Detection and quantification of modified proteins

Abstract

The invention provides a method detecting and quantifying proteins by mass spectrophotometric analysis using peptide internal standards and provides a highly sensitive way of detecting protein modifications. In one aspect, the invention provides a method for determining a site of ubiquitination in a polypeptide and for evaluating ubiquitination targets in a population of polypeptides. In this way, a proteome ubiquitination map can be obtained which comprises information relating to the ubiquitination states of a plurality of cellular polypeptides. Maps can be obtained for a variety of different types of cells and cell states. For example, ubiquitination targets in normal and diseased cells can be evaluated. Preferably, the map is stored as data files in a database. Individual ubiquitinated polypeptides identified can be used to generate molecular probes diagnostic of a cell state and / or can serve as targets for agents that modulate one or more cellular processes.

Description

RELATED APPLICATIONS[0001]This application is a divisional application of U.S. utility application Ser. No. 10 / 506,877 filed Jul. 29, 2005 which claims the benefit of the PCT / US03 / 07527 filed Mar. 11, 2003 which claims the benefit of the U.S. provisional application Ser. No. 60 / 363,179 filed Mar. 11, 2002, each of which is hereby incorporated by reference herein in its entirety.GOVERNMENT GRANTS[0002]At least part of the work contained in this application was performed under government grant HG00041 from the National Institutes of Health, U.S. Department of Health and Human Services. The government may have certain rights in this invention.FIELD OF THE INVENTION[0003]This invention provides methods, reagents and kits for obtaining absolute quantification of proteins and their modifications directly from cell lysates. In particular, the invention provides peptide internal standards for use in high performance liquid chromatography (HPLC) with online detection by multistage mass spect...

AI Technical Summary

Benefits of technology

[0009]The present invention provides reagents, kits, and methods for accurate quantification of proteins and methods for using the same. In particular, the method is useful for detecting and quantitating modified proteins and identifying sites of protein modification, such as sites of ubiquitination. The reagents, kits, and methods of the invention are useful for rapid, high throughput analysis of proteomes.

[0021]The invention also provides a method of determining the presence of and / or quantity of a modification in a target polypeptide. Preferably, the label in the internal standard is part of a peptide comprising a modified amino acid residue or to an amino acid residue which is predicted to be modified in a target polypeptide. In one aspect, the presence of the modification reflects the activity of a target polypeptide and the assay is used to detect the presence and / or quantity of an active polypeptide. The method is advantageous in enabling detection of small quantities of polypeptide (e.g., about 1 part per million (ppm) or less than about 0.001% of total cellular protein).

Problems solved by technology

In addition, many antibodies only recognize proteins in an unfolded (denatured) form, cross-reactivity can be severely limiting, and quantification is generally relative.

However, while these approaches dramatically accelerate protein identification, quantities of the analyzed proteins cannot be easily determined, and these methods have not been shown to substantially alleviate the dynamic range problem also encountered by the 2DE / MS / MS approach.

Therefore, low abundance proteins in complex samples are also difficult to analyze by the microcapillary LC / MS / MS method without their prior enrichment.

The major drawback to this technique are 1) quantification is only relative; 2) specialized chemistry is required, and 3) database searches are hindered by the presence of the large ICAT reagent molecule, and 4) relative amounts of posttranslationally modified (e.g., phosphorylated) proteins are transparent to analysis.

In a further aspect, the site of ubiquitination is correlated with disease and detection of ubiquitination at the site is associated with risk of the disease.

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