Thermo Scientific™ SMART Digest™ Immunoaffinity (IA) Protein G Kits

Yes, perform additional enzymatic digest reactions after digestion with the SMART Digest ImmunoAffinity (IA) Kits.

No, the kits are not compatible with gels as the beads are unable to penetrate gels and start digestion.

The current digest buffer contains amines so it is not compatible with isobaric tagging. However, amine-free buffers are available. For more information contact Technical Support at www.thermofisher.com/chromexpert

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

The resin is a 20 µm PS-DVB core made hydrophilic with a two-tailed coating.

Yes, our extensive studies have shown that complete sample digestion is achieved in as little as 5 minutes for simple, mono-protein samples, and 3.5 hours for complex matrices, such as plasma.

The immobilization of trypsin in SMART Digest ImmunoAffinity (IA) Kits 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 MS compatible surfactant that does not negatively impact trypsin activity. It is not charged, so it does not impact MS ionization. It also exists as one molecular weight, so it does not result in multiple background peaks.

In comparison to in-solution digests, a comparable number of 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 IA 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 we recommend that you perform reduction and alkylation after digestion. Denaturants and reducing reagents can negatively impact digestion using the SMART Digest IA kits.

The pH of the SMART Digest IA buffer is approximately 7.2.

The SMART Digest buffer in the SMART Digest ImmunoAffinity (IA) Kits contains about 0.5M salts. These salts enable quick digestion at high temperatures. We recommend using valve switching or Thermo Scientific SOLAµ SPE cleanup for desalting.

The SMART Digest IA 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. Temperature and incubation times can be adjusted for optimal results. We recommend the following process for screening digestion time of your protein:
1. Create a method in your heater/shaker and set the temperature to 70°C and RPM to 1400.
2. Allow the temperature to reach equilibrium for at least 5 minutes.
3. Prepare eight 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. Add them to individual SMART Digest wells.
4. Add 150 µL of SMART Digest IA buffer to each well and cap.
5. Place all wells firmly into the pre-heated heater/shaker.
6. Periodically remove a sample from the strip (every 5 to 15 minutes).
7. Centrifuge, filter or perform an SPE process with a SOLAµ HRP plate (Cat. No. 60209-001) and then analyze the samples to determine the extent of digestion.

Recommended digestion time for 200 µL protein solution (100µg/mL) at 70°C:
(Protein - digestion time)
• 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 a result, there is no need to vary the amount of trypsin used for any given sample.

Every 30 µL of beads contains 14 µg of immobilized trypsin.

We recommend a digesting material ranging from 200 pg to 3.5 mg.

Following the immunoaffinity enrichment and wash steps, the sample volume is generally reduced to 50 µL or less. For every 1 µL of sample remaining, 3 µL of digest buffer should be added. The minimum recommended digestion volume is 50 µL (12.5 µL sample post wash, 37.5 µL digestion buffer) and the maximum recommended volume is 400 µL. Generally, 200 µL is used (50 µL sample post wash and 150µL digestion buffer). Refer to the SMART Digest ImmunoAffinity (IA) Kit User Manual for additional information).

We have successful applications in mouse, monkey, beagle and human plasmas. We also have successful applications in cell lysates, urine and cerebral spinal fluid.

We do not recommend using your standard PCR instrument because the shaking is necessary to avoid any diffusion limitations.

Uniform heating is key to sample reproducibility; shaking is a necessity to avoid any diffusion limitations. A heater shaker device with the following features is required:
• Heating block capable of uniformly heating samples to 70 °C
• Heated lid
• Shaking

Volumes ranging from 5 to 1000 µL of sample per well are readily processed.

Samples can be added directly to the SMART Digest IA kit, Protein A and G beads for bulk fractionation of immunoglobulins. For this workflow, no cross-linking is required. Be sure to take into account the capacity of the beads when developing a bulk fractionation workflow.

The literature shows that many crosslinking methods are effective. We recommend using a glutaraldehyde based cross-linking method. For additional details refer to the cross-linking procedure outlined in the SMART Digest ImmunoAffinity (IA) Kit User Manual.

We did not observe any reduction in loading capacity in tests where the resin was prewashed with plasma, compared to those were it was loaded with antibody first.

The digestion is temperature and buffer-dependent. Because the affinity step is performed at lower temperatures (room temperature), in an optimal binding/suboptimal digestion buffer, no digestion is normally observed during the affinity step. Once the buffer is changed to the digestion buffer, which is designed to enhance digestion, and the sample is heated, digestion will proceed rapidly.

We recommend using a 30 µL aliquot of beads per sample. The loading capacity for 30 µL of beads is at least 5 µg of antibody or more.