Table 1.
Features of different protein digestion methods.
| Method | Feature | Ref. | |
|---|---|---|---|
| ISD | Urea-based ISD | Proteins are not separated from contaminants during reduction, alkylation, and digestion; not applicable for detergent-containing samples; sample loss; large starting amount (>100 µg protein); long digestion time; low throughput and reproducibility | [97] |
| SCASP | Use cyclodextrin to remove SDS before digestion | [98] | |
| Simultaneous lysis, reduction, and alkylation | Use TCEP and CAA; reduce time and sample loss | [99] | |
| SPEED | Use pure TFA to dissolve cells and tissues without strong detergent; use tris(hydroxymethyl)-aminomethane to neutralize sample and precipitate proteins; reduce time and sample loss | [101] | |
| In-gel digestion | Use polyacrylamide gel electrophoresis to separate proteins; cut, digest, and analyze gel spots separately; low throughput and reproducibility | [53] | |
| On-membrane digestion | FASP | Use a membrane (3000 or 10,000 Da) to separate proteins from detergents and contaminants; on-membrane digestion; tolerant to strong detergent; long centrifugal time; reduced performance with samples of low protein amount (<20 μg) | [54,102,106] |
| N-Glyco-FASP | Use lectins to enrich and PNGase F to deglycosylate N-glycopeptides | [161] | |
| MED-FASP | Use multiple enzymes for digestion | [162] | |
| iFASP | Combine FASP with TMT or iTRAQ | [107] | |
| eFASP | Use 0.2% deoxycholic acid instead of urea; increase efficiency of trypsin digestion | [108] | |
| Express eFASP | Use TCEP and 4-vinylpyridine for simultaneous reduction and alkylation | [108] | |
| MicroFASP | Use a filter with surface area of ∼0.1 mm2 to process low amount samples; applicable to samples of 100 cells or 1 μg protein | [112] | |
| μFASP | Use 96-well plates with small filter area (∼0.8 mm2) to process low amount samples; applicable to samples of 0.4 μg protein | [113] | |
| MStern | Use a membrane with 0.45 µm pore size to reduce processing time; high number of missed cleaved proteins | [114] | |
| fa-SPEED | Use pure TFA (similar to SPEED); use acetone to facilitate the protein aggregation; use 0.2 µm spin filter to reduce centrifugation time; quick hands-on time (~22 min, excluding digestion) | [101] | |
| Bead-based digestion | Proteomic reactor | Use SCX or SAX beads to bind proteins; all steps are performed in a small volume (~50 nL) of a capillary; applicable to samples of low protein amount (<10 μg) | [24,116] |
| SP3 | Use hydrophilic carboxylate-coated paramagnetic beads to bind proteins; use a magnetic rack to separate proteins from contaminants; applicable to low protein amount (~100 ng); bead clumping and aggregation | [56,121] | |
| USP3 | Use TFA to hydrolyze DNA and RNA; use TCEP and CAA; reduce time | [124] | |
| C4-tip | Use C4 RP resin tip to entrap proteins; use 30% acetonitrile in digestion buffer to increase peptide recovery and reduce missed cleavage percentage | [125] | |
| IMERs | Flow-through devices contain entrapped enzymes; less reagent consumption; fast reaction rate; applicable to low protein amount (<1 μg); integration with fractionation and LC-MS/MS; complex instrumental setups | [127,128] | |
| S-Trap | Use methanol to precipitate proteins; use a quartz or borosilicate glass depth filter to trap proteins; quick centrifugal time (~1 min per step, total process time ~20–30 min); integration with RP fractionation | [55,115] | |
| On-slide digestion | Applicable to FFPE and fresh frozen tissue slides; suitable for MALDI IMS | [143,144] | |
| Integrated methods | iST | Combine C18 membrane (filter) and SCX or SAX disks (fractionation); all steps are performed in a device; sample loss due to C18 material-protein binding; applicable to low protein amount (~1 µg) | [99,106] |
| Micro-FASP + RPLC | Combine micro-FASP and C18 microreactor (sample preparation and fractionation); chemical solution volumes ~5 µL | [176] | |
| RCPR | Use SCX column for cell loading, protein reduction, alkylation, and digestion; combine the SCX column and a C18 RP column for 2D-LC | [177,178] | |
| SISPROT | Integrate SCX or SAX or SCX+SAX beads with C18 disks in a pipet tip for digestion and fractionation (1 or 2 dimensions); applicable to low protein amount (~1 µL serum) | [23,32,34] | |
2D, 2-dimensional; CAA, 2-chloroacetamide; eFASP, enhanced filter-aided sample preparation; fa-SPEED, FASP by easy extraction and digestion; FFPE, formalin-fixed paraffin-embedded; iFASP, isobaric labeling with FASP; IMERs, immobilized enzyme reactors; ISD, in-solution digestion; iST, in-StageTip; LC–MS/MS, liquid chromatography–tandem mass spectrometry; MALDI IMS, matrix-assisted laser desorption/ionization imaging mass spectrometry; MED-FASP, multiple enzyme digestion with FASP; RCPR, rare cell proteomic reactor; RP, reserved-phase; SAX, strong anion exchange chromatography; SCASP, sodium dodecyl sulfate (SDS)–cyclodextrin-assisted sample preparation; SCX, strong cation exchange chromatography; SP3, single-pot solid-phase-enhanced sample preparation; SPEED, sample preparation by easy extraction and digestion; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; TMT, tandem mass tag; iTRAQ, isobaric tag for relative and absolute quantitation; USP3, universal solid-phase protein preparation.