Thermo Scientific™ SMART Digest™ Trypsin Kits

Catalog No.	Format	Type	Includes
03-150-404	Resin in Pre-filled PCR Tubes	Trypsin Kit	Resin, Collection Plate
03-251-893	Bulk Low pH Resin	Trypsin Kit	Resin, Collection Plate
03-251-897	Resin in Pre-filled PCR Tubes	Trypsin Kit	Low pH Resin, Collection Plate
03-150-406	Resin in Pre-filled PCR Tubes	Trypsin Kit	Resin, Collection Plate, SOLAμ SPE Plate
03-251-894	Bulk Low pH Magnetic Resin	Trypsin Kit	Resin, Collection Plate
03-150-405	Resin in Pre-filled PCR Tubes	Trypsin Kit	Resin, Collection Plate, Filter Plate
03-375-364	Bulk Non-magnetic Resin	Trypsin Kit	Resin, Buffer
03-253-935	Soluble Trypsin	Soluble Trypsin Kit - Low pH	Soluble Trypsin, Low pH Buffer, Collection Plate
03-252-203	Bulk Magnetic Resin	Trypsin Kit	Resin, Buffer
03-252-204	Bulk Non-magnetic Resin	Trypsin Kit	Resin, Buffer, Collection Plate, Filter Plate
13-622-452	Bulk Magnetic Resin	Trypsin Kit	Resin, Buffer, Collection Plate, Filter Plate
03-252-205	Bulk Non-magnetic Resin	Trypsin Kit	Resin, Buffer, Collection Plate, SOLAμ SPE Plate
03-251-711	Soluble Trypsin	Soluble Trypsin Kit	Soluble Trypsin, Buffer, Collection Plate
03-252-206	Bulk Magnetic Resin	Trypsin Kit	Resin, Buffer, Collection Plate, SOLAμ SPE Plate
03-170-489	Soluble Trypsin	Soluble Trypsin Kit - Low Salt	Soluble Trypsin, Buffer, Collection Plate

Catalog No. 03-150-404 Supplier Thermo Scientific™ Supplier No. 60109101

Description

Obtain fast, simple, and highly reproducible trypsin protein digestion for peptide characterization and quantitation in biopharmaceutical protein research.

Obtain high quality analytical results from protein digests using Thermo Scientific™ SMART Digest™ Kits. A significant advance in sample preparation for biopharmaceutical protein research, the kits provide fast and simple protein digestion with high reproducibility, high sensitivity, and high levels of data quality in a format that's compatible with automation. The SMART Digest Kits are a significant improvement over current in-solution protein digestion technologies, which are not reproducible, have poor sensitivity, and employ protracted methodologies not amenable to automation.

Trypsin Protein Digestion

SMART Digest kits enable you to characterize and quantitate proteins faster, easier, and quicker than ever before. This version of the kit uses trypsin, which cleaves peptides on the C-terminal side of lysine and arginine amino acid residues.

Easy and simple

• Significantly increased speed of sample processing
• Simple process
• Easily automate entire protocol for high-throughput processing

Reliable Results

• Increased reproducibility over existing protocols
• Higher sensitivity
• Higher data confidence

Significantly Reduces Sample Preparation Time

Typically it takes less than 60 minutes (dependent on sample complexity) to achieve full digestion, whereas an in-solution digest can take up to 24 hours.

Cleaner Samples

Reduction in the number of chemicals used and the immobilized trypsin design helps mitigate against chemically induced post translational modifications (PTMs) and autolysis. This results in results which are easier to interpret.

Couple with SOLAμ SPE Plates for Desalting

When used with the SMART Digest kits for desalting, Thermo Scientific™ SOLAμ™ SPE solid phase extraction plates provide a fast, easy-to-use, robust, and easily automatable sample preparation solution for analysis of biomolecules.

Specifications

• Type: Trypsin Kit
• Description: SMART Digest Trypsin Kit, with Collection plate
• Format: Resin in Pre-filled PCR Tubes
• Enzyme: Trypsin
• Cleavage: C-terminal side of lysine and arginine amino acids
• Capture: None
• Product Line: SMART Digest
• For Use With (Application): Peptide Mapping
• Includes: Resin, Collection Plate

Frequently Asked Questions (FAQs)

How compatible is the SMART Digest Trypsin kit with detergents (e.g. CHAPS, OGS, TWEEN and RIPA)?

• 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.

How much urea can I use with the SMART Digest Trypsin Kit?

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.

What is the composition of the SMART Digest buffer that comes with 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%

Why don't I need to quantitate the protein for the SMART Digest Trypsin Kit?

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.

Why don't I need to use urea to unfold the protein in the SMART Digest Trypsin Kit?

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.

Can I use the SMART Digest Trypsin Kit with Lys-C or other enzymes?

Yes, the SMART Digest Trypsin Kit can be used with Lys-C and other enzymes. We recommend to perform the additional steps post-digestion.

Is the SMART Digest Trypsin Kit compatible with gels?

No the SMART Digest Trypsin Kit is not compatible with gels, as the beads are unable to penetrate gels and start digestion.

Is the SMART Digest Trypsin Kit compatible with isobaric tagging (e.g. SILAC, ITRAQ, etc.)?

Yes, the SMART Digest Trypsin Kit is compatible with isobaric tagging. We recommend to perform the tagging post-digestion.

Why do I need to perform reduction and alkylation post digestion for the SMART Digest Trypsin Kit?

The reducing agent lowers digestion efficiency and adds extra steps unless you are specifically looking for disulphides.

What is the composition of the resin to which the trypsin is coupled in the SMART Digest Trypsin kit?

The resin is made of 10 µm PS-DVB core made hydrophilic with a two-tailed coating.

Is complete sample digestion achieved with the SMART Digest Trypsin Kit?

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.

What helps mitigate against trypsin self-digestion (autolysis) in the SMART Digest Trypsin Kit?

The immobilization of trypsin prevents it from attacking and digesting itself, contrary to what happens in an in-solution digestion.

Is the trypsin in the SMART Digest Trypsin Kit mutated to be heat stable? Would this affect my sample?

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.

Can I use surfactants with the SMART Digest Trypsin kit?

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.

Does digestion using the SMART Digest Trypsin kit at high temperatures result in an increase in post-translational modifications?

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.

Will disulfide bonds scramble during digestion with the SMART Digest Trypsin Kit?

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.

Do I have to reduce and alkylate my protein for use with the SMART Digest Trypsin Kit?

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.

What is the pH of the SMART Digest buffer that comes in the SMART Digest Trypsin Kit?

The pH of the SMART Digest buffer is approximately 7.2.

Are there salts in the SMART Digest buffer that comes with the SMART Digest Trypsin Kit?

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.

Do I have to use the SMART Digest buffer with the SMART Digest Trypsin Kit?

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.

What is a typical digestion time for the SMART Digest Trypsin Kit?

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

Can I vary the amount of trypsin used for the SMART Digest Trypsin Kit depending on protein load?

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.

How much trypsin is there in each well of the the SMART Digest Trypsin Kit?

Each well contains 14 µg of immobilized trypsin.

What amount of materials can I digest with the SMART Digest Trypsin Kit?

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.

What kind of samples have you worked with for the SMART Digest Trypsin Kit?

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.

Can I use my standard PCR instrument for the SMART Digest Trypsin Kit?

Unfortunately, you cannot use a standard PCR instrument. Shaking is a necessity to avoid any diffusion limitations.

What heater shaker device should I use for the SMART Digest Trypsin Kit?

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