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A rapid and preparative method for the separation of yeast ribosomal proteins by using high-performance liquid chromatography.

ABSTRACT: Ribosomal proteins from the yeast Saccharomyces cerevisiae were separated, on a preparative scale, by ion-exchange h.p.l.c. Proteins from the small and large ribosomal subunits were resolved, respectively, into 33 and 23 peaks, and most of the proteins present in these peaks were identified by using one- and two-dimensional gel electrophoresis. Several of the peaks appeared to contain a single protein uncontaminated by other species. Ribosomal proteins were also separated by using reverse-phase h.p.l.c. Analysis of the peaks resolved indicated that the order of elution for the proteins of both ribosomal subunits is, in certain cases, different for each of the two h.p.l.c. techniques used. Thus a combination of the two chromatographic methods employed here has the potential to facilitate the rapid and preparative separation of each of the proteins present in yeast ribosomes.

SUBMITTER: Threadgill GJ

PROVIDER: S-EPMC1148027 | biostudies-other | 1987 Jun

REPOSITORIES: biostudies-other

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Project description:Improvements in sample preparation, separation, and mass spectrometry continue to expand the coverage in LC-MS based lipidomics. While longer columns packed with smaller particles in theory give higher separation performance compared to shorter columns, the implementation of this technology above commercial limits has been sparse due to difficulties in packing long columns and successfully operating instruments at ultrahigh pressures. In this work, a liquid chromatograph that operates up to 35?kpsi was investigated for the separation and identification of lipid species from human plasma. Capillary columns between 15-50?cm long were packed with 1.7?µm BEH C18 particles and evaluated for their ability to separate lipid isomers and complex lipid extracts from human plasma. Putative lipid class identifications were assigned using accurate mass and relative retention time data of the eluting peaks. Our findings indicate that longer columns packed and operated at 35?kpsi outperform shorter columns packed and run at lower pressures in terms of peak capacity and numbers of features identified. Packing columns with relatively high concentration slurries (200?mg/mL) while sonicating the column resulted in 6-34% increase in peak capacity for 50?cm columns compared to lower slurry concentrations and no sonication. For a given analysis time, 50?cm long columns operated at 35?kpsi provided a 20-95% increase in chromatographic peak capacity compared with 15?cm columns operated at 15?kpsi. Analysis times up to 4?h were evaluated, generating peak capacities up to 410?±?5 (n?=?3, measured at 4?) and identifying 480?±?85 lipids (n?=?2). Importantly, the results also show a correlation between the peak capacity and the number of lipids identified from a human plasma extract. This correlation indicates that ionization suppression is a limiting factor in obtaining sufficient signal for identification by mass spectrometry. The result also shows that the higher resolution obtained by shallow gradients overcomes possible signal reduction due to broader, more dilute peaks in long gradients for improving detection of lipids in LC-MS. Lastly, longer columns operated at shallow gradients allowed for the best separation of both regional and geometrical isomers. These results demonstrate a system that enables the advantages of using longer columns packed and run at ultrahigh pressure for improving lipid separations and lipidome coverage.

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A rapid and preparative method for the separation of yeast ribosomal proteins by using high-performance liquid chromatography.

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