S-Trap vs FASP vs in-solution digestion
Three ways to get from protein to peptide, compared on the criteria that matter at the bench: detergent tolerance, recovery, time, throughput and where each one fails.
The three methods in one paragraph each
In-solution digestion is the oldest route: denature (urea or a mild detergent), reduce, alkylate, dilute, digest, then desalt. It is cheap and needs no device, but SDS is incompatible and must be removed before digestion, usually by precipitation, which costs material and reproducibility.
FASP (filter-aided sample preparation) traps protein on a molecular-weight cut-off ultrafiltration membrane so that SDS can be exchanged for urea and washed away before digestion on the filter (Wiśniewski et al., Nat Methods 2009). It made SDS lysis compatible with proteomics, at the cost of long centrifugation steps and variable recovery that depends on the membrane.
Suspension trapping (S-Trap™) keeps SDS lysis but replaces the membrane with a depth filter: the acidified, methanol-diluted lysate forms a fine protein suspension that is captured in the filter, SDS is washed through, and protease is added into the trap (Zougman et al., Proteomics 2014; HaileMariam et al., J Proteome Res 2018). Digestion is typically complete in about an hour.
Side by side
| Criterion | S-Trap™ | FASP | In-solution |
|---|---|---|---|
| SDS tolerance | Yes, removed in the trap | Yes, exchanged on the filter | No, must be removed first |
| Hands-on time | Low | Moderate to high | Low to moderate |
| Digestion time | ~1 h | Overnight (typical) | Overnight (typical) |
| Recovery | High and consistent in our hands | Membrane-dependent, variable | Variable; precipitation losses |
| Low input | Micro format to ~1 µg | Poor below ~20 µg | Possible with care |
| 96-well format | Yes | No | Yes |
| Difficult matrices (FFPE, membrane, lipid-rich) | Strong | Workable | Weak |
| Consumable cost | Highest per sample | Moderate | Lowest |
Where S-Trap™ is not the answer
If you already get clean, reproducible data from in-solution digestion of a simple cell lysate, suspension trapping costs more per sample for little gain. Its case is strongest for matrices where SDS is the only lysis buffer that works, for low-input samples where every loss step matters, and for plate-format throughput. It is weakest as a drop-in replacement for a workflow that is not broken.
A note on the bead-based alternative: SP3 (Hughes et al., Mol Syst Biol 2014; detailed protocol in Nat Protoc 2019) uses carboxylate-coated magnetic beads to capture protein by aggregation, and is excellent at very low input and for automation. Its characteristic failure is beads carried into the LC, which is what bead-removal plates exist for.
References
- Wiśniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods 2009;6:359-362. PubMed · DOI
- Zougman A, Selby PJ, Banks RE. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis. Proteomics 2014;14:1006. PubMed · DOI
- HaileMariam M et al. S-Trap, an ultrafast sample-preparation approach for shotgun proteomics. J Proteome Res 2018;17:2917-2924. PubMed · DOI
- Hughes CS et al. Ultrasensitive proteome analysis using paramagnetic bead technology. Mol Syst Biol 2014;10:757. Open access PubMed · DOI · Full text (PMC)
- Hughes et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc 2019. PubMed · DOI
Citations verified against PubMed records; “Open access” marks papers whose full text is freely available via PubMed Central or the journal.
Written by the omicslink proteomics team. Reviewed November 2026.
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