# Proteomics in Forensic Analysis: Applications for Human Samples
## 2 Proteomics Methods: Sample Preparation Techniques, Data Acquisition, and Data Analysis  
Proteomics can be separated into top-down, middle-down, and bottom-up approaches. In top-down proteomics, intact proteins extracted from samples are directly separated and analyzed by LC-MS/MS. This allows the identification of proteoforms with post-translational modifications (PTMs) [53]. The disadvantages of top-down proteomics include difficulties in protein separation, protein solubility, MS analysis, and quantification [54]. The top-down approach is widely used in sports anti-doping to identify banned peptides or proteins [20]. In contrast, in bottom-up proteomics, proteins are digested into thousands of peptides by enzymes such as trypsin and LysC. The resulting peptides are then smaller and easier to analyze using LC-MS/MS. Prior to this analysis, peptide mixtures are usually separated into several fractions to reduce sample complexity [55]. In middle-down proteomics, protein digestion is also carried out, but the aim is to yield relatively larger peptides (2.5-10 kDa) compared with those in bottom-up proteomics. It requires special proteases, such as OmpT, Sap9, and IdeS [56]. Middle-down proteomics can reduce the complexity of the digests and enable the identification of proteoforms. The sequence coverage of the proteins can also be increased. [57]. Among three approaches, bottom-up proteomics is more feasible and applicable than top-down and middle-down proteomics, and has been widely applied in forensics. Thus, this section introduces and discusses the methods used in bottom-up proteomics. Methods and considerations of the top-down  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Sample Type** & **Applications** \\ \hline Hair & Identify ethnic groups [18; 36; 37] \\  & Distinguish gender [26] \\  & Distinguish individuals [27; 38; 39] \\ \hline \multirow{3}{*}{Bone} & Estimate biological age [29; 40; 41; 42] \\  & Estimate post-mortem interval (PMI) [29; 30] \\  & Distinguish individuals [43] \\ \hline Body fluid and tissue & Identify body fluids using biomarkers [12; 14; 44] \\  & Identify tissues using biomarkers [45; 46] \\ \hline \multirow{3}{*}{Urine and blood} & Identify and quantify illegal peptides and small protein \\  & hormones for sport doping [28; 47; 48] \\ \hline Fingermail & Identify body fluids and tissues trapped under fingernails [34] \\ \hline Muscle & Estimate mid-PMI [31] \\ \hline \multirow{3}{*}{Brain and cerebrospinal fluid} & Estimate PMI [32] \\  & Identify diagnostic biomarkers for sudden infant death \\  & syndrome (SIDS) [49] \\ \hline \multirow{3}{*}{Blood} & Identify biomarkers in the postmortem \\  & diagnosis of drowning [50] \\ \hline Decomposition fluid & Estimate PMI [33] \\ \cline{2-2}  & Identify traces of body fluids [35] \\ \cline{1-1}  & Determine age-markers of fingermarks [51] \\ \hline Epidermal corneocyte & Distinguish individuals [52] \\ \hline \hline \end{tabular}
\end{table}
Table 1: Applications of forensic proteomics using human samples.  
and middle-down proteomics can be found in some literature reviews (e.g., [58; 59] for top-down and [56] for middle-down proteomics). An overview of general methods for bottom-up proteomics is presented in Figure 1[23]. A bottom-up proteomic study includes three main parts: sample preparation, data acquisition, and data analysis.