# Proteomics in Forensic Analysis: Applications for Human Samples
## 2 Proteomics Methods: Sample Preparation Techniques, Data Acquisition, and Data Analysis
### Sample Preparation
#### 2.1.3 Proteolytic Digestion  
Typical protein digestion methods that have been widely used are in-gel, in-solution, on-bead, on-slide tissue digestion, filter-aided sample preparation (FASP), and suspension trapping (S-Trap). In in-solution digestion, protein solutions or pellets are usually mixed with 8 M urea. Urea and thiourea can increase protein solubility and denature protein three-dimensional structures. Proteins are then reduced (e.g., with dithiothreitol or tris(2-carboxyethyl)phosphine) and alkylated (e.g., with iodoacetamide or chloroacetamide). These steps are necessary to reduce intra- and intermolecular disulfide bonds within and between proteins [82]. Subsequently, the proteins are digested with enzymes (e.g., trypsin and LysC). One of the most frequently used enzymes is sequencing-grade modified porcine trypsin. The typical trypsin: protein ratio is 1:20 to 1:50. Trypsin cleaves peptide bonds at the amino acid residues arginine and lysine. The resultant peptides had an average size of 800-2000 Da, which is the preferred mass range for MS/MS sequencing. These MS/MS spectra are information-rich and easily interpretable for peptide and protein identification [83]. A procedure of in-gel digestion is relatively similar to in-solution digestion. Briefly, proteins are separated by 1 or 2 dimensional (1D or 2D) polyacrylamide gel electrophoresis. The gel slice is then cut into small pieces (approximately 1 \(\times\) 1 mm). These pieces are destained with 100 mM ammonium bicarbonate/acetonitrile (1:1, _v/v_) prior to optional reduction and alkylation. After tryptic digestion, peptides are usually extracted from gel pieces using 50% ACN/5% formic acid [84]. The in-gel approach is more laborious and time-consuming than in-solution digestion. It can process only a small number of protein spots at a time. A typical example of on-bead digestion is single-pot solid-phase-enhanced sample preparation (SP3), which allows proteomic sample preparation to be carried out in a single tube with high throughput, speed, efficiency, scalability, and flexibility [85]. A complete proteomic workflow can be performed in a single SP3 tube, including protein and peptide enrichment, cleanup, digestion, chemical isotope labeling, and fractionation. In brief, after reduction and alkylation, proteins are mixed with SP3 beads for protein-bead binding. After the binding is complete, a magnetic rack is used to induce the migration of the beads to the tube wall, and unbound supernatant is removed. The proteins bound to beads are then digested using proteolytic enzymes. Subsequently, the tube is centrifuged, and a magnetic rack is used to settle the beads onto the tube wall, which allows the separation of peptides. This method has some limitations, such as difficulties in the recovery of intact proteins from the paramagnetic beads, bead clumping, and aggregation [86]. On-slide tissue digestion is a straightforward approach typically applied for formalin-fixed paraffin-embedded (FFPE) tissue section [87]. It can be used in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). After sections are cut from FFPE tissue blocks, they are washed with xylene to remove paraffin and then rehydrated in a series of ethanol washes. Subsequently, antigen retrieval is carried out in Tris\(-\)HCl (pH 8.0, 95 \({}^{\circ}\)C, 20 min). On-slide tissue digestion is conducted using spray technologies [88]. FASP consists of several ultrafiltration steps to retain proteins over ultrafiltration membranes and remove contaminants by stepwise elution. After enzymatic protein cleavage, the membranes allow separation of peptides from undigested materials [89]. In FASP, protein pellets are mixed with a buffer solution containing sodium dodecyl sulfate for solubilization, then reduced, alkylated, and added to a 30 kDa molecular weight cut-off(MWCO) FASP spin column. Samples are centrifuged to remove waste and contaminants, whereas proteins remain on the membrane.