Institute for Systems Biology

University of Washington

Applera Corporation

Other

The University of British Columbia

Rudolf Hans Aebersold

Beate Rist

Michael H Gelb

Frantisek Turecek

C Ronald Scott

George J Vella

Steven P Gygi

Scott A Gerber

Subhasish Purkayastha

Sasi K Pillai

Hui Zhang

Huilin Zhou

Timothy K Nadler

Yulin Huang

Michael E Wright

Patricia Q Bottari

Marcus B Smolka

Kenneth C Parker

Lawrence N Amankwa

The invention provides a method for diagnosing or predicting susceptibility to a prostate neoplastic condition in an individual. The method involves (a) determining a level of RDC1 in a sample from the individual, and (b) comparing the level of RDC1 in the sample to a reference level of RDC1, wherein a level of RDC1 in the sample 2-fold or more higher than the reference level indicates the presence of, or susceptibility to, a prostate neoplastic condition in the individual.

Methods to determine androgen responses in prostate cells

The invention provides methods of detecting polypeptides in a sample. The method can include the steps of cleaving polypeptides in a test sample to generate peptides; adding a predetermined amount of isotopically labeled peptide standards to the cleaved test sample, wherein the peptide standards correspond to peptides cleaved with the same reagent used to cleave the test sample; contacting the cleaved test sample containing peptide standards with an array of immobilized binding agents specific for the peptide standards; washing the array to remove unbound peptides, thereby retaining affinity captured sample peptides and standard peptides; analyzing the affinity captured peptides using mass spectrometry; and determining the presence of bound test peptides and standard peptides. The method can further include the step of quantifying the amount of the test peptides by comparing the ratio of test peptide to corresponding standard peptide.

Affinity capture of peptides by microarray and related methods

The invention provides methods for identifying and quantifying polypeptides in a sample. The methods include the steps of labeling peptides in a polypeptide sample with an isotope tag; adding a plurality of peptide standards to the polypeptide sample, wherein the peptide standards are labeled with an isotopically distinct version of the isotope tag; resolving the labeled sample and standard peptides into a plurality of fractions; analyzing the resolved fractions using mass spectrometry; identifying an isotope-tagged sample peptide in an analyzed fraction; and determining the amount of the identified isotope-tagged sample peptide in the analyzed fraction by comparison to the amount of isotope tagged standard peptide in the same fraction.

Methods for high-throughput and quantitative proteome analysis

Analytical reagents and mass spectrometry-based methods using these reagents for the rapid, and quantitative analysis of proteins or protein function in mixtures of proteins. The methods employ affinity labeled protein reactive reagents having three portions: an affinity label (A) covalently linked to a protein reactive group (PRG) through a linker group (L). The linker may be differentially isotopically labeled, e.g., by substitution of one or more atoms in the linker with a stable isotope thereof. These reagents allow for the selective isolation of peptide fragments or the products of reaction with a given protein (e.g., products of enzymatic reaction) from complex mixtures. The isolated peptide fragments or reaction products are characteristic of the presence of a protein or the presence of a protein function in those mixtures. Isolated peptides or reaction products are characterized by mass spectrometric (MS) techniques. The reagents also provide for differential isotopic labeling of the isolated peptides or reaction products which facilitates quantitative determination by mass spectrometry of the relative amounts of proteins in different samples. The methods of this invention can be used for qualitative and quantitative analysis of global protein expression profiles in cells and tissues, to screen for and identify proteins whose expression level in cells, tissue or biological fluids is affected by a stimulus or by a change in condition or state of the cell, tissue or organism from which the sample originated.

Rapid quantitative analysis of proteins or protein function in complex mixtures

The invention provides a reagent comprising an affinity tag, a detectable moiety, a linker, an isotope tag and a reactive group. The invention also provides methods of using a reagent of the invention. The methods can be used to label a polypeptide in a sample by contacting a sample with a reagent of the invention under conditions allowing the reactive group to bind to one or more polypeptides in the sample. The invention additionally provides methods of isolating, identifying and quantifying a polypeptide in a sample. The invention further provides methods of diagnosing a disease using a reagent of the invention.

Chemical reagents and methods for detection and quantification of proteins in complex mixtures

The invention provides methods for labeling a molecule by contacting a sample molecule with a solid support coupled to a chemical group comprising a cleavable functional group, one or more functional groups, and a reactive group for the sample molecule, under conditions allowing the sample molecule to covalently bind to the reactive group; and cleaving the cleavable functional group, thereby releasing the sample molecule comprising the one or more functional groups, which can be a tag. The invention also provides a solid support covalently coupled to a chemical group comprising a cleavable functional group, a mass spectrometry tag and a reactive group for covalently attaching a sample molecule, wherein the cleavable functional group, the tag and the reactive group are positioned relative to each other to allow transfer of the tag to the sample molecule upon cleavage of the cleavable functional group.

Methods for isolating and labeling sample molecules

Methods for isolation and labeling of sample molecules

The invention provides a method for identifying and quantifying polyglycopeptides in a sample. The method can include the steps of immobilizing glycopolypeptides to a solid support; cleaving the immobilized glycopolypeptides, thereby releasing non-glycosylated peptides and retaining immobilized glycopeptides; releasing the glycopeptides from the solid support; and analyzing the released glycopeptides. The method can further include the step of identifying one or more glycopeptides, for example, using mass spectrometry.

Methods for quantitative proteome analysis of glycoproteins

Methods using gel electrophoresis and mass spectrometry for the rapid, quantitative analysis of proteins or protein function in mixtures of proteins derived from two or more samples in one unit operation are disclosed. In one embodiment the method includes (a) preparing an extract of proteins from each of at least two different samples; (b) providing a set of substantially chemically identical and differentially isotopically labeled protein reagents; (c) reacting the extract of proteins from different samples of step (a) with a different isotopically labeled reagent from the set of step (b) to provide two or more sets of isotopically differentially labeled proteins; (d) mixing each of the two or more sets of isotopically labeled proteins to form a single mixture of isotopically differentially labeled proteins; (e) electrophoresing the mixture of step (d) by an electrophoresing method capable of separating proteins within the mixture; (f) digesting at least a portion of one or more separated proteins of step (e) and (g) detecting the difference in the expression levels of the proteins in the two samples by mass spectrometry based on one or more peptides in the sample of labeled peptides. The analytical method can be used for qualitative and particularly for quantitative analysis of global protein expression profiles in cells and tissues, i.e. the quantitative analysis of proteomes.

Process for analyzing protein samples

Analytical reagents and mass spectrometry-based methods using these reagents for the rapid, and quantitative analysis of proteins or protein function in mixtures of proteins. The methods employ affinity labeled protein reactive reagents having three portions: an affinity label (A) covalently linked to a protein reactive group (PRG) through a linker group (L). The linker may be differentially isotopically labeled, e.g., by substitution of one or more atoms in the linker with a stable isotope thereof. These reagents allow for the selective isolation of peptide fragments or the products of reaction with a given protein (e.g., products of enzymatic reaction) from complex mixtures. The isolated peptide fragments or reaction products are characteristic of the presence of a protein or the presence of a protein function in those mixtures. Isolated peptides or reaction products are characterized by mass spectrometric (MS) techniques. The reagents also provide for differential isotopic labeling of the isolated peptides or reaction products which facilitates quantitative determination by mass spectrometry of the relative amounts of proteins in different samples. The methods of this invention can be used for qualitative and quantitative analysis of global protein expression profiles in cells and tissues, to screen for and identify proteins whose expression level in cells, tissue or biological fluids is affected by a stimulus or by a change in condition or cell state of the cell, tissue or organism from which the sample originated.

The present invention provides analytical devices for the characterization of the primary structure of proteins and peptides, comprising a microenzyme reactor, a separation device, an interface between the microenzyme reactor and the separation device, a mass spectrometer, and an interface between the separation device and the mass spectrometer. Also provided are methods for characterizing a protein or peptide utilizing such devices.

Growing community of inventors

Mercer Island, WA, United States of America

Rudolf Hans Aebersold