High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites.

High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites. Research Abstract Details 

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High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites. Abstract Text:

Jianru Stahl-Zeng,Vinzenz Lange,Reto Ossola,Katrin Eckhardt,Wilhelm Krek,Ruedi Aebersold,Bruno Domon,

The detection and quantification of plasma (serum) proteins at or below the ng/ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers. This has been achieved either by the development of high sensitivity ELISA or other immunoassays for specific proteins or by the extensive fractionation of the plasma proteome followed by the mass spectrometric analysis of the resulting fractions. The first approach is limited by the high cost and time investment for assay development and the requirement of a validated target. The second, although reasonably comprehensive and unbiased, is limited by sample throughput. Here we describe a method for the detection of plasma proteins at concentrations in the ng/ml or sub-ng/ml range and their accurate quantification over 5 orders of magnitude. The method is based on the selective isolation of N-glycosites from the plasma proteome and the detection and quantification of targeted peptides in a quadrupole linear ion trap instrument operated in the multiple reaction monitoring (MRM) mode. The unprecedented sensitivity of the mass spectrometric analysis of minimally fractionated plasma samples is the result of the significantly reduced sample complexity of the isolated N-glycosites compared with whole plasma proteome digests and the selectivity of the MRM process. Precise quantification was achieved via stable isotope dilution by adding (13)C- and/or (15)N-labeled reference analytes. We also demonstrate the possibility of significantly expanding the number of MRM measurements during one single LC-MS run without compromising sensitivity by including elution time constraints for the targeted transitions, thus allowing quantification of large sets of peptides in a single analysis.

High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites. Publishing Authors By Initials

J Stahl-Zeng,V Lange,R Ossola,K Eckhardt,W Krek,R Aebersold,B Domon,

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High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Molecular & cellular proteomics : MCP

VOLUME: 6

Page Numbers: 1809-17

Journal Abbreviation: Mol. Cell Proteomics

ISSN: 1535-9476

DAY: 20

MONTH: 07

YEAR: 2007

High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites. Information

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LANGUAGE: eng

NlmUniqueID: 101125647

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Grant and Affiliation Information for High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites.

AFFILIATION: Applied Biosystems, 64293 Darmstadt, Germany.

Country: United States

AGENCY: United States NHLBI

GRANT: N01-HV-28179

ACRONYM: HV

MEDLINETA: Mol Cell Proteomics

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