Quantitative improvements in peptide recovery at elevated chromatographic temperatures from microcapillary liquid chromatography-mass spectrometry analyses of brain using selected reaction monitoring.
Elevated chromatographic temperatures are well recognized to provide beneficial analytical effects. Previously, we demonstrated that elevated chromatographic temperature enhances the identification of hydrophobic peptides from enriched membrane samples. Here, we quantitatively assess and compare the recovery of peptide analytes from both simple and complex tryptic peptide matrices using selected reaction monitoring (SRM) mass spectrometry. Our study demonstrates that elevated chromatographic temperature results in significant improvements in the magnitude of peptide recovery for both hydrophilic and hydrophobic peptides from both simple and complex peptide matrices. Importantly, the analytical benefits for quantitative measurements in mouse whole brain matrix are highlighted, suggesting broad utility in the proteomic analyses of complex mammalian tissues. Any improvement in peptide recovery from chromatographic separations translates directly to the apparent sensitivity of downstream mass analysis in microcapillary liquid chromatography-mass spectrometry (muLC-MS) based proteomic applications. Therefore, the incorporation of elevated chromatographic temperatures should result in significant improvements in peptide quantification as well as detection and identification.
Farias
Santiago E
SE
Department of Pharmacology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado 80045, USA.
Kline
Kelli G
KG
Klepacki
Jacek
J
Wu
Christine C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D006358:Hot Temperature; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008832:Microchemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics
D010455:Peptides