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Title: Structural determination of intact proteins using 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.

Inventors:
 [1];  [2];  [3]
  1. San Francisco, CA
  2. Oakland, CA
  3. Livermore, CA
Publication Date:
Research Org.:
Sandia National Laboratories (SNL-CA), Livermore, CA
Sponsoring Org.:
USDOE
OSTI Identifier:
936326
Patent Number(s):
7,368,290
Application Number:
10/437,268
Assignee:
Sandia National Laboratories (Livermore, CA) ALO
DOE Contract Number:
AC04-94AL85000
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Kruppa, Gary, Schoeniger, Joseph S, and Young, Malin M. Structural determination of intact proteins using mass spectrometry. United States: N. p., 2008. Web.
Kruppa, Gary, Schoeniger, Joseph S, & Young, Malin M. Structural determination of intact proteins using mass spectrometry. United States.
Kruppa, Gary, Schoeniger, Joseph S, and Young, Malin M. 2008. "Structural determination of intact proteins using mass spectrometry". United States. doi:. https://www.osti.gov/servlets/purl/936326.
@article{osti_936326,
title = {Structural determination of intact proteins using mass spectrometry},
author = {Kruppa, Gary and Schoeniger, Joseph S and Young, Malin M},
abstractNote = {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.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = 2008,
month = 5
}

Patent:

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  • We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection of intact proteins from a Shewanella oneidensis MR-1 cell lysate. 715 intact proteins were detected and the combined results from the WAX fractions and the unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post translational modifications were assigned for ~10% of the detected proteins by comparing intact protein mass measurements to proteins identified in peptide MS/MS analysis of an aliquot of the same fraction.more » Intact proteins were also detected for S. oneidensis lysates obtained from cells grown on 13C, 15N depleted media under aerobic and sub-oxic conditions. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification complement. The strategy can be readily applied for measuring differential protein abundances, and provides a platform for high-throughput selection of biologically relevant targets for further characterization.« less
  • A method for obtaining protein molecular masses with an accuracy of approximately {plus minus}0.01% by matrix-assisted laser desorption using an internal calibrant is described. The technique allows accurate mass determinations of protein sample sizes as small as 1 pmol. High concentrations of organic and inorganic contaminants (e.g. 1 M urea) do not strongly affect either the signal intensity or the mass assignment. The ability to assign an accurate molecular mass to a protein is contingent on the observation of clearly resolved protonated molecule ions in the mass spectrum.
  • Tandem mass spectrometry has been used to obtain information related to portions of the primary sequence for an intact protein, bovine ribonuclease A. Multiply charged molecular ions, generated by electrospray ionization, were collisionally dissociated at low energies in a triple quadrupole mass spectrometer to yield singly and multiply charged fragment ions that can be assigned to the known sequence of the protein. Dissociation of the highly charged molecular ions resulted in pairs of complementary product ions. The higher order (gas-phase) protein structure affects the dissociation processes, as observed in comparisons of tandem mass spectra of the native and disulfide-reduced formsmore » of ribonuclease A. 22 refs., 4 figs.« less
  • In this paper, Fourier transform mass spectrometry is applied to study the structures of NiC/sub n/H/sub 2n/+ complexes generated from reactions of Ni+ with a variety of hydrocarbons and demonstrate that different isomeric structures can readily be distinguished both by a collision-induced dissociation and by specific ion-molecule reactions. Evidence is provided for four unique structures of NiC/sub 4/H/sub 8/+ generated from reactions of Ni+ with butane, hexane, 2,2-dimethylpropane, and cyclopentanone. Dehydrogenation reactions of Ni+ with n-alkanes yield bis(olefin) complexes that are distinguishable from their isomeric monoalkene complexes. Ni+ is found to be highly selective for cleaving internal bonds and highlymore » selective against insertion into terminal C-C bonds. Dehydrogenation becomes more competitive with alkane elimination when ..beta..-hydride transfers from secondary carbons are involved instead of from primary carbons.« less