# Proteomics in Forensic Analysis: Applications for Human Samples
## 4 Challenges, Considerations, and Future of Forensic Proteomics  
Various studies have reported potential applications of proteomics in forensic sciences. However, forensic proteomics may face several difficulties. Proteomic samples in other fields, such as microbiology, molecular biology, plant sciences, marine sciences, and food sciences, are generally clear for researchers (e.g., type of cell, tissue, or organism; species; sample matrices). In forensic proteomics, samples are sometimes unknown. In other fields of forensic science, such as identification of pathogenic species, sample preparation is generalized to the case of an uncharacterized sample. Taxonomy of the species can be narrowed down or determined using several methods [22; 176]. For human-related samples, researchers have to identify which body fluids or tissues exist in the samples [45; 149]. Another challenge in forensic proteomics is that samples may contain contaminants that are unknown to researchers. This causes many difficulties in defining which procedure to apply. One of the biggest challenges in forensic proteomics is that the sample amount from trace evidence may be very low, making sample preparation difficult. That is the case of body fluids and tissues deposited in cloth, floor, the surface of solid materials (e.g., rock, plastic), under fingernails, or on skin. When the sample amount is low, low-abundance proteins cannot be identified. However, bottom-up proteomics has been developed to allow analysis of small sample amounts, such as a single hair of approximately 1 mm length [94], epidermal corneocytes from human skin [150], and debris deposited in fingernails [141]. Another challenge of forensic proteomics relates to sample preservation. In all cases, samples have to be collected and preserved in an appropriate manner to minimize sample degradation and contamination. Usually, sampling approaches include protocols for quality assurance and quality control. After collection, samples are tightly sealed and stored in short-term (at 4 \({}^{\circ}\)C) or long-term (at \(-20^{\circ}\)C or \(-80~{}^{\circ}\)C), except for hair and nail [62]. The legal aspect is an important issue to consider in the development of proteomic methods for applications in forensics. The United States Supreme Court issued guidelines for the admissibility of scientific testimony in 1993, which is now called the Daubert standard [177]. One of the important requirements is that only scientific evidence obtained from scientific methods and principles is admissible, and the methods employed should have been tested, peer-reviewed, and widely accepted by the scientific community. In addition, methods and principles must be properly applied with suitable control samples, and error rates must be known or estimated [178]. Forensic proteomics, as a member of forensic science, should be developed and applied with sufficient consideration regarding legal issues. The proteomic method development process includes proof-of-concept, developmental verification and validation, and routine implementation (Figure 7). The proof-of-concept includes initial studies to develop protocols, determine sensitivity and specificity, and identify limitations. A standard operating protocol (SOP) constructed in this stage will be used in the second stage, developmental verification, and validation. This stage is performed with formal verification and validation experiments to determine the final SOP, identify operational limitations, and validate the methods in forensic samples with calibration and quality-control practices. All of these elements are essential for establishing reporting guidelines and obtaining certification from the International Organization for Standardization (ISO) or other accreditation prior to the application of the methods in routine operation [178]. In proteomics, a number of standard protocols and methodologies have been developed and widely applied [1; 2; 70; 116]. They have also been used in forensicscience for various applications [12; 13; 14; 15; 16; 17; 18].