trypsin is that there is reduced autolytic activity. As such, there is no need to vary the amount trypsin used for any given sample.
Each well contains 14 µg of immobilized trypsin.
Up to 50 µL of plasma (approx. 3.5 mg) and as little as 200 pg can be digested with the SMART Digest Trypsin Kits.
To date we have successful applications in mouse, monkey, beagle and human plasma samples. We also have successful applications in cell lysate, urine and cerebral spinal fluid samples.
Unfortunately, you cannot use a standard PCR instrument. Shaking is a necessity to avoid any diffusion limitations.
The PCR format is key to sample reproducibility; shaking is a necessity to avoid any diffusion limitations. A heater shaker device with the following features is required:
• PCR heating block
• Heated lid
• Shaking