Peptide is a compound formed by linking α-amino acids together with peptide bonds, which is an intermediate product of protein hydrolysis. The compound formed by dehydration condensation of two amino acid molecules is called dipeptide, and by the same analogy, there are tripeptide, tetrapeptide, pentapeptide, etc. Peptides composed of three or more amino acid molecules are called polypeptides. There are also references to peptides consisting of 2-10 amino acids as oligopeptides (small molecule peptides); peptides consisting of 10-50 amino acids are called polypeptides; peptides consisting of more than 50 amino acids are called proteins.
Thousands of natural peptides have been identified and recognized, and they play an important role in human physiological processes, including as hormones, neurotransmitters, growth factors, ion channel ligands or anti-infectives. Peptide drugs are considered to be highly selective and effective, and, at the same time, relatively safe and well-tolerated. As a result, there is a growing interest in peptides for drug research and development (R&D), and hundreds of peptides are being evaluated in clinical trials.
Mass spectrometry-based peptide sequence analysis
There are two common methods for sequence determination of peptides: database search and de novo sequencing method. For relatively short peptides, such as less than 30 amino acids, Creative Proteomics adopts secondary fragmentation to get the raw data of mass spectrometry, and the theoretical sequence can be obtained by database search or de novo sequencing.
For longer peptides (>30 amino acids), Creative Proteomics will use three proteases to enzymatically cleave and identify the target peptide separately to obtain fragmented peptide fragments, and then complete the determination of 100% of the peptide sequence after peptide splicing, i.e. by ab initio sequencing. For peptide mixtures, sequence analysis can also be performed based on the above method.
Creative Proteomics uses Obitrap Fusion Lumos mass spectrometer combined with Nano-LC nanoliter chromatography technology to provide mass spectrometry-based peptide sequence analysis services and peptide de novo de novo sequencing services, ensuring high resolution and high sensitivity.
Workflow of Peptide Sequencing Service
Recommendations for sample preparation:
- Prepare a sample of 100 ug as much as possible: prepare a sample of at least 10-25 ug for a sample of<30 and="" prepare="" a="" purified="" sample="" of="" at="" least="">50 ug for a sample of >30 aa;
- Use the purest possible reagents, rather than molecular biology grade reagents;
- Use the smallest centrifuge tubes possible;
- Try to avoid contaminants, or concentrate proteins in solution.
Full protein sequence determination
The application of peptide drugs lies mainly in the following areas.
1. cytokine peptide mimetics
The screening of cytokine peptide mimetics from the peptide library using known receptors of cytokines has become a hot research topic in recent years. The amino acid sequence of these peptide mimetics is different from that of their corresponding cytokines, but they have the activity of cytokines and small molecular weight. These cytokine peptide mimetics are in the preclinical or clinical research stage.
2. Antibacterial active peptides
When insects are stimulated by the external environment, a large number of cationic polypeptides with antibacterial activity are produced. At present, more than 100 kinds of antimicrobial peptides have been screened. In vitro and in vivo experiments have confirmed that many antimicrobial peptides not only have strong bactericidal ability but also kill tumor cells.
3. Peptide for diagnosis
The most important use of peptides in diagnostic reagents is as antigens to detect antibodies to microorganisms such as viruses, cells, mycoplasma, helminths and parasites such as cysts and trypanosomes. Peptide antigens are more specific than natural microbial or parasite protein antigens and easy to prepare, so antibody detection reagents assembled with peptide antigens have low false negative rates and background reactions for detecting antibodies, which are easy for clinical application. The antibody detection reagents assembled with peptide antigens include: A, B, C, G or liver virus, HIV, human cytomegalovirus, herpes simplex virus, rubella virus, syphilis spirochetes, cysticercus, trypanosomes, Lyme disease and rheumatoid etc. Most of the peptide antigens used are obtained from analytical screening within the natural proteins of the corresponding pathogens, and some are brand new small peptides screened from peptide libraries.
4. Other small peptides for pharmaceutical use
In addition to the above mentioned aspects, small peptide drugs have made great progress in many other fields. For example, Stiernberg et al. found that a synthetic peptide (TP508) peptide could promote the regeneration of wound blood vessels and accelerate the healing of deep skin wounds. pfister et al. found that a small peptide could prevent the infiltration of inflammatory cells in alkali-damaged cornea and inhibit the inflammatory response. carron et al. confirmed that two synthetic peptides they screened could inhibit bone resorption by osteoclasts.
Peptide drug research has great potential, such as discovery of new peptides, more targeted peptide library and sequence optimization design, research and development of new formulations, research and development of multifunctional peptides and complexes, etc. And to realize these researches, identification of peptides and analysis of peptide sequences are the first step to drive the research.