Immunoprecipitation

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Studying proteins in their natural environment can be a complex and challenging task. Proteins in a cell are surrounded by millions of other molecules that may interfere with the analysis. Using the unique specificity that antibodies have for their antigen in a technique called immunoprecipitation (IP) one may capture and concentrate a protein from complex solutions which contain thousands of other proteins such as body fluids or cell and tissue lysates. The principle of IP has been applied to improve sensitivity in MS-based detection and quantification by mean of peptide or protein enrichment from high volumes of solutions. Moreover, IP provides a possibility to study interactions between proteins since other molecules in complex with the targeted protein will also be precipitated. Co-immunoprecipitation (Co-IP) aims specifically to investigate complexes and cross-linking and FLAG expression systems may be used to stabilize the complexes and simplify the purification. In particular, Co-IP have been extensively used to study protein-protein, DNA-protein (Chromatin Immunoprecipitation, ChIP), as well as RNA-protein (RNA Immunoprecipitation, RIP) interactions in living cells.

Technology

The basic principle of IP is outlined in Figure 1 (Bonifacino, Dell'Angelica, & Springer, 2001). An antibody that is highly specific to the target protein is incubated with the sample, for instance a cell lysate. The antibody can either be pre-immobilized on a solid support or used free in solution and later captured by the addition of antibody-binding proteins (e.g. protein A/G) bound to solid matrices, e.g. agarose, sepharose, or magnetic beads. Using the solid support allows the immune complex to be separated from the sample, washed, and finally released from the support in order to be analyzed. A variety of ways may be used for the detection of the precipitated protein such as gel electrophoresis, Western Blotting, or mass spectrometry. In this way, information on the level of expression, the molecular weight, which post-translational modifications the protein has etc. may be gained. Using the same principle, one may study the interaction partners of the target protein through co-immunoprecipitation (Co-IP) as any molecule bound to the target protein will follow the immune complex and will thus be co-purified in the process (Moresco, Carvalho, & Yates, 2010). A significant advantage of immunoprecipitation with antibodies targeting directly the protein of interest is that one may study the native protein with its natural functionality. When using techniques such as pull-down, where the addition of a tag allows for affinity purification, there is always the risk of the tag altering expression or function of the protein. Using highly specific antibodies, minimizing unspecific binding of protein to the matrix and the antibody, as well as including control experiments using a non-specific antibody is essential for the quality and interpretation of the results.

Figure 1. An antigen immunoprecipitation experiment. The antibody is either pre-immobilized to a solid support or immobilized using antibody binding proteins after incubation with the sample. Immobilization allows the immune complex to be extracted from the complex sample, washed and eluted providing a high enrichment of the protein under investigation.

Immunoprecipitation has been extensively used to study interactions between proteins and genomic DNA in vivo through a technique referred to as chromatin immunoprecipitation (ChIP), Figure 2. DNA-binding proteins are cross-linked in the cell to the DNA they are binding through treatment with formaldehyde or UV-light (specific photo-reactive groups are needed to activate cross-linking with UV-light). After cell lysis the DNA is broken down to smaller pieces using sonication or enzyme. Thereafter, using an antibody specific for the protein under investigation the complex is immunoprecipitated. The cross-linking is thereafter reversed, with heat in the case of formaldehyde cross-linking, and the protein broken down enzymatically using a proteinase (e.g. proteinase K). The isolated DNA can then be purified and identified through micro-arrays (ChIP-chip) or, more commonly, sequencing (ChIP-seq).

Immunoprecipitation can similarly to ChIP be used to study RNA-binding proteins in their native cellular context through the use of antibodies specific to RNA-binding proteins, RNA Immunoprecipitation (RIP).

Figure 2 Chromatin immunoprecipitation workflow.

Specific examples

Immunoprecipitation used in combination with mass spectrometry provides a powerful tool for protein analysis and several methods have been developed for this (Ten Have, Boulon, Ahmad, & Lamond, 2011). IP is a fundamental part of a technique called Stable Isotope Standards with Capture by Anti-Peptide Antibodies (SISCAPA) (Anderson et al., n.d.). Using SISCAPA a protein in a complex sample can be quantified with the help of immunoprecipitation of specified peptides. The method involves several steps of which IP is an essential one. Firstly the sample is broken down to peptides using proteases and then internal standard peptides labeled with stable isotopes are added. Low-abundance peptides are then enriched by immunoprecipitation using peptide specific antibodies followed by quantitation using MS. Likewise, Mass spectrometric immunoassays (MSIA) exploit antibodies to enrich the target of interest but in this case proteins are captured in their native form and later digested in peptides and quantified by mean of internal standard. MSIA assays have been developed for example to measure lung cancer biomarkers (Torsenetes et al.,2014a; Torsenetes et al.,2014b).

Protein interactions are fundamental in biology and co-IP followed by MS analysis is a good complement to other techniques such as the yeast-2-hybrid system to analyze protein complexes in for instance humans as well as C. elegans (Malovannaya et al., 2010). The technique has been applied in large-scale using several thousands of antibodies and IP experiments to generate a better understanding of protein complex components and cellular protein networks (Malovannaya et al., 2011).

ChIP has been used to generate genome-wide maps of specific protein-DNA interactions in cells as well as become an important tool to map epigenetic modifications in the genome. It has been one of the fundamental techniques used in the Encyclopedia of DNA Elements projects ENCODE and modENCODE that aims to map all functional elements encoded in the genomes of man and model organisms (Bernstein et al., 2012; Gerstein et al., 2010; Roy et al., 2010). Along with other techniques to study the elements of the human genome their ChIP-seq data on the binding location of more than hundred different DNA-binding proteins has revealed that a very much larger part of the genome is involved in gene regulation than that representing only the protein-coding exons.

References and Links

• Anderson, N. L., Anderson, N. G., Haines, L. R., Hardie, D. B., Olafson, R. W., & Pearson, T. W. (n.d.). Mass spectrometric quantitation of peptides and proteins using Stable Isotope
  Standards and Capture by Anti-Peptide Antibodies (SISCAPA). Journal of Proteome Research, 3(2), 235-44. Retrieved from PubMed: 15113099


• Bernstein, B. E., Birney, E., Dunham, I., Green, E. D., Gunter, C., & Snyder, M. (2012). An integrated encyclopedia of DNA elements in the human genome.
  Nature, 489(7414), 57-74. DOI: 10.1038/nature11247. PubMed: 22955616


• Bonifacino, J. S., Dell'Angelica, E. C., & Springer, T. A. (2001). Immunoprecipitation.
  Current Protocols in Immunology / Edited by John E. Coligan ... [et Al.], Chapter 8, Unit 8.3. DOI: 10.1002/0471142735.im0803s41. PubMed: 18432858.


• Gerstein, M. B., Lu, Z. J., Van Nostrand, E. L., Cheng, C., Arshinoff, B. I., Liu, T., ... Waterston, R. H. (2010). Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project.
  Science (New York, N.Y.), 330(6012), 1775-87. DOI: 10.1126/science.1196914. PubMed: 21177976.


• Malovannaya, A., Lanz, R. B., Jung, S. Y., Bulynko, Y., Le, N. T., Chan, D. W., ... Qin, J. (2011). Analysis of the human endogenous coregulator complexome.
  Cell, 145(5), 787-99. DOI: 10.1016/j.cell.2011.05.006. PubMed: 21620140.


• Malovannaya, A., Li, Y., Bulynko, Y., Jung, S. Y., Wang, Y., Lanz, R. B., ... Qin, J. (2010). Streamlined analysis schema for high-throughput identification of endogenous protein complexes.
  Proceedings of the National Academy of Sciences of the United States of America, 107(6), 2431-6. DOI: 10.1073/pnas.0912599106. PubMed: 20133760.


• Moresco, J. J., Carvalho, P. C., & Yates, J. R. (2010). Identifying components of protein complexes in C. elegans using co-immunoprecipitation and mass spectrometry.
  Journal of Proteomics, 73(11), 2198-204. DOI: 10.1016/j.jprot.2010.05.008. PubMed: 20546956.


• Roy, S., Ernst, J., Kharchenko, P. V, Kheradpour, P., Negre, N., Eaton, M. L., ... Kellis, M. (2010). Identification of functional elements and regulatory circuits by Drosophila modENCODE.
  Science (New York, N.Y.), 330(6012), 1787-97. DOI: 10.1126/science.1198374. PubMed: 21177974.


• Ten Have, S., Boulon, S., Ahmad, Y., & Lamond, A. I. (2011). Mass spectrometry-based immuno-precipitation proteomics - the user's guide. Proteomics, 11(6), 1153-9. DOI: 10.1002/pmic.201000548. PubMed: 21365760.


• Torsetnes S.B., Broughton M.N., Paus E., Halvorsen T.G., & Reubsaet L. (2014). Determining ProGRP and isoforms in lung and thyroid cancer patient samples: comparing an MS method with a routine clinical immunoassay. Analytical and Bioanalytical Chemistry, 406(11), 2733-8. DOI: 10.1007/s00216-014-7634-x. PubMed: 24518900.


• Torsetnes S.B., Levernæs M.S., Broughton M.N., Paus E., Halvorsen T.G., & Reubsaet L. (2014). Multiplexing determination of small cell lung cancer biomarkers and their isovariants in serum by immunocapture LC-MS/MS. Analytical Chemistry, 86(14), 6983-92. DOI: 10.1021/ac500986t. PubMed: 24945626.


• The ENCyclopedia Of DNA Elements (the ENCODE project) – to identify all functional elements in the human genome sequence:
  http://www.genome.gov/10005107


• Model Organism ENCyclopedia Of DNA Elements (modENCODE project) - Identification of All Functional Elements in Selected Model Organism Genomes:
  http://www.genome.gov/26524507


• Antibodypedia - An open-access database of publicly available antibodies and their usefulness in various applications:
  http://www.antibodypedia.com