Expression quantification using mass spectrometry

Abstract

In various aspects, the present teachings provide systems, methods, assays and kits for the absolute quantitation of protein expression. In various aspects, the present teachings provide methods of determining the concentration of one or more proteins of interest in one or more samples of interest. In various aspects, the present teachings provide methods of determining the absolute concentration of one or more isoforms of a protein using standard samples of signature protein fragments and parent-daughter ion transition monitoring (PDITM). In various embodiments, the absolute concentration of multiple isoforms of a biomolecule in a sample, multiple proteins in a biological process, a combination of multiple samples, or combinations thereof, can be determined in a multiplex fashion using the present teachings. In various aspects, provided are methods of assessing the response of a biological system to a chemical agent.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a continuation-in-part of and claims the benefit of and priority to copending U.S. application Ser. No. 11 / 134,850, entitled “Expression Quantification Using Mass Spectrometry”, filed May 19, 2005, which claims the benefit of and priority to U.S. Provisional Application No. 60 / 572,826, entitled “Expression Quantification Using Mass Spectrometry”, filed May 19, 2004, the entire disclosures of both of which are herein incorporated by reference.INTRODUCTION [0002] Understanding protein expression is important to understanding biological systems. Unlike mRNA, which only acts as a disposable messenger, proteins implement almost all controlled biological functions and, as a result, are integral to such functions as normal cell activity, disease processes, and drug responses. However, protein expression is not reliably predictable. First, protein expression is not predictable from mRNA expression maps because mRNA tr...

AI Technical Summary

Benefits of technology

[0018] A control sample can be provided in a variety of ways. For example, a control sample can comprise, for example, a normal sample, a pooled reference standard from all or some of the samples to be analyzed, or combinations thereof. For example, in various embodiments, a control sample comprises a normal patient sample that can serve as an internmal standard to determine if samples under investigation differ from the normal sample, and thus, e.g., providing a potential indication of a disease state for a disease state. In various embodiments, the control sample is mixed into every sample to be analyzed at a substantially fixed ratio. In various embodiments, a fixed ratio of about 1:1 is used and, for example, can facilitate observation of both up-regulated and down-regulated peptides, proteins or both.

[0020] In various embodiments, the present teachings facilitate identifying therapeutic candidate compounds, including antibodies and cellular immunotherapies. In various embodiments, the present teachings facilitate the study of drug metabolizing enzymes, (for example, cytochromes P450, uridine 5′-triphosophate glucuronosyltransferases, etc.). For example, the cytochrome P450 protein family of mono-oxygenases is responsible for the regulation of drug elimination in the liver and the formation of toxic drug metabolites. There are four major families of P450 isoforms with about 25 different isoforms, each with different substrate specificities inducible by different drugs or chemicals. This enzymatic behavior can make this family of proteins important in drug development. For example, the changes in expression of the different P450 proteins can provide information on the toxicity of different drugs and the possibility of forming dangerous drug metabolites. A system, method or assay to screen for multiple P450 isoforms could be of value in drug development, particularly if it yielded quantitative data relating to expression changes for individual isoforms.

[0030] As will be appreciated more fully from the following description in conjunction with the drawings, various embodiments of the present teachings can provide methods that facilitate the discovery, verification and / or validation of biomarkers; that facilitate the elucidation of basic biology and cell signaling; that facilitate drug discovery, or combinations thereof.

[0031] In various embodiments the present teachings can elucidation of basic biology and cell signaling, for example, by facilitating the ability to quantitatively measure amount of a protein or proteins involved in a pathway; e.g., a labeled control standard being created from a “resting state” sample and being added into labeled perturbed state samples to facilitate quantitatively measuring changes in protein expression between resting and perturbed states.

[0032] In various embodiments the present teachings can facilitate drug discovery, for example, by facilitating the determination of the biological pathways effected by an agent. For example, various embodiments of the present teachings can be used to investigate a panel of proteins that represent good, or potential, drug targets. The method could be used to analyze samples that have been treated with a drug candidate to determine if any pathways have been affected, e.g., advantageous, negatively (e.g., toxic effect), or both. In various embodiments, a panel of proteins can be chosen to cover a broad spectrum of cellular pathways; and, for example, the qualitative and / or quantitative changes in protein expression used to obtain a greater understanding of the mode of action of the candidate therapeutic, the actual target, etc.

Problems solved by technology

However, protein expression is not reliably predictable.

First, protein expression is not predictable from mRNA expression maps because mRNA transcript levels are not always strongly correlated with protein levels.

Second, proteins are dynamically modified in biological systems by environmental factors in ways which are not predictable from genetic information.

Although absolute quantitation typically provides more information than relative quantitation, it has traditionally been more difficult to implement.

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