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Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR.

ABSTRACT: Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by "mass locking" with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations.

SUBMITTER: Lee SW

PROVIDER: S-EPMC122881 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR.

Lee Sang-Won SW Berger Scott J SJ Martinović Suzana S Pasa-Tolić Ljiljana L Anderson Gordon A GA Shen Yufeng Y Zhao Rui R Smith Richard D RD

Proceedings of the National Academy of Sciences of the United States of America 20020401 9

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by "mass locking" with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational m ...[more]

PMID: 11983894

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Project description:With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-alpha hCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-alpha hCG, using capillary electrophoresis (CE) with a separation time under 20 min, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometry (FT-MS) allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-alpha hCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-alpha hCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for final product characterization.

Dataset Information

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR.

Publications

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR.

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