Immunoaffinity isolation of modified peptides from complex mixtures

Inactive Publication Date: 2011-03-03
CELL SIGNALING TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0015]The method of the invention enables the rapid, efficient, and direct isolation (and subsequent characterization) of modified peptides from complex mixtures, such as crude cell extracts, without the need for costly and timely pre-purification of desired peptides or proteins. The method enables the single-step immunoaffinity isolation, and subsequent characterization of multiple different modified pe

Problems solved by technology

In spite of the importance of protein modification, it is not yet well understood at the molecular level.
The reasons for this lack of understanding are, first, that the cellular modification system is extraordinarily complex, and second, that the technology necessary to unravel its complexity has not yet been fully developed.
Although several methods for purifying phosphopeptides have been described, these methods have significant limitations that render them unsuitable for the isolation or purification of modified peptides from complex mixtures of peptides on a genome- or cell-wide basis.
The reversed-phase HPLC method has several important limitations that render it unsuitable for the purification of modified peptides from complex mixtures of peptides, e.g. cellular digests.
The method cannot be applied to biological samples that cannot be radioactively labeled, such as tissue biopsy samples.
The HPLC method is often unsuccessful when applied to proteins that are modified at low levels, for example, where only a small percentage (less than 10%) of the protein is phosphorylated at one site.
This problem results from the difficulty of purifying a phosphopeptide to homogeneity against a high background of nonphosphorylated peptides, and the need for a nearly homogenous phosphopeptide during chemic

Method used

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  • Immunoaffinity isolation of modified peptides from complex mixtures
  • Immunoaffinity isolation of modified peptides from complex mixtures
  • Immunoaffinity isolation of modified peptides from complex mixtures

Examples

Experimental program
Comparison scheme
Effect test

Example

Example I

A. Isolation of Phosphotyrosine-Containing Peptides from a Peptide Mixture

[0162]To establish that phosphopeptides can specifically be purified from complex mixtures without contamination from nonphosphorylated peptides, the method of the invention was used to isolate phosphotyrosine (p-Tyr)-containing peptides from a mixture of phosphorylated and nonphosphorylated synthetic peptides. A phosphotyrosine peptide mix comprising 5 phosphotyrosine-containing peptides and their 5 nonphosphorylated partner peptides was prepared (Table 1); note the nonphosphorylated peptides have the same sequences as the phosphorylated peptides but are not phosphorylated, that is, they contain tyrosine instead of phosphotyrosine. Peptides were synthesized by Fmoc chemistry on a Rainin / Protein Technologies Symphony peptide synthesis instrument and using Fmoc-Tyr(PO(OBzl)OH)—OH as the phosphotyrosine monomer. See Perich, Lett. Pept. Sci. 6: 91 (1999). The peptide mixture covers a broad mass range des...

Example

Example II

A. Isolation of Phosphotyrosine-Containing and Phospho-Akt Substrate Peptide Subsets from a Digested Crude Cell Extract

[0179]Example I demonstrates that several phosphorylation-specific antibodies can be employed in the method of the invention to selectively separate desired phosphopeptides from non-phosphopeptides. The antibodies may be general modification-specific antibodies or motif-specific, context-independent antibodies that recognize a short non-unique motif comprising several invariant residues, which motif is present on a plurality of different peptides or proteins within a genome. As shown in Example I, the antibodies can distinguish phosphopeptides from non-phosphopeptides even when the only difference between the peptides is the presence or absence of a phosphate group. In the present Example, it is shown that desired phosphopeptides may be selectively isolated by the method of the invention from a complex mixture containing phosphopeptides of different types....

Example

Example III

Isolation of Phosphotyrosine-Containing Peptides from an Extract of Cells

Overexpressing Epidermal Growth Factor Receptor

[0190]The selective isolation of modified peptides from a complex mixture according to the method of the invention was further demonstrated using a digested whole cell extract and a general phosphotyrosine antibody to isolate known phosphopeptides. A model system, the A431 epidermoid carcinoma cell line overexpressing the human epidermal growth factor receptor (EGFR), was selected since the modification (phosphorylation) of sites on this protein is well-studied. Activation of EGFR family members is associated with many tumors. Five sites of in vivo autophosphorylation have been identified in EGFR: three major sites (Tyr-1068, Tyr-1148, and Tyr-1173) and two minor sites (Tyr-992 and Tyr-1086) (Downward et al., J. Biol. Chem. 260: 14538-546 (1985); Hsuan et al., Biochem. J. 259: 519-27 (1989); Margolis et al., EMBO J. 9: 4375-380 (1990); Walton et al., J. ...

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Abstract

The invention provides methods for isolating a modified peptide from a complex mixture of peptides, the method comprising the steps of: (a) obtaining a proteinaceous preparation from an organism, wherein the preparation comprises modified peptides from two or more different proteins; (b) contacting the preparation with at least one immobilized modification-specific antibody; and (c) isolating at least one modified peptide specifically bound by the immobilized modification-specific antibody in step (b). The method may further comprise the step of (d) characterizing the modified peptide isolated in step (c) by mass spectrometry (MS), tandem mass spectrometry (MS-MS), and/or MS3 analysis, or the step of (e) utilizing a search program to substantially match the spectra obtained for the modified peptide during the characterization of step (d) with the spectra for a known peptide sequence, thereby identifying the parent protein(s) of the modified peptide. Also provided are an immunoaffinity isolation device comprising a modification-specific antibody, and antibodies against novel UFD1 and PTN6 phosphorylation sites.

Description

RELATED APPLICATIONS[0001]This application claims priority to U.S. Ser. No. 10 / 777,893, filed Feb. 12, 2004, presently pending, the disclosures of which are hereby incorporated by reference herein.FIELD OF THE INVENTION[0002]The invention relates to peptides and methods of isolating and characterizing the same.BACKGROUND OF THE INVENTION[0003]The activation of proteins by modification represents an important cellular mechanism for regulating most aspects of biological organization and control, including growth, development, homeostasis, and cellular communication. For example, protein phosphorylation plays a critical role in the etiology of many pathological conditions and diseases, including cancer, developmental disorders, autoimmune diseases, and diabetes. In spite of the importance of protein modification, it is not yet well understood at the molecular level. The reasons for this lack of understanding are, first, that the cellular modification system is extraordinarily complex, ...

Claims

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Application Information

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IPC IPC(8): C12N11/00C12S3/14G01N33/566C07K1/14C07K16/18C07K16/00C07K16/40C07K1/22H01J49/26G01N30/72B01D11/02
CPCB01D15/3809B01D15/3828C07K1/22C07K16/44G01N30/7233C07K16/18G01N33/6848G01N33/6854G01N2030/8831G01N2440/14G01N33/6842
Inventor RUSH, JOHNZHANG, HUIZHA, XIANGMINGCOMB, MICHAEL J.TAN, YI
Owner CELL SIGNALING TECHNOLOGY
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