title: RePLiCal: A QconCAT Protein for Retention Time Standardization in Proteomics Studies.
abstract: 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.
authors.affiliation: Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB, United Kingdom.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Holman
authors.affiliation: Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB, United Kingdom.
authors.forename: Lynn
authors.initials: L
authors.lastname: McLean
authors.affiliation: Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB, United Kingdom.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Eyers
mesh_terms: D000595:Amino Acid Sequence; D056148:Chromatography, Reverse-Phase / Q000592:standards*; D029968:Escherichia coli Proteins / Q000737:chemistry; D040901:Proteomics / Q000592:standards*; D012015:Reference Standards; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D029968:Escherichia coli Proteins; D029701:Saccharomyces cerevisiae Proteins
keywords: LC; QconCAT; SRM; calibration; mass spectrometry; proteomics; quality control; retention time; standardization