Orbitrap Illustration

Top-Down Proteomics


The main advantages of the top-down approach include the ability to detect degradation products, sequence variants, and combinations of post-translational modifications. 


Complex research endeavors such as the investigation of cell signaling pathways, disease state characterization, and biomarker discovery have all benefited from advances in mass spectrometry.  Such pursuits often employ mass spectrometry-based bottom-up protein identification techniques. These same research endeavors would, in many cases, also benefit from high-throughput top-down mass spectrometric analyses. The main advantages of the top-down approach include the ability to detect degradation products, sequence variants, and combinations of post-translational modifications.  In a Nature publication by Neil Kelleher and co-workers, over one thousand unique gene products were identified from human cells in a single experiment. This accounted for over three thousand differentially modified species. High-resolution mass spectrometry is not only essential to resolve co-eluting intact proteins but also to resolve the isotopic peaks of the highly charged large molecules for charge state determination and accurate mass determination.  Additionally, due to the number of product ions generated during fragmentation of intact proteins, high-resolution mass analysis is required for accurate detection and assignment of product ions in the resultant complex MS/MS spectra.
 

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Workflow Overview for Top-Down Proteomics


High throughput top down proteomics involves identifying proteins in complex mixtures without prior digestion into their corresponding peptide species.  For high throughput protein top down characterization, scientists can extract the proteins from their sample, fractionate using a variety of techniques including GELFREE™ fractionation by molecular weight or immunoprecipitation, and then analyze the resulting fractions by high resolution, accurate mass MS/MS on the Orbitrap-based platforms with online chromatography using data dependent acquisition methods with CID, HCD, and ETD fragmentation.  The key benefits to high throughput top down analysis of complex mixtures are the ability to detect degradation products, sequence variants, and combinations of post-translational modifications.






 


NEW WEBSITE: The Consortium for Top Down Proteomics
 

LITERATURE HIGHLIGHT


Mapping intact protein isoforms in discovery mode using top-down proteomics

Tran JC, Zamdborg L, et al.
Nature. 2011 Oct 30;480(7376):254-8.