Yes, but for some workflows, reduction and alkylation are not required (see below). If reduction and alkylation are required or helpful, we recommend the following protocol.

1) Add DTT (5mM final concentration) to sample in 75% SDB and incubate for 30 minutes at 60°C

2) Add iodoacetamide (15mM final concentration) to sample and incubate for 30 minutes at room temperature.

3) Add SMART Digest Trypsin and digest at 70°C.

As an alternative, we recommend the following protocol:

1)  Add TCEP (2.5mM final concentration) to sample in 75% SDB and incubate for 30 minutes at 60°C

2)  (Optional when using TCEP) Add iodoacetamide (7.5mM final concentration) to sample and incubate for 30 minutes at room temperature.

3)  Add SMART Digest Trypsin and digest at 70°C

Examples of reduction and alkylation being performed as part of a SMART Digest workflow can be found in the following publications:

• Rauniyar, N., et al. (2024) Comparative analysis of Herceptin N-Linked glycosylation by HILIC-FLD and LC-MS/MS methods. Journal of Pharmaceutical and Biomedical Analysis, 244, 116123.

• Kristensen, D. B., et al. (2022). Optimized Multi-Attribute method workflow addressing missed cleavages and chromatographic Tailing/Carry-Over of hydrophobic peptides. Analytical Chemistry, 94(49), 17195–17204.

The use of chaotropes is not recommended. Guanidine is incompatible with SMART Digest at most concentrations while SMART Digest is compatible with 0.5M urea and Soluble SMART Digest is compatible with 1M urea.

The SMART Digest kits are thermally stable. When operated at high temperatures denaturation and digestion occur 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 as part of your workflow.

Any device capable of performing uniform heating is compatible with the SMART Digest Kits. We generally recommend either PCR formats, or deepwell plates with seals (like the Sepra Seals Thermo Part No. 4463). For the SMART Digest and SMART Digest Immunoaffinity kits, shaking is a necessity to avoid any diffusion limitations.

A heater/shaker device with the following features is recommended:

• PCR or deepwell plate heating block

• Heated lid

• Shaking

When using the Soluble SMART Digest Kits with soluble samples, shaking is not required.

All proteins vary with regards to digestion; adjust temperature and incubation time accordingly.

A recommend strategy for screening digestion time is outlined below.

1. Create a method in your heater/shaker−set temperature to 70 ºC and RPM to 1400.

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 wells.

4. Add 150 μL of SMART Digest buffer and the SMART Digest enzyme to each well then 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. When using Soluble SMART Digest, samples can be analyzed directly. For SMART Digest, centrifuge, filter or perform an SPE process with a SOLAμ HRP plate (60209-001) then analyze the samples to determine the extent of digestion.

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.

Yes, the SMART Digest buffer contains 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.

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.

See “SMART Digest compared to classic in-solution digestion of rituximab for in-depth peptide mapping characterization” for additional details.

The results of compatibility testing of CHAPS, OGS, TWEEN and RIPA are summarized below:

• CHAPS – Reduction ~ 30% in digest efficiency.

• OGS – no reduction in digestion efficiency.

• TWEEN – no reduction in digestion efficiency.

• RIPA – 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. 20% reduction in digestion efficiency.

Additional surfactants may be compatible as well. It should be noted that SMART Digest is a highly active enzyme, so that small reductions in digestion efficiency do not always impact the overall workflow.