title: Comparative studies of peak intensities and chromatographic separation of proteolytic digests, PTMs, and intact proteins obtained by nanoLC-ESI MS analysis at room and elevated temperatures.
abstract: This work demonstrates that the chromatographic separation performed at highly stabilized elevated temperature results in significant improvements in sensitivity, quantitative accuracy, chromatographic resolution, and run-to-run reproducibility of nanoLC-MS analysis of complex peptides mixtures. A newly developed platform was shown to provide conditions for accurate temperature stabilization and temperature homogeneity when performing nanoLC-ESI MS analysis. We quantitatively assessed and compared the recovery of peptides and small proteins from nanoLC columns at room and elevated temperatures. We found that analyses performed at highly stabilized elevated temperatures led to improved detection sensitivity, reproducibility, and chromatographic resolution in reversed-phase LC separation of unmodified peptides (both hydrophilic and hydrophobic), post-translationally modified peptides (O-phosphorylated), and small intact proteins. The analytical benefits of elevated temperatures for qualitative and quantitative proteomic LC-MS profiling were demonstrated using mixtures of synthetic peptides, tryptic digests of mixtures of model proteins, and digested total lysates of isolated rat kidney mitochondria. The effect of elevated temperature on the ion suppression was also demonstrated. Graphical Abstract A fragment of overlaid LC retention time-m/z planar views demonstrates the improved separation performance in the analysis of a complex peptide mixture at elevated temperature. Retention time-m/z 2D peptide features detected at 60 °C (magenta) were matched and aligned with features detected at room temperature (green).
authors.affiliation: MassTech Inc, 6992 Columbia Gateway Drive, Columbia, MD, 21046, USA. moskovets@apmaldi.com.
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Moskovets
authors.affiliation: Northeastern University, Barnett Institute, 360 Huntington Avenue, Boston, MA, 02115, USA. a.ivanov@neu.edu.
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Ivanov
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D004063:Digestion; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D013696:Temperature
chemical_list: D010455:Peptides; D011506:Proteins
keywords: NanoLC-MS analysis; Proteins and protein digests; Separations at elevated temperature