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Rapid, Accurate ESI-IT MS Method for Proteomics

MCW #1330

 

 Key Inventor

Brian Halligan, PhD

 State-of-the-Art

The main goal of comparative proteomics is the quantitation of the differences in abundance of many proteins between two different biological samples in a single experiment. By differentially labeling the peptides from the two samples and combining them in a single analysis, relative ratios of protein abundance can be accurately determined.

 Problem

Stable isotope labeling with (18)O is a promising technique for obtaining both qualitative and quantitative information from a single differential protein expression experiment. The small 4 Da mass shift produced by incorporation of two molecules of (18)O, and the lack of available methods for automated quantification of large data sets has limited the use of this approach with electrospray ionization-ion trap (ESI-IT) mass spectrometers.

 Solution

Researchers at MCW have developed a method of acquiring ESI-IT mass spectrometric data that provides accurate calculation of relative ratios of peptides that have been differentially labeled using(18)O. The method utilizes zoom scans to provide high resolution data. This allows for accurate calculation of (18)O/(16)O ratios for peptides even when as much as 50% of a (18)O labeled peptide is present as the singly labeled species. The use of zoom scan data also provides sufficient resolution for calculating accurate ratios for peptides of +3 and lower charge states. Sequence coverage is comparable to that obtained with data acquisition modes that use only MS and MS/MS scans.

 Benefit

ZoomQuant is a stand-alone computational tool that analyzes the mass spectra of (18)O labeled peptides from ion trap instruments and determines relative abundance ratios between two samples. Starting with a filtered list of candidate peptides that have been successfully identified by Sequest, ZoomQuant analyzes the isotopic forms of the peptides using high-resolution zoom scan spectrum data. The theoretical isotope distribution is determined from the peptide sequence and is used to deconvolute the peak areas associated with the unlabeled, partially labeled, and fully labeled species. The ratio between the labeled and unlabeled peptides is then calculated using several different methods. ZoomQuant's graphical user interface allows the user to view and adjust the parameters for peak calling and quantitation and select which peptides should contribute to the overall abundance ratio calculation. Finally, ZoomQuant generates a summary report of the relative abundance of the peptides identified in the two samples.

 

ZoomQuant main window displays the proteins and peptides identified by the peptide identification program and the data for individual scans. Users can choose individual proteins and scans to analyze. Users also can perform ratio calculations and generate a report for the experiment.

 
The peaks window displays the spectra, the peaks identifications and quantization. Users can split, combine, or delete peaks. All small peaks can be removed or the group of peaks to be used in the ratio calculations can be changed. Ovals indicate the peaks selected for ratio calculations. The optional ruler indicates the expected peak widths based on the ion charge.

 Stage of Development

The invention has been tested in a research setting. Results have been published in the Journal of the American Society for Mass Spectrometry Volume 16, Issue 3, March 2005, Pages 302-306 and Volume 16, Issue 6, June 2005, Pages 916-925.

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Office of Technology Development

MEDICAL COLLEGE OF WISCONSIN
8701 Watertown Plank Road
Milwaukee, WI 53226

Summary Information

Patent Status:
60/537,605

Patent Coverage Type:
Method of Use

Geographical Coverage:
US Patent

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