Differential labeling for quantitative analysis of complex protein mixtures

a technology of complex protein mixture and quantitative analysis, applied in the direction of assay labels, peptide preparation methods, material testing goods, etc., can solve the problems of difficult to explain the mechanism of biological processes by genomic analysis alone, no protein analytical technology approaches the throughput and level of automation of genomic technology, and achieves high specificity

Inactive Publication Date: 2008-04-03
SYNGENTA PARTICIPATIONS AG
View PDF0 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The interpretation of the data obtained by these techniques in the context of the structure, control and mechanism of biological systems has been recognized as a considerable challenge.
In particular, it has been extremely difficult to explain the mechanism of biological processes by genomic analysis alone.
At present no protein analytical technology approaches the throughput and level of automation of genomic technology.
However, the sequential manner in which samples are processed limits the sample throughput, the most sensitive methods have been difficult to automate and low abundance proteins, such as regulatory proteins, escape detection without prior enrichment, thus effectively limiting the dynamic range of the technique.
While these approaches accelerate protein identification, the quantities of the analyzed proteins cannot be easily determined, and these methods have not been shown to substantially alleviate the dynamic range problem also encountered by the 2DE / (MS)n approach.
Therefore, low abundance proteins in complex samples are also difficult to analyze by the μLC / MS / MS method without their prior enrichment.
It is therefore apparent that current technologies, while suitable to identify a portion of the components of protein mixtures, are neither capable of measuring the quantity nor the state of activity of the protein in a mixture.
Even improvements of the current approaches are unlikely to advance their performance sufficiently to make routine quantitative and functional proteome analysis a reality.
While these techniques are quite sensitive, they do not necessarily provide chemical separation of products and may, as a result, be difficult to use for assaying several proteins or enzymes simultaneously in a single sample.
Current methods may not distinguish among aberrant expression of different enzymes or their malfunctions which lead to a common set of clinical symptoms.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Differential labeling for quantitative analysis of complex protein mixtures
  • Differential labeling for quantitative analysis of complex protein mixtures
  • Differential labeling for quantitative analysis of complex protein mixtures

Examples

Experimental program
Comparison scheme
Effect test

example 1

Synthesis of Peptide Labeling Moiety (or Peptide Encoded Tags, “PEPTags”)

[0421]A pair of PEPTags, described generally above, was synthesized from peptides with following sequences: Ac-AYPYDVPDYASENLYFQGK (SEQ ID NO: 39) and AYPYDVPDYASENLYFQGOrn (SEQ ID NO: 40). In dry DMF containing excess (2-3 molar equivalents) DIEA, each of the peptides was mixed with two molar equivalents of iodoacetic anhydride for 10 min at room temperature under N2 gas, to give Lys-PEPTag and Orn-PEPTag, respectively. The reaction was terminated by adding acetic acid. Solvent was removed by vacuum centrifugation, and the product was purified by reverse-phase FPLC, and analyzed by MAILDI MS (TofEpec 2E, Micromass, Beverly, Mass.) and ESI MS / MS (API 3, PE Sciex, Foster City, Calif.).

[0422]In order to demonstrate that the mass spectrometric ionization efficiency of the two synthesized peptide tags was essentially equal, the two products were mixed in different ratios and analysed by LC-MS. The ratio of the meas...

example 2

PEPTag Qualitative Protein Analysis

Simplification of Complex Mixtures

[0423]We tested the PEPTag method, described generally herein, on Bovine Serum Albumin (BSA). 200 μL BSA (0.25 mg / mL) was denatured and reduced in a solution containing 0.1% SDS, 5 mM tributyl phosphine and 50 mM Tris buffer (pH 8.5) for 3 min at 100° C. and for 1 hour at 37° C. The side chains of cysteinyl residues were derivatized with a tenfold molar excess of Lys-PEPTag. Tagged protein was digested by trypsin overnight at 37° C. Trypsin activity was quenched with trypsin inhibitor and the peptide mixture bound to anti-HA affinity matrix for 2 hours at 4° C. The anti-HA resin with bound peptides was washed in equilibration-buffer (20 mM Tris, pH 7.5; 0.1 M NaCl; 0.1 mM EDTA), 3×10 min. at 4° C. The bound peptides were cleaved from the matrix by incubation with TEV protease for 6 hours at 30° C. The cleaved peptides were analyzed by either Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS), ...

example 3

PEPTag Quantitative Protein Analysis

Differential Labeling

[0426]We tested the PEPTag quantitative strategy on two mixtures containing the same two proteins at different concentrations. Mixture 1 had 500 pmol BSA (0.1 mg / mL) and 400 pmol β-lactoglobulin (0.1 mg / mL) and was reacted with 9 mmol Lys-PEPTag. Mixture 2 had 250 pmol BSA (0.05 mg / mL) and 800 pmol β-lactoglobulin (0.2 mg / mL) and was reacted with 9 nmol Orn-PEPTag. Protein denaturation, reduction, tagging, and digestion were the same as described above. The two samples were combined after tryptic digestions, and bound to anti-HA matrix. TEV digestion and MS analysis were as described in Example 2. Peptides were quantified by measuring, in the MS mode, the relative signal intensities for pairs of peptide ions of identical sequence, tagged with Lys or Orn-PEPTags, respectively. The results are shown in FIGS. 6, 7, and 8 and the following table.

Peptide sequenceObservedExpectedProteinidentifiedratioMean ± S.D.ratioBovineSLHTLFGDEL...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
molecular weightsaaaaaaaaaa
Phaaaaaaaaaa
Login to view more

Abstract

The disclosed subject matter relates to a method of simultaneously identifying and determining the levels of expression of cysteine-containing proteins in normal and perturbed cells, a method for determining peptide expression levels between a first biological sample and a second biological sample, and a method for quantitative proteomic analysis of two or more peptide populations, and compounds and reagents related thereto.

Description

RELATED APPLICATIONS[0001]The present application is a division of, and claims priority to, U.S. application Ser. No. 10 / 212,628, filed Aug. 1, 2002, entitled “DIFFERENTIAL LABELING FOR QUANTITATIVE ANALYSIS OF COMPLEX PROTEIN MIXTURES”, which in turn is a continuation-in-part of U.S. application Ser. No. 10 / 057,789, filed Jan. 25, 2002 and entitled “DIFFERENTIAL LABELING FOR QUANTITATIVE ANALYSIS OF COMPLEX PROTEIN MIXTURES”, which in turn claims priority to U.S. Provisional Application Ser. No. 60 / 305,232 filed Jul. 13, 2001 and entitled “DIFFERENTIAL LABELING FOR QUANTITATIVE ANALYSIS OF COMPLEX PROTEIN MIXTURES”, and to U.S. Provisional Application Ser. No. 60 / 264,576 filed Jan. 26, 2001 and entitled “DIFFERENTIAL LABELING FOR QUANTITATIVE ANALYSIS OF COMPLEX PROTEIN MIXTURES”, all of which are incorporated by reference herein in their entirety including any drawings.BACKGROUND OF THE INVENTION[0002]Genomic technology has advanced to a point at which, in principle, it has become...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/53C07K1/13C07K14/395G01N33/68
CPCC07K1/13C07K14/395G01N33/6803G01N2550/00G01N33/6842G01N33/6848G01N2458/15G01N33/6815
Inventor HAYNES, PAULWEI, JINGYATES, JOHNANDON, NANCYWASHBURN, MICHAEL P.DECIU, COSMINULASZEK, RYAN U.
Owner SYNGENTA PARTICIPATIONS AG
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap