Structural determination of intact proteins using mass spectrometry

• US 20040229369A1
• Filed: 05/12/2003
• Published: 11/18/2004
• Est. Priority Date: 05/12/2003
• Status: Active Grant

• Current Assignee: National Technology & Engineering Solutions Of Sandia LLC (Honeywell International Inc.)
• Original Assignee: Sandia National Laboratories (Government of the United States of America)
• Inventors: Kruppa, Gary, Schoeniger, Joseph S.
• Granted Patent: US 7,368,290 B2
• Time in Patent Office: Days
• US Class Current: 436/86
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry

Abstract

The present invention relates to novel methods of determining the sequence and structure of proteins. Specifically, the present invention allows for the analysis of intact proteins within a mass spectrometer. Therefore, preparatory separations need not be performed prior to introducing a protein sample into the mass spectrometer. Also disclosed herein are new instrumental developments for enhancing the signal from the desired modified proteins, methods for producing controlled protein fragments in the mass spectrometer, eliminating complex microseparations, and protein preparatory chemical steps necessary for cross-linking based protein structure determination.

Additionally, the preferred method of the present invention involves the determination of protein structures utilizing a top-down analysis of protein structures to search for covalent modifications. In the preferred method, intact proteins are ionized and fragmented within the mass spectrometer.

Claims

1. A method for determining the structure of a protein in a sample, said method comprising the steps of:

exposing said sample to a chemical cross-linker;

cleaning said cross-linked sample;

introducing the sample into a mass spectrometer;

isolating said cross-linked species in the gas phase;

fragmenting said cross-linked species; and

identifying said cross-linked fragments to determine distances between atoms of said protein.

2. A method for determining protein structure by determining the distance between atoms or amino acid residues in a protein;

said method comprising the steps of;

preparing a sample containing a protein in an active, functional form;

exposing said sample to a cross-linker such that internal cross-linking of the protein occurs;

terminating the cross-linking reaction;

removing unreacted cross-linker;

dissolving said cross-linked sample in a solvent;

introducing said cross-linked sample into a mass spectrometer via an ionization technique;

isolating specific cross-linked protein species;

fragmenting said cross-linked species;

determining the mass-to-charge ratios of said fragments;

identifying said fragments by comparing the mass-to-charge ratios of said fragments to a database of mass-to-charge ratios for known protein fragments.