Development of peptide spectrometry using the multi-solvent mixing capability of the ACQUITY UPLC H-Class system
aims The ACQUITY UPLC ® H-Class system uses a quaternary solvent blending automation optimization scheme to systematically optimize peptide mapping conditions. background Peptide mapping is used to identify the primary structure of a protein, identify post-translational modifications (PTM), and analyze impurities. Any protein structural differences are reflected in changes in the retention time of the peptide containing the modification. The complete structure of a protein needs to be able to resolve 100 or more polypeptide resolutions. These molecules have a wide range of molecular sizes and molecular properties. When developing separation conditions for the separation of these peptides, changes in mobile phase composition and organic solvent composition are altered. Moreover, each measurement condition requires the preparation of a pair of mixed solvents involving two or three accurate measurements. The quaternary solvent is mixed using the ACQUITY UPLC H-Class system, which is produced by mixing the system from a pure solvent bottle. This approach reduces the amount of work required to prepare many solvent mixtures and minimizes the possibility of error. At the same time, the intermediate component can be easily determined. The ACQUITY UPLC H-Class system simplifies the development and optimization of peptide mapping methods that can be used to characterize the structure of proteins. The ACQUITY UPLC H-Class System and its quaternary solvent blending capabilities are designed to help customers focus on the quality of their analysis and efficiently achieve reliable results. Solution The MassPREP(TM) enolase trypsin-enzymatic peptide standard was separated using an ACQUITY UPLC BEH 300 C18 column and multiple mobile phase solvents. In the ACQUITY UPLC H-Class system, we set the water to solvent A, acetonitrile to solvent B, isopropanol to solvent C, and 1% aqueous solution of TFA as solvent for solvent D. In order to evaluate the effect of acid concentration, three identical gradients of acetonitrile elevated elution gradients were run at 2.5%, 5%, and 10% D, respectively, such as the corresponding TFA concentrations of 0.025%, 0.05%, and 0.1, respectively. %. Enolase peptide digestion standard Column: peptide separation technology BEH 300 C 18 , 2.1X 100mm , 1.7 μm Solvent: A: water, B: acetonitrile, C: isopropanol, D: 1% TFA Figure 1. Solvent composition can be easily adjusted using the Aut o · Blend TM function of the ACQUITY UPLC H-Class system. In this case, the TFA optimal concentration of the peptide map was determined only by changing the percentage change in the flow rate taken from the D line. It is not necessary to prepare more bottles of solvent, and the median can be measured with minimal labor. Figure 2. Using the automatic mixing function of the ACQUITY UPLC H-Class system, pure solvent is extracted from a pure solvent bottle and mixed into a solvent mixture. The chromatographic results in Figure 1 show a significant change in chromatographic selectivity as the acid concentration changes. This experiment can be extended by repeating the same experiment but using an incremental isopropanol gradient, which can be conveniently carried out by running a gradient of solvent A (water) to solvent C (isopropyl alcohol) while the concentration of solvent D remains constant, Consistent with the concentrations in previous experiments. The change of solvent from acetonitrile to isopropanol shortens the retention time and also significantly changes the selectivity. The system experiment evaluated six different mobile phase formulations to confirm the best peptide profile. Selective changes can be used to obtain the optimal resolution of key polypeptides in the mixture. If a binary system is used or an on-off valve is used for these experiments, scientists need to prepare 12 bottles of mobile phase solution, requiring at least 24 volume measurements. When using the ACQUITY UPLC H-Class system, only one mixture was used, using two metric volumes associated with three bottles of pure solvent (Figure 2). The ACQUITY UPLC H-Class system reduces the amount of work, time spent, and the possibility of errors to achieve optimal separation conditions. to sum up Biochemists are able to efficiently develop peptide mapping methods for qualitative proteins using the ACQUITY UPLC H-Class system. The system uses a convenient four-solvent mixing capability to assist the user in focusing on the quality of the analysis, enabling it to efficiently develop complete, reliable separation methods. The ACQUITY UPLC H-Class system combines UPLC separation principles with flexible equipment operation for optimal bioseparation results. Fei County Mingtao Electronic Co.,Ltd , https://www.yourbeautyassistant.com
