Replical: engineered protein for improved proteomics workflows
Holman SW, McLean L, Eyers CE. (2016) RePLiCal: A QconCAT Protein for Retention Time Standardization in Proteomics Studies. J Proteome Res. 2016 Mar 4;15(3):1090-102. doi: 10.1021/acs.jproteome.5b00988
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.
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.