Long, shallow gradients are typically used to resolve complex samples such as peptide digests. For these particular samples, each combination of protein, digest reagent, and method conditions, among other variables, will result in a unique peptide mapping fingerprint. Thus, a peptide mapping separation can be used to identify, track, or validate processes. In addition, the complexity of the resulting peptide digest will depend on multiple factors including initial protein size and digestion enzyme. For very complex peptide digests, where the separation of many peaks is required, run-to-run retention time reproducibility is of the utmost importance for tracking peaks.
However, the reproducibility of any method is dependent on many factors, including sample preparation, column, LC method conditions, as well as the LC instrumentation used. In order to focus on the impact of the LC instrument in this study, all other variables were controlled. For example, the sample consisted of a purchased standard and a single lot of columns were used for the study. To reduce the impact of solvent evaporation, low-evaporative bottle caps were used. Lastly, to ensure proper performance of the instrument, the system was qualified prior to the study.
As stated in the introduction, due to the complexity of peptide maps, LC methods typically consist of very shallow gradients run at low flow rates in an effort to separate as many components as possible, leading to improvements in characterization. In addition, conditions commonly start with a low % of organic solvent, often times less than 5%, to retain highly polar or early eluting peptides. It is commonly known that binary pumps, which utilize high pressure mixing, offer better compositional accuracy and precision than do their low pressure mixing counterparts. The compositional accuracy and precision of a binary pump depends only on the flow accuracy for each pump head. Conversely, for a tertiary or quaternary mixing pump, the compositional precision and accuracy depends on the ability of the gradient proportioning valve or apparatus to perform in a consistent and correct manner. For these reasons, the binary ACQUITY UPLC I-Class PLUS System was selected for the study.
Prior to running analysis on the ACQUITY UPLC I-Class PLUS System, a generic peptide mapping liquid chromatography method was developed for the analysis of the MassPREP Enolase Digestion Standard. The method was designed to impart a 0.66% change in organic solvent per column volume of mobile phase delivered. A flow rate of 0.200 mL/min was employed to account for the relatively slow diffusion of peptides. Finally, to ensure maximum peptide map coverage, a gradient method was employed to start at 1% organic to retain highly polar or early eluting peptides. Because the conditions chosen include the use of TFA, an ion-pairing reagent, as an additive, columns were equilibrated by running the method gradient for 24 hours. Each individual batch was comprised of two blank injections (mobile phase A) followed by eight to twelve replicate injections of the reconstituted enolase digest (Figure 1).
