# Proteomics in Forensic Analysis: Applications for Human Samples
## 3 Applications of Forensic Proteomics for Human Samples
### Identification of Body Fluids and Tissues  
Conventional DNA analysis can determine the donor's identity from human body fluids or tissues collected at crime scenes. However, a genetic profile alone cannot distinguish the body fluid or tissue type of samples (e.g., whether the fluids are blood, saliva, vaginal fluid, semen, or menstrual blood). Identifying specific body fluids or tissue sources can provide critical information on the context in which the samples may have been deposited [149]. Forensic serology is the study of classifying human body fluids, which relies on chemical reaction assays, enzyme activity assays, immunosassays, and microscopy-based assays [150]. For example, specific antibodies can determine the hemoglobin of blood, alpha amylase of saliva, semenogelin, and other prostate-specific antigens of semen. However, these tests are often presumptive, and they are usually unable to make a definite statement about the body fluid or tissue origin of a sample [12]. To achieve the final conclusion of the human body fluid type, a sample may be subjected to multiple tests, which is challenging for a limited sample amount [150]. Thus, different novel technologies have been developed, including the use of mRNA and miRNA, epigenetics, spectroscopic techniques (Raman spectroscopy, Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and fluorescence spectroscopy), biosensors, and proteomics [151]. Proteomics has emerged as a powerful and feasible strategy, allowing detection of various proteins in a single experiment and requiring only a small amount of sample. Notably, sample preparation procedures for targeted proteomics and conventional DNA analysis are highly compatible [152]. To determine the identity of body fluids, untargeted proteomics is performed, as depicted in Figure 4a. This workflow includes the extraction of proteins from materials, protein digestion, and LC-MS/MS analysis of peptides. Based on the proteomic results, one can use a decision tree (Figure 4b) developed by [PERSON] et al. in 2003 to identify which body fluids are available [14]. This decision tree is based on a set of marker proteins for different biological fluids. Hemoglobin (alpha and beta subunit) is present in blood in large amounts and with high specificity, and is thus used as a biomarker of blood [153]. In addition, proteomic analysis can determine species-specific peptides, which allow distinguishing between different species of origin. A previous study tested three species, including _Homo sapiens_, _Canis familiaris_, and _Bos taurus_, and unambiguously identified species-specific peptides [154]. Marker proteins of semen are semenogelin 1 and 2, prostate-specific antigen, and prostatic acid phosphatase, while cornulin, involucrin, and cornifin are vaginal biomarkers. Alpha-amylase 1 is present in saliva, and alpha-amylase 2 is present in semen and vaginal secretion; thus, alpha-amylase 1 is chosen as a salivary marker protein because of its high abundance and specificity. Biomarkers of menstrual blood are hemoglobin from blood and cornulin from vaginal secretions [14]. Plunc-protein is specific for nasal secretions and is therefore selected as a biomarker for this matrix [155]. Uromodulin is the most abundant protein in normal human urine, whereas alpha-1-microglobulin/bikunin precursor (AMBP) can also be detected in urine. Since these proteins are found in blood, their presence in the absence of hemoglobin can be used to identify urine [156; 157]. However, it should be noted that uromodulin and AMBP are sometimes present in urine at low concentrations and cannot be detected by proteomic analysis. Immunoglobulins can be detected in feces and can be used as a marker protein of this matrix together with the absence of hemoglobin [14]. Using this decision tree, [PERSON] et al. were able to correctly identify menstrual blood, peripheral blood, semen, vaginal fluid, saliva, nasal secretion, urine, and feces.