# Nanoformulation of Peptides for Pharmaceutical Applications: In Vitro and In Vivo Perspectives
## 2 Overview of Peptides
### Membrane Filtration
#### 2.2.2 Characterization of Peptides  
Most researchers perform peptide sequencing or amino acid sequence for structural characterization to obtain the database of the targeted protein. The peptide sequencing measures the peptide length, which is essential to determine the bioactivity of a specific peptide [83]. The main tools for protein identification and characterization are mass spectrometry (MS), electrospray ionization (ESI), and matrix-assisted laser desorption ionizations (MALDI) [84]. Liquid chromatography equipped with tandem mass spectrometry detection (LC-MS/MS) is also useful in peptide sequence detection, while matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) helps in generating peptide profiles [85].  
Using the protein sequencer and LC/MSD, [PERSON] et al. (2016) identified that the sequence of the purified peptide from sandfish was Ala-Thr-Ser-His-His. The high antioxidant activity of the peptide was due to the two histidine residues and the presence of alanine residues might act as inhibitors in the radical-mediated peroxiding chain reaction [71]. Further, [PERSON] et al. (2020) used UPLC-Q-TOF-MS and identified that the molecular weight(s) (MW) of those eight antioxidant peptides from mung beans were in the range of 300 to 800 Da [86]. An antioxidant peptide usually contains 2 to 10 amino acid residues with a MW less than 1500 Da [87].  
[PERSON] et al. (2013) isolated and purified antimicrobial peptides from bacteria (SK.DU.4) using a combination of chemical extraction and chromatographic techniques, SEC, and RP-HPLC. However, UF was not used as the pre-purification step [88]. Characterization of the antimicrobial peptide was performed using MALDI-TOF-MS to determine the MW, GC-MS to identify the fatty acid, SDS PAGE to detect the bacteriocin activity, and the agar/broth dilution method to determine the minimum inhibitory (MIC) concentration. The results revealed that 5.3 kDa of isolated antimicrobial peptides are similar to the lipoproteins _of Methylomicobium album_, which is an enzyme exhibiting antimicrobial activity via degradation of the structural cell wall component; the narrow spectrum antimicrobial activity against Gram-positive bacteria, low molecular weight, and resistance to high temperature suggested the bacteriocin-like antimicrobial activity of the peptide [89].  
It is noteworthy that the selection of a suitable and proper purification method is extremely significant as it is the rudiment of peptide characterization. As mentioned earlier, good characterization is critical as the results provide information within the isolated peptide. The purification and characterization choices depend on the original source of the peptide type. The majority of researchers have combined distinct purification methods or repeated the same purification steps to obtain better results. Thus, the best combination of a purification method is UF and RP-HPLC, as both are proven to be efficient in purification. Further, they are affordable and require less time for high quality purification. UF can be used as the first step of the peptide purification before further purification via RP-HPLC. Hence, the selection of the characterization result is highly based on the desire of the researchers to explore the interior components of the purified peptides.