The previous 30 days or so have been focused on making new peptide amphiphiles and purifying them. This is a straightforward process but can be highly time-consuming. Since I have two projects and both revolved around purification and verification, I will be looping them together.
To purify a peptide, a crude peptide can be produced via a PS3 peptide synthesizer. Peptides with specific sequences can also be purchased from various online companies. Once a peptide is synthesized, it is usually contaminated with debris or unbound peptides, etc. A crude peptide will not be 100% pure, so the next step is to obtain a pure peptide source so that future steps are accurate in concentration. To purify a peptide, reverse-phase high-performance liquid chromatography (HPLC) with a C18 column is used. The basic idea of using HPLC is that the peptide will be separated from other molecules by running down a column so that more dissolvable smaller molecules will be separated from larger molecules. Once the peptide is dissolved in Milli-Q water and injected into the HPLC, a peak can be detected and collected. These fractions are then put under a fume hood to allow the acetonitrile (ACN) to evaporate. To verify that each fraction contains pure peptide, mass spectrometry is used. Briefly, 0.8 ul of sample is spotted onto a target plate and 0.8 ul of a matrix is added. The spots can then be analyzed via MS. Once the ACN is evaporated, the fractions can be placed into pre-weighed tubes and stored in a -80C freezer for 48 hours. Once completely frozen, the fractions can be placed into the lyophilizer, which freeze-dries the sample.
The following peptides MGSHIEPGGC (AC1), KMGGTNHPEC (AC2), and CKDSPKSSKSIRFIPVST (CKD) were dissolved in Milli-Q water and purified using a reverse-phase high-performance liquid chromatography system with a C18 column. The m/z was characterized by MALDI-TOF and the expected peak is [M+H]+ = 1029, 1115, and 2021, respectively.