# Proteomics in Forensic Analysis: Applications for Human Samples
## 2 Proteomics Methods: Sample Preparation Techniques, Data Acquisition, and Data Analysis
### Sample Preparation
#### 2.1.1 Sampling and Sample Pretreatment  
Samples can be collected from human bodies and crime scenes. Body fluids are usually collected using wide-bore pipettes or disposable hypodermic syringes with appropriate needle gauges and lengths. Antemortainilippings are obtained from all fingers and toes using Teflon-coated stainless steel scissors, whereas post-mortem nails are lifted from all fingers and toes. Most of the samples can be stored in appropriate plastic containers with screw caps [60]. Sample collection devices in crime scenes include wipes, dry swabs, premoistened swabs, aspirating needles, high-efficiency particulate air vacuums, and filters [61]. After collection, hair and nail samples can be stored at room temperature. Other samples should be stored in tightly-sealed containers at 4 \({}^{\circ}\)C for short-term storage and at \(-20\)\({}^{\circ}\)C or preferably at \(-80\)\({}^{\circ}\)C for long-term storage. Plasma and serum required for analysis should be separated before storage [62]. Sample pretreatment methods are selected depending on the sample type. If samples containing proteins are culture media, exosomes, or body fluids such as serum, plasma, saliva, tears, nasal fluids, urine, or aqueous humor, they do not require lysis pretreatment. In these cases, an appropriate amount of buffer is added to favor subsequent enzymatic digestion steps [63; 64]. If samples are cells, a lysis step is applied to break the cell membrane. Lysis is usually performed using lysis buffer and optional sonication [65]. Probe sonication is one of the most common methods of cell disruption.  
Figure 1: Overview of bottom-up proteomics.  
In the case of solid samples such as human tissue and bone, the samples are usually disrupted by mortar and pestle, cryogenic grinding, cryo-pulverization (e.g., using Covaris CP02 cryoPrep automated dry pulverizer), and bead beating. Bone powder can be treated with a weak acid for partial demineralization prior to digestion [66]. Soft tissues such as the brain and liver are appropriate for manual pestle homogenization or bead homogenization. In manual pestle homogenization, tissues are placed into tubes (e.g., Potter homogenizer) and manually homogenized with a pestle while the tubes are kept on ice [67]. A lysis buffer is added before the homogenization. Homogenization is completed when no large pieces of tissue are observed [68]. In bead homogenization, tissues are placed in pre-chilled tubes with tungsten carbide, stainless steel, or glass beads and subjected to high-speed shaking [69]. Hard tissues (bone and fingernails) and those with tough connective fibers are appropriate for cryo-pulverization. Typically, tissues are quickly frozen in liquid nitrogen and disrupted by force [70]. For example, the Covaris CP02 cryoPrep automated dry pulverizer allows users to control the force for disruption. It is equipped with tissue tubes (strong and flexible plastic devices developed to resist extreme temperature fluxes) to prevent sample loss. Tissues were placed in these tubes, frozen, and pulverized to powder [71]. Lysis buffer solutions were added to the tubes to collect all the samples for further steps. If this system is not available, the samples are placed in a mortar with liquid nitrogen and gently ground with a pestle. Sometimes, mortar and pestle are also used for lyophilized tissues without liquid nitrogen [72; 73]. Generally, the resultant samples undergo a lysis step with lysis buffer and sonication.  
In many cases, samples are bound to matrices such as cloth, cotton swab, filter surface, or rock, and thus should be solubilized in an appropriate buffer solution (e.g., triethylammonium bicarbonate (TEAB), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), and ammonium bicarbonate with or without detergents). Sample-bearing materials were placed inside a tube with a buffer solution and subjected to sonication or vortexing to collect the targeted samples.