English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Immunoproteomics Using Polyclonal Antibodies and Stable Isotope-labeled Affinity-purified Recombinant Proteins

Edfors, F., Boström, T., Forsström, B., Zeiler, M., Johansson, H., Lundberg, E., et al. (2014). Immunoproteomics Using Polyclonal Antibodies and Stable Isotope-labeled Affinity-purified Recombinant Proteins. MOLECULAR & CELLULAR PROTEOMICS, 13(6), 1611-1624. doi:10.1074/mcp.M113.034140.

Item is

Files

show Files

Locators

show

Creators

show
hide
 Creators:
Edfors, Fredrik1, Author
Boström, Tove1, Author
Forsström, Bjoern1, Author
Zeiler, Marlis2, Author              
Johansson, Henrik1, Author
Lundberg, Emma1, Author
Hober, Sophia1, Author
Lehtiö, Janne1, Author
Mann, Matthias2, Author              
Uhlen, Mathias1, Author
Affiliations:
1external, ou_persistent22              
2Mann, Matthias / Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565159              

Content

show
hide
Free keywords: PEPTIDE IMMUNOAFFINITY ENRICHMENT; SPECTROMETRY-BASED QUANTIFICATION; TANDEM MASS-SPECTROMETRY; HUMAN SERUM SAMPLES; ABSOLUTE QUANTIFICATION; STATISTICAL-MODEL; PROTEOME ANALYSIS; MAGNETIC BEADS; IDENTIFICATION; SISCAPA
 Abstract: The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

Details

show
hide
Language(s): eng - English
 Dates: 2014-06
 Publication Status: Published in print
 Pages: 14
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: ISI: 000337239500018
DOI: 10.1074/mcp.M113.034140
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: MOLECULAR & CELLULAR PROTEOMICS
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3996 USA : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Pages: - Volume / Issue: 13 (6) Sequence Number: - Start / End Page: 1611 - 1624 Identifier: ISSN: 1535-9476