RePLiCal: A QconCAT Protein for Retention Time Standardization in Proteomics Studies

This
study introduces a new reversed-phase liquid chromatography
retention time (RT) standard, RePLiCal (Reversed-phase liquid chromatography calibrant),
produced using QconCAT technology. The synthetic protein contains
27 lysine-terminating calibrant peptides, meaning that the same complement
of standards can be generated using either Lys-C or trypsin-based
digestion protocols. RePLiCal was designed such that each constituent
peptide is unique with respect to all eukaryotic proteomes, thereby
enabling integration into a wide range of proteomic analyses. RePLiCal
has been benchmarked against three commercially available peptide
RT standard kits and outperforms all in terms of LC gradient coverage.
RePLiCal also provides a higher number of calibrant points for chromatographic
retention time standardization and normalization. The standard provides
stable RTs over long analysis times and can be readily transferred
between different LC gradients and nUHPLC instruments. Moreover, RePLiCal
can be used to predict RTs for other peptides in a timely manner.
Furthermore, it is shown that RePLiCal can be used effectively to
evaluate trapping column performance for nUHPLC instruments using
trap-elute configurations, to optimize gradients to maximize peptide
and protein identification rates, and to recalibrate the m/z scale of mass spectrometry data post-acquisition.