title: Improved Reversed Phase Chromatography of Hydrophilic Peptides from Spatial and Temporal Changes in Column Temperature.
abstract: Reversed phase chromatography is an established method for peptide separation and frequently coupled to electrospray ionization-mass spectrometry for proteomic analysis. Column temperature is one parameter that influences peptide retention and elution, but it is often overlooked as its implementation requires additional equipment and method optimization. An apparatus that allows temperature manipulation in three areas of a two-column setup was evaluated for improvements in chromatography. Using commercially available standards, we demonstrate that a low column temperature (0 °C) during sample loading enhances the peak shape of several bovine serum albumin hydrophilic peptides. For digested HeLa lysates, approximately 15% more peptide identifications were obtained by increasing the precolumn temperature to 50 °C after the 500 ng sample was loaded at a low temperature. This method also identified additional early eluting peptides with grand average of hydropathicity values less than -2. We also investigated the effect of cooler column temperatures on peptides with post-translational modifications. It was possible to minimize the coelution of an isoaspartylated peptide and its unmodified version when the analytical column temperature was decreased to 5 °C. Aside from demonstrating the utility of lower temperatures for improved chromatography, its application at specific locations and time points is critical for peptide detection and separation.
authors.affiliation: The Novo Nordisk Foundation Center for Protein Research, Proteomics Program, Faculty of Health and Medical Sciences, University of Copenhagen , Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
authors.forename: Clifford
authors.identifier: 0000-0002-3945-286X
authors.initials: C
authors.lastname: Young
authors.affiliation: Evosep Biosystems , Thriges Plads 6, DK-5000 Odense, Denmark.
authors.forename: Alexandre V
authors.initials: AV
authors.lastname: Podtelejnikov
authors.affiliation: The Novo Nordisk Foundation Center for Protein Research, Proteomics Program, Faculty of Health and Medical Sciences, University of Copenhagen , Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Nielsen
mesh_terms: D000818:Animals; D002417:Cattle; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D010455:Peptides / Q000032:analysis*; D012710:Serum Albumin, Bovine; D062211:Spatio-Temporal Analysis; D013696:Temperature
chemical_list: D010455:Peptides; D012710:Serum Albumin, Bovine
keywords: hydrophilic peptide; isoaspartic acid; liquid chromatography; mass spectrometry; temperature