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Mass spectrometry, also referred to as mass spec or MS, is an analytical technique that is becoming increasingly important in bioscience research. This article will introduce you to mass spectrometry in biological research, explain how it works, and how it could be useful in your research.

What Is Mass Spectrometry?

In a nutshell, mass spectrometry accurately measures the mass of different molecules within a sample. Even large biomolecules like proteins are identifiable by mass, which means that biologists can perform some very interesting experiments using mass spectrometry, potentially adding a new dimension to their research.[1]

Mass Spectrometry in Biological Research - is it Useful?

Yes, if you want to:

Identify molecules in a mixture;

Detect impurities in a sample;

Analyze a purified protein;

Study the protein content of a sample of cells.

How Does Mass Spectrometry Work?

Before you get started using mass spectrometry in biological research, you should make sure you understand the basics of how the technique works. There are three key stages to a mass spectrometer, and we discuss each in more detail below.

1. Ionization

Molecules in a sample are vaporized (converted to the gas phase by heating). Then, an electron beam bombards the vapors, which converts the vapors to ions. Because mass spectrometry measures the mass of charged particles, only ions will be detected, and neutral molecules will not be seen.

Ions are created by giving electrons to a molecule (producing a negatively charged ion) or taking electrons away from a molecule (producing a positively charged ion).

Note: A sample can only be analyzed by mass spectrometry if it can be vaporized without decomposing.

2. Acceleration and Deflection

Next, the ions are sorted according to mass in two stages - acceleration and deflection.

Acceleration is simply attraction. The positive ions created in the ionization stage accelerate towards negative plates at a speed dependent on their mass. In other words, lighter molecules move quicker than heavier ones.

Deflection is the magnetic bit. Here, the ions are deflected by a magnetic field, and the extent of deflection is again dependent on mass. So, ions of different masses travel through the spectrometer at different speeds.

3. Detection

Ions of increasing mass eventually reach the detector one after another, and then it's over to the computer to provide a spectrum. The output will look something like in Figure 1.

Note: This simplified mass spectrum has been drawn using data from the Spectral Data Base System for Organic Compounds (SDBS) at the National Institute of Materials and Chemical Research in Japan.

Omission of minor lines with peak heights of 2 % or less of the base peak (the tallest peak) provides clarity.

Reading The Spectrum

First, you should note that the x-axis denotes not just 'mass' but ' m/z', where m is the mass and z is the charge. What the spectrometer actually measures is the mass/charge (m/z) ratio. Sounds complicated, but it's not - because since the majority of ions produced during mass spectrometry have a charge of 1, m/z is normally equal to the mass of the molecule. Simple!

Is Mass Spectrometry in Biological Research Worth It?

In a word, yes! Mass spectrometry provides accurate weight measurements for your bio- (or other) molecules, which can be used to:

Give a good estimate of the purity of the sample (i.e. whether there are one or more molecular species in your sample and what ratio those species are in)

Monitor reactions, sequence amino acids, and oligonucleotides as well as give information on protein structure

Identify and quantify samples

Mass spectrometry has enabled biology to move from identifying single proteins to proteome-wide characterization and quantification. With the development of this tool, researchers can now feasibly begin defining biochemical pathways on a kinetic basis, which will help us understand the mechanistic response of cells to changing environments.

Practical Uses of Mass Spectrometry in Biological Research

Drug companies and researchers are using the technique for drug discovery, for gaining information on drug metabolism, and for pharmacokinetic studies.[2] The study of proteins through mass spectrometry is now aided by information obtained from genome sequencing projects.[3] The technique has also found medical uses, such as neonatal screening, blood analysis, and drug testing. These uses also make mass spectrometry (when coupled with other analytical techniques) a powerful tool in forensic analysis.

How Do I Go About Using Mass Spectrometry in Biological Research?

If you feel that mass spectrometry is something that could be of use in your research, check whether your department or college has mass spectrometers available for use. This might be either as a 'walk-up' service where you book a slot and run your own samples or as a technician-run service. Training will always be provided for these services so ask around.

While the number of uses is large, the instrument itself is not - normally the machine can fit on a benchtop.

But Remember This Before You Use a Communal Mass Spectrometer

The most important factor in getting accurate results is keeping it clean and free of contaminants. As many a chemist will testify, this is often an issue with communal mass spectrometers - especially if the user before you did not clean the column properly. It's wise to bring your own solvent to clean the column before (and after!) use.

On the other hand, also note that accidental contamination during sample preparation can mess up your results, and keeping a tab of these mass spec contaminants can help your experiment.

Want to Know More?

This article was a brief introduction to the basics of mass spectrometry, but it doesn't end here! There are various ways of producing the required ions, and the method chosen depends on the nature of the sample molecule. You can find out more information in the references below. Try out the technique and let us know what new applications it finds in your research.
References

1. Baker M. (2010) Mass spectrometry for biologists. Nature Methods. 7:157.

2. Ashcroft AE. An Introduction to Mass Spectrometry.

3. Yates JR. Mass spectrometry in biology. In: Encyclopedia of Life Sciences. pp. London: Nature Publishing Co.; 2001. 1-5.

Originally published February 16, 2011. Reviewed and republished June 2021.

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Mass Spectrometry in Biological Research: An Easy Intro

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Mass spectrometry, also referred to as mass spec or MS, is an analytical technique that is becoming increasingly important in bioscience research. This article will introduce you to mass spectrometry in biological research, explain how it works, and how it could be useful in your research.

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This protocol guides you through the western blot procedure using chemiluminescent and fluorescent detection.

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This sketch video about epigenetics was created by Armando Hasudungan, in collaboration with Professor Susan Clark and Dr Kate Patterson at the Garvan Institute of Medical Research. It has been created for a broad, non-expert audience to highlight key messages about the role epigenetics plays in biological processes like development and diseases such as cancer.

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Methods in Enzymology - ScienceDirect.com

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The online version of Methods in Enzymology at ScienceDirect.com, the world's leading platform for high quality peer-reviewed full-text journals.

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This chromatin remodeling tutorial explains the chromatin immunoprecipitation assay to find out the exact structure of a nucleosome complex in eukaryotes. For more information, log on to- http://shomusbiology.weebly.com/ Download the study materials here- http://shomusbiology.weebly.com/bio-materials.html PPT source- Dr. Anindita Banerjee, Department of Microbiology, St. Xavier's college

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There are two problems that pathologists face today. The first is, that a pathologist's diagnosis using tissue sections is becoming more complex. There are many more biomarkers, and clinicians now ask for a more precise diagnosis. Secondly, the number of pathologists worldwide is declining every day, as the profession is often overlooked in favor of other specializations.

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DNA methylation is an epigenetic mechanism used by cells to control gene expression. A number of mechanisms exist to control gene expression in eukaryotes, but DNA methylation is a commonly used epigenetic signaling tool that can fix genes in the "off" position.

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Methylated DNA Immunoprecipitation and Micro-array Based Analysis: Detection of DNA Methylation in Breast Cancer Cell Lines

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Methylated DNA Immunoprecipitation and Micro-array Based Analysis: Detection of DNA Methylation in Breast Cancer Cell Lines

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Y. Weng, T.H. Huang and P.S. Yan
Methods Mol Biol: 2009
doi: 10.1007/978-1-60327-378-7_10

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Methylated DNA immunoprecipitation(MeDIP) - Dna methylation | Diagenode

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Methylated DNA immunoprecipitation method is based on the affinity purification of methylated DNA using an antibody directed against 5 methylcytosine (5-mC).

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Assessing the efficiency and significance of Methylated DNA Immunoprecipitation (MeDIP) assays in using in vitro Methylated Genomic DNA

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Assessing the efficiency and significance of Methylated DNA Immunoprecipitation (MeDIP) assays in using in vitro Methylated Genomic DNA

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J. Jia, A. Pekowska, Sebastien Jaeger, T. Benoukraf, P. Ferrier, S. Spicuglia
BMC Res Notes: 2010 Sept.
doi: 10.1186/1756-0500-3-240

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MeDIP

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Next Generation Sequencing - Advances, Applications and Challenges

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Buy Next Generation Sequencing - Advances, Applications and Challenges by Jerzy K. Kulski (ISBN: 9789535122401) from Amazon's Book Store. Everyday low prices and free delivery on eligible orders.

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Immediate chromatin immunoprecipitation and on-bead quantitative PCR analysis: a versatile and rapid ChIP procedure

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Immediate chromatin immunoprecipitation and on-bead quantitative PCR analysis: a versatile and rapid ChIP procedure

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K.M Harmeyer, P.F South, B. Bishop, J.Ogas an S.D Briggs.
Nucleic Acids Res. 2014 Dec
DOI: 10.1093/nar/gku1347

Links to PCR
Highlights issues of time restraints and improvements for this.

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Chromatin Immunoprecipitation and Microarray Analysis Suggest Functional Cooperation between Kaposi's Sarcoma-Associated Herpesvirus ORF57 and K-bZIP

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Chromatin Immunoprecipitation and Microarray Analysis Suggest Functional Cooperation between Kaposi's Sarcoma-Associated Herpesvirus ORF57 and K-bZIP

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O.V. Hunter, E. Sei, R. Black Richardson and N.K. Conrad
J Virol. 2013 doi: 10.1128/JVI. 03459

ORF57 regulatory gene link to K-bZIP protein, highlights modes of coregulation.

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Bio-Rad Introduces SureBeads™ Magnetic Bead Immunoprecipitation System | News & Events | Life Science Research | Bio-Rad

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Home >Life Science Research >News & Events >News > Bio-Rad Introduces SureBeads™ Magnetic Bead Immunoprecipitation System

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An old adage says: "Show me your friends, and I'll know who you are." In the same way, finding interaction partners for a protein can reveal its function. To that end, researchers are now building entire networks of protein-protein interactions. Unlike biological pathways, which represent a sequence of molecular interactions leading to a final result - for example, a signalling cascade - networks are interlinked. Represented as starbursts of protein 'nodes' linked by interaction 'edges' to form intricate constellations, they provide insight into the mechanisms of cell functions. Furthermore, placing proteins encoded by disease genes into these networks will let researchers determine the best candidates for assessing disease risk and targeting with therapies.

K. ONO/UC SAN DIEGO/CYTOSCAPE

"This is the next step after the Human Genome Project," says Trey Ideker, a systems biologist at the University of California, San Diego, and principal investigator at the National Resource for Network Biology, which provides open-source software for network visualization. "That effort identified 30,000 genes, but that is not the end goal. How the genes work in pathways and how these pathways function in disease states and development is the end goal. To accomplish this we will need to systematically map gene and protein interactions."

Unlike the genome, the interactome - the set of protein-to-protein interactions that occurs in a cell - is dynamic. Many interactions are transient, and others occur only in certain cellular contexts or at particular times in development. The interactome may be tougher to solve than the genome, but the information, researchers say, is crucial for a complete understanding of biology.

The right partners

At any time, a human cell may contain about 130,000 binary interactions between proteins. So far, a mere 33,943 unique human protein-protein interactions are listed on BioGRID ( http://thebiogrid.org), a database that stores interaction data. Clearly, there is work to do.

There are two main approaches for detecting interacting proteins: techniques that measure direct physical interactions between protein pairs - binary approaches - and those that measure interactions among groups of proteins that may not form physical contacts - co-complex methods (see 'Tools for the search').

The most frequently used binary method is the yeast two-hybrid (Y2H) system. It has variations involving different reagents, and has been adapted to high-throughput screening. The strategy interrogates two proteins, called bait and prey, coupled to two halves of a transcription factor and expressed in yeast. If the proteins make contact, they reconstitute a transcription factor that activates a reporter gene.

Another method for identifying binary interactions is luminescence-based mammalian interactome mapping (LUMIER), a high-throughput approach developed by Jeff Wrana at the Samuel Lunefeld Research Institute in Toronto, Canada. This strategy fuses Renilla luciferaze (RL) enzyme, which catalyses light-emitting reactions, to a bait protein, which is expressed in a mammalian cell along with candidate protein partners tagged with a polypeptide called Flag. Researchers use a Flag antibody to immunoprecipitate all proteins with the Flag tag, along with any that interact with them. Interactions between the RL-fused bait and the Flag-tagged prey are detected when light is emitted. Other binary methods include the mammalian protein-protein interaction trap and techniques based on proteome chips.

The most common co-complex method is co-immunoprecipitation (coIP) coupled with mass spectrometry (MS). In this approach, a protein bait is tagged with a molecular marker. Several types of tags are commercially available; each requires a distinct biochemical technique to recognize the tag and fish the bait protein out of the cell lysate, bringing with it any interacting proteins. These are then identified by MS.

In addition to these empirical methods, researchers have used computational techniques to predict interactions on the basis of factors such as amino-acid sequence and structural information. "People ask 'Why are you predicting interactions when you can just do the experiment?'" says Gary Bader, a bioinformatician at the University of Toronto. "But experimental techniques fail for some proteins."

False readings

Every step of a procedure to detect protein-protein interactions - from the reagents used to the cell types and experimental conditions - influences the proteins that are identified. Two studies this year used similar methods to identify interacting proteins in transcription factors in embryonic stem cells; there was incomplete overlap between the resulting data sets. "If you use the same protocol you will get reproducible lists of proteins. But different labs use different protocols, which affects the end result," says Raymond Poot, a cell biologist at Erasmus MC hospital in Rotterdam, the Netherlands, and lead author of one of the studies.

In his protocol, Poot pulled interacting proteins from cells using nuclear extracts expressing different Flag-tagged transcription factors. He added a nuclease to his reactions to remove DNA and eliminate possible artefacts caused by proteins binding to it. "Transcription factors bind to DNA so you are likely to pull out DNA-binding factors that are not directly interacting," he explains. Purifying many different transcription factors with the same protocol also enabled the researchers to determine which interactions were most likely to be specific. For example, proteins that consistently co-purified with all transcription factors would be treated as unlikely to indicate a genuine interaction.

Calling out false positives - reported interactions that don't actually occur - and false negatives - interactions that do occur but are not picked up by the experimental protocol or are discarded - is one of the main challenges in the field. "Normally when you do a coIP followed by MS you will get hundreds of protein candidates interacting with any one bait," says Wade Harper, a cell biologist at Harvard Medical School in Boston, Massachusetts. "When you weed out all the stochastic and non-specific interactions you end up with many fewer proteins. Some proteins in large complexes might have 30-50 partners, others only 4-5."

One way in which researchers increase the accuracy of their results is to use more than one method (for example, Y2H plus LUMIER) to detect the interactions. But the definition of a 'real' interaction depends on the context. "Does a real interaction mean that two proteins interact if they are placed next to each other in a test tube, or that they must interact in a cell? Or does real mean that the interaction should have a biological function?" asks Ideker. Researchers can home in on functional interactions by combining data on interactions with other types of biological information, such as genetic interactions, protein localizations or gene expression. For instance, proteins whose genes are co-expressed are likely to interact with each other or to be part of the same complex or pathway.

Many tools are available on the web for integrating different types of information about a given protein or gene. One is GeneMANIA, developed by Bader's group in collaboration with Quaid Morris, a computational biologist also at the University of Toronto. A user enters the gene names into GeneMANIA; the program provides a list of genes that are functionally similar or have shared properties, such as similar expression or localization, and then displays a proposed interaction network, showing relationships among the genes and the type of data used to gather that information. The user can click on any node to obtain information about the gene and on any link to obtain information about their relationship (such as citations for any published studies or other sources of data). "It's like a Google for genetic and protein information," says Bader.

Other web-based interfaces that predict gene functions include STRING ( http://string-db.org) developed at the European Molecular Biology Laboratory in Heidelberg, Germany. It hunts for protein interactions on the basis of genomic context, high-throughput experiments, co-expression and data from the literature.

Keeping score

To select real protein-protein interactions, Harper and some members of his lab, Matt Sowa and Eric Bennett, developed a software platform called CompPASS to assign confidence scores to an interaction detected by MS. CompPASS takes data sets of interacting proteins (including those identified in experiments) and measures frequency, abundance and reproducibility of interactions to calculate the score.

This year, Harper used CompPASS to identify interactions among proteins involved in autophagy, the process by which cellular proteins and organelles are engulfed into vesicles and delivered to the lysosome to be degraded. Starting with 32 proteins known to have a role in autophagy, they identified 2,553 interacting proteins using coIP-MS. CompPASS then narrowed the list down to 409 high-confidence interacting proteins with 751 interactions.

Ideker's group used a different approach to map interactions among human mitogen-activated protein kinases (MAPKs), which respond to external stimuli and regulate cell function. Having used Y2H to identify more than 2,000 interactions among known MAPKs, Ideker used evidence including conservation of interactions among different species to winnow that down to a core network of 641 high-confidence interactions.

K. ONO/UC SAN DIEGO/CYTOSCAPE

For some of the proteins there was no previous evidence of interactions with MAPKs. Ideker and his colleagues knocked down the expression of these proteins using RNA interference, then looked for the effect of the knock-downs on proteins known to be activated by MAPKs. This allowed them to confirm that about one-third of their interactions had a role in MAPK signalling.

These methods are helping to weed out false positives and provide associated confidence scores, but the problem of false negatives persists. "With these assays we try to get false positives down to zero. The hit you take is on false negatives. So now you can be highly confident of your data but you are probably probing only about 20% of the interactome," says Ideker. "We would like to get every interaction but we do not get even close with current technologies."

New methods may become available to identify interactions that escape detection by current techniques (see 'Real-time analysis'). In the meantime, one way to address the problem is to combine procedures for detecting interactions, each sampling a different portion of the interactome. The interaction data obtained in an experiment can also be combined with that available in public databases, thus providing a more complete picture, says Bader.

From data to networks

Protein-protein interactions are only the raw material for networks. To build a network, researchers typically combine interaction data sets with other sources of data. Primary databases that contain protein-protein interactions include DIP ( http://dip.doe-mbi.ucla.edu), BioGRID, IntAct ( http://www.ebi.ac.uk/intact) and MINT ( http://mint.bio.uniroma2.it). These databases have committed to making records available through a common language called PSICQUIC, to maximize access.

Other types of data that can be combined with protein-protein interactions include information on gene expression, cellular co-localization of proteins (based on microscopy), genetic information, metabolic and signalling pathways, and data from high-throughput assays.

"One challenge computationally is integrating heterogeneous data sets to build a network model," says Ilya Shmulevich, a professor at the Institute for Systems Biology in Seattle, Washington. The second challenge is to decide on a modelling approach. "It will depend on what kind of data you have available and how you will be using the model," says Shmulevich.

Several bioinformatic tools have been developed to model and represent networks. The most widely used ones are associated with Cytoscape ( http://www.cytoscape.org), an open-source program for visualizing networks and for integrating them networks with other types of data. Several Cytoscape plug-ins allow users to download and explore databases.

Commercial packages with similar functions include MetaCore from GeneGO in St Joseph, Michigan; Pathway Analysis from Ingenuity Systems in Redwood City, California; and Pathway Studio from Ariadne Genomics in Rockville, Maryland. These can access public sources of data as well as the company's proprietary databases. "One of the unique features of Pathway Studio is the openness of our system and the ability to integrate many different kinds of data," says David Denny, director of marketing and product management at Ariadne.

Guilt by association

One reason for developing networks is to help assign functions to proteins through guilt by association. But "a huge slice of the proteome consists of proteins that no one knows what they do or interact with", says Benjamin Cravatt, a chemical physiologist at the Scripps Research Institute in San Diego, California.

For proteins not yet assigned to a portion of the human interaction network, Cravatt's group developed a technology for assigning protein functions by exploiting an interaction between enzymes and chemical reagents dubbed activity-based probes. These probes consist of a reactive group that binds the active sites of many members of an enzyme family, and a reporter tag that is used for the detection and identification of the probe-labelled enzymes.

Because these probes bind only to enzymes that are active, they can give insights into the enzymes' functions. For example, if a probe binds to a set of enzymes in a cancer cell but not in a normal cell, it means that these enzymes become more active in the cancer cell and so may have a role in cell growth. The activity probes can also serve as assays for the discovery of inhibitors for a particular enzyme, which may help researchers to understand the role of that enzyme. "You can develop an inhibitor for an enzyme before ever knowing what the actual substrate is," says Cravatt.

This year, he developed another strategy that not only determines differences in enzyme activities in different cells, but also pinpoints where in the protein these differences occur, providing a more quantitative measure of the differences. The activities of many families of enzymes are regulated or fine-tuned by cysteine modifications. By looking specifically for changes in cysteine modifications across the proteome, he found 'hyper-reactive' cysteine residues in several proteins of unknown function, which suggests that they probably have roles in signalling pathways.

One challenge in defining protein-protein interaction networks is that interactions vary depending on the type of cell and the cellular environment. For example, Wrana mapped the protein-protein interaction network for TGF-β, a growth factor that regulates cell functions, and found that two proteins that pass on the signals from the factor inside the cell - Smad2 and Smad4 - interact with one another only when the cells are stimulated with TGF-β. If the cells are not stimulated, these two proteins don't come into contact.

Bennett, Harper and Steven Gygi, a cell biologist also at Harvard Medical School, developed a proteomics platform centred around a technology called multiplex absolute quantification (AQUA) to look at dynamic changes in protein interaction networks. AQUA uses synthetic peptides that contain stable isotopes as internal standards for the native peptides that are produced when proteins from a cell lysate are digested. Using tandem MS, researchers can compare the levels of native and synthetic peptides in a cell to obtain a measure of the amount of native proteins present. Synthetic peptides can also be prepared with modifications, such as extra phosphate groups, to measure the number of post-translationally modified proteins. "We are pursuing the dynamics of protein networks by quantifying changes in the amount of proteins present in specific protein complexes," says Harper. "Techniques such as AQUA provide an accurate and sensitive measure of how the stoichiometry of components within complexes that make up a network are altered in response to a stimulus."

The team used the approach to describe the rearrangements that occur in the protein network of cullin-RING ubiquitin ligases, enzymes that regulate protein turnover, under various cellular conditions.

Developments in diagnostics

Changes in protein-protein interaction networks may provide information about the mechanisms of disease. Last year, Wrana applied the network approach to the diagnosis of breast cancer. He used microarrays to measure genome-wide protein expression in the tumours of people with breast cancer, and then overlaid the expression data on the network diagram of the human interactome.

Wrana had noted that 'hub' proteins, defined as those that interact with more than four others, can be grouped into two categories depending on whether they are expressed at the same time as the proteins with which they interact. When they looked at breast-cancer samples, Wrana and his colleagues found that certain hub proteins were in a different category in breast-cancer patients with a good prognosis than in those with a poor prognosis.

Thus, by overlaying the expression pattern of a cancer cell from an individual patient onto the human interactome network, Wrana could predict a patient's prognosis. "We found that the detection of global changes in network organization is more predictive of outcome than is gene expression alone," says Wrana. "We have now applied this method to other tumour models and obtained similar results."

KAYAK (kinase activity assay for protein profiling) is another approach to developing diagnostic tools for cancer on the basis of the functional consequences of the interaction between a protein, in this case a kinase, and its substrate. In this method, up to 90 peptide substrates for kinases are used to simultaneously measure the addition of phosphate groups to proteins in a cell lysate - in essence providing a 'phosphorylation signature' for that particular cell. "The readout is so sensitive and so quantitative that even small differences are teased out," says Gygi, who helped to develop the method.

According to Gygi, the biggest application of KAYAK might be in tumour classification. "Biopsies or excised tissues can be profiled for kinase activities with pinpoint accuracy. These patterns could contribute towards personalized drug treatments based on dysregulated kinase pathways," he says.

The combination of different types of data and technologies should continue to fill in the empty spaces of the current human interactome map. The picture may never be complete, but it will continue to provide insights into cellular mechanisms of health and disease. "I think that the network we have is dense enough for us to start doing studies to classify disease states," says Wrana. "As the networks become better and coverage improves, the accuracy of diagnosis will also improve."

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An old adage says: "Show me your friends, and I'll know who you are." In the same way, finding interaction partners for a protein can reveal its function. To that end, researchers are now building entire networks of protein-protein interactions.

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Co-Immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

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Co-Immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

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J. Verhelst, Dorein De Vlieger and Xavier Saelens.
J Vis EXP 2015. doi: 10.37891/52871

Provides in depth protocol.
Talks about disadvantages.
Interaction of antiviral Mx1 and nucleoprotein.

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Immunoprecipitation (IP) is the small-scale affinity purification of antigens using a specific antibody that is immobilized to a solid support such as magnetic particles or agarose resin. Immunoprecipitation is one of the most widely used methods for isolation of proteins and other biomolecules from cell or tissue lysates for the purpose of subsequent detection by western blotting and other assay techniques.

What is immunoprecipitation? How does IP work?

Immunoprecipitation was first developed as an adaptation of traditional column affinity chromatography, which involves allowing sample, wash, and other solutions to pass through a column that is packed with porous resin (typically beaded agarose) onto which a target-specific antibody has been immobilized. However, instead of using a packed column, immunoprecipitation uses a small amount of resin in a microcentrifuge tube, and incubation steps are performed in a batch-wise manner. For each step, solution is added to the beads, which are then mixed and incubated together as a slurry (i.e., the beads are suspended in the solution). At the end of each incubation step, the beads are pelleted to the bottom of the tube by centrifugation (or a magnet; see below) so that the solution can be removed using a pipet.

Unlike column affinity chromatography, the goal of immunoprecipitation is to isolate just enough protein to be able to measure it by western blotting or other semi-quantitative or quantitative assay methods. Usually treated and untreated samples are compared to assess the relative amount of the protein of interest. The basic protocol for performing an IP is diagrammed below, where the order (sequence) of steps can be done in two different ways.
Diagram of immunoprecipitation (IP) using either pre-immobilized or free antibodies. Each step involves incubation, followed by bead collection (centrifugation or magnetic) and removal of the solution. Wash steps are also included after each incubation step. Elution during the final step typically involves heating the beads in sample loading buffer for polyacrylamide gel electrophoresis (SDS-PAGE), which results in denaturation of the proteins (including the antibody) and irreparable damage to the beads, which are discarded.
In one sequence (left), an antibody (monoclonal or polyclonal) against a specific protein is pre-immobilized onto an insoluble support, such as agarose or magnetic beads, and then incubated with a cell lysate containing the target protein. During the incubation period, gentle agitation of the lysate allows the target antigen to bind to the immobilized antibody. The immobilized immune complexes are then collected from the lysate, eluted from the support and analyzed based on the nature of the target antigen.

Alternatively (right), free, unbound antibody is allowed to form immune complexes in the lysate and the complexes are then retrieved by the beads. While the pre-immobilized antibody approach is more commonly used for IP, using free antibody to form immune complexes is beneficial if the target protein is present in low concentrations, the antibody has a weak binding affinity for the antigen or the binding kinetics of the antibody to the antigen are slow

Magnetic beads vs. agarose resin for immunoprecipitation

Because it developed as an adaptation of column affinity chromatography, the IP technique was first done using small aliquots (10-25 µL) of agarose resin in microcentrifuge tubes. Agarose beads are sponge-like structures of varying shapes and sizes (50 to 150 μm diameter). Centrifugation must be used to separate the beads from sample and buffer solutions, either by pelleting the translucent beads and carefully pipetting off the solution or by using a microcentrifuge filter cup to retain the beads and collect the solution in the tube upon centrifugation. These characteristics limit the extent to which agarose-based IP procedures can be scaled down or automated.

Magnetic particles, such as Dynabeads and Pierce magnetic beads, have largely replaced agarose beads as the preferred support for immunoprecipitation and other micro-scale affinity purification procedures. Magnetic particles are solid and spherical, and antibody binding is limited to the surface of each bead. While they do not have the advantage of a porous center to increase the binding capacity, magnetic beads are significantly smaller than agarose beads (1 to 4 μm diameter), which collectively give them sufficient surface area for high-capacity antibody binding.

High-power magnets are used to localize magnetic beads to the side of the incubation tube and out of the way to enable cell lysate aspiration without the risk of also aspirating immune complexes bound to the beads. Magnetic separation avoids centrifugation, which can break weak antibody-antigen binding and cause loss of target protein. They make it easier to manually pipet solutions from the beads, and instrumentation is available to fully automate magnetic bead procedures, even in 96-well microplate formats.

They make it easier to manually pipet solutions from the beads, and instrumentation is available to fully automate magnetic bead procedures, even in 96-well microplate formats.

The performance advantages of magnetic beads compared to agarose resin for immunoprecipitation procedures are summarized in the following table and described in detail thereafter. These advantages are supported by current publication trends (see summary of data here), which show a clear migration from resin-based to magnetic bead methods for IP.
Performance of agarose resin vs magnetic beads in immunoprecipitation (IP) applications. This diagram compares and contrasts the distinct advantages and disadvantages of using agarose resin vs magnetic beads for immunoprecipitation.
Details concerning the advantages of magnetic beads for IP

Capacity and yield -Because beaded agarose resin is porous (sponge-like), it has a large surface-area-to-volume ratio and therefore a high binding capacity for antibody. Magnetic beads are nonporous with a smooth outer surface. Consequently, although they are much smaller than agarose beads, magnetic beads have a lower theoretical binding capacity than agarose. However, individual antibodies immobilized on the sponge-like agarose aren't always accessible to target proteins (which often exist as large protein complexes) in a sample, and antibody may be lost during the washing steps (centrifugation). By contrast, antibodies bound to the surface of magnetic beads are all accessible to antigen, and the antibody is less likely to be lost during the gentle washing steps (on a magnet). Therefore, even if the antibody-binding capacity is lower compared to agarose, the final antigen yield is often the same or greater with magnetic beads.

Reproducibility and purity -With agarose beads, it is difficult to thoroughly remove buffer without also disturbing and drawing up some of the pelleted resin. With magnetic beads and a magnet, all the beads are held firmly to the side of the tube, so all of the buffer can be removed without touching the bead-pellet. Furthermore, agarose typically requires longer incubation times (to enable diffusion of solutions to the internal spaces within) and pre-clearing (to control non-specific bind of unwanted proteins). Because all interactions occur on the outer smooth surface of the magnetic beads, and because they are much more uniform in size, magnetic beads generally provide higher reproducibility and purity compared to agarose. Pre-clearing is usually not necessary with magnetic beads.

Ease of use, speed, and automation -Considered together, the aforementioned differences between agarose and magnetic beads explain why magnetic beads are now preferred for immunoprecipitation. Because it requires longer incubation times, pre-clearing steps, and several centrifugation steps, agarose-based IP requires considerable manual handling and a total time of 1-1.5 h to perform. By contrast, an individual magnetic bead IP experiment can be completed in about 30 min.

Furthermore, magnetic bead separation lends itself to processing multiple samples easily and rapidly. For example, with a DynaMagTM-2 Magnet and accompanying sample rack, one can easily process 16 samples at the same time using magnetic beads in microcentrifuge tubes. Also available is a  magnet for 96-well plates. In fact, entire IP protocols can be automated with magnetic beads using small benchtop instrumentation, such the Thermo Scientific Kingfisher Flex Magnetic Bead Processor.

Summary of bead options for IP

Use magnetic beads for immunoprecipitation  (i.e., when the sample size is < 2 mL). Magnetic beads provide the best balance of capacity/yield, reproducibility, purity, and cost savings for routine small-scale isolation of specific proteins and protein complexes. Magnetic beads are best for manual and automated standard IP, Co-IP, ChIP, ChIP-Seq, RIP, and pull-down reactions for immediate assay analysis.

Use agarose resin for protein purification  (i.e., when the sample size is > 2 mL). Agarose is most appropriate for column affinity chromatography or individual, large-scale spin cup IP reactions when sufficient antibody is plentiful at a low cost and where the goal is to purify a sufficient amount of target protein for multiple downstream assays.

Types of immunoprecipitation

In its simplest form, IP is used to isolate a single protein (the target antigen of the antibody) to investigate its identity, structure, expression, activation or modification state. Variations of IP are also used to study the interactions of the primary antigen protein with other proteins or nucleic acids. In these methods, the goal is to study the interactors or associated cellular components that are bound to the primary antigen.

Co-immunoprecipitation is a popular technique for protein interaction discovery. Co-IP is conducted in essentially the same manner as an IP, except that the target antigen precipitated by the antibody is used to co-precipitate its binding partner(s) or associated protein complex from the lysate. The assumption that is usually made when associated proteins are co-precipitated is that these proteins are related to the function of the target antigen at the cellular level. This is only an assumption, however, that is subject to further verification.

Chromatin immunoprecipitation (ChIP) and RNA immunoprecipitation (RIP)

Chromatin immunoprecipitation (ChIP) assays are performed to identify regions of the genome with which DNA-binding proteins, such as transcription factors and histones, associate. In ChIP assays, proteins bound to DNA are temporarily crosslinked and the DNA is sheared prior to cell lysis. The target proteins are immunoprecipitated along with the crosslinked nucleotide sequences, and DNA is then removed and identified by PCR, sequenced, applied to microarrays or analyzed in some other way. RNA immunoprecipitation (RIP) is similar to ChIP, except that RNA-binding proteins are immunoprecipitated instead of DNA-binding proteins. Immunoprecipitated RNAs can then be identified by RT-PCR and cDNA sequencing.

Tagged-protein IP or tag-based pull-down

A key limitation of the previously-described IP approaches is their dependence upon the availability of antibodies that specifically recognize the target protein with little or no cross-reactivity with other cellular targets. Due to this limitation, many proteins are unable to be immunoprecipitated because of the lack of an available antibody.

To circumvent this problem, proteins can be tagged with an epitope to which a high-affinity antibody is available and ectopically expressed in the cell of interest. Today, this approach is commonplace for all types of immunoprecipitations in molecular biology research. These tags can be either short peptide sequences or fluorescent proteins, including:

FLAG; peptide sequence DYKDDDDK

c-Myc; peptide sequence EQKLISEEDL

Hemagglutinin (HA); peptide sequence YPYDVPDYA

V5; peptide sequence GKPIPNPLLGLDST

Green fluorescent protein (GFP)

One downside of using tagged proteins is that the overexpressed tagged protein, not the endogenous protein, is immunoprecipitated, which limits the applicability of any findings using this approach to true biological relevance. Additionally, tagging the protein may interfere with protein function.

However, growth factors and conditions affecting a specific protein-protein interaction in cultured cells can be measured very precisely using a quantitative immunoprecipitation (qIP) method based on co-expression of epitope-tagged and luciferase-tagged proteins.

Strategies for antibody immobilization

Antibody-binding proteins

Protein A, Protein G, Protein A/G and Protein L are immunoglobulin (Ig)-binding proteins that, when attached to beaded support and used as affinity ligands, comprise the most popular antibody-binding platforms for IP applications. As shown in the diagram below, Proteins A and G both show specificity for the heavy chains on the Fc region of antibodies, which effectively orients the immobilized antibodies with antigen-binding sites facing outward; Protein G also shows some affinity for Fab fragments (1,3). Protein L binds to light chains, but because of specific binding characteristics, Protein L is only used for limited applications.

Most immunoprecipitations are performed with Protein A, Protein G or Protein A/G, which is an engineered recombinant protein combining four Protein A and two Protein G antibody binding sites. Protein A and G both show high affinity for antibodies of multiple, but not necessarily identical, subclasses and Ig species, while Protein A/G binds all of the subtypes to which Protein A and G individually bind.

Immobilized Protein A, G and A/G (hereafter collectively called "Protein A/G") are effective tools for attaching antibodies to a beaded support for IP applications. Innovations in manufacturing of prepared Protein A/G resins and beads have yielded commercially available supports that have very high binding capacities, enabling excellent immunoprecipitation results to be obtained with very small volumes of beads.
Diagram of Protein A/G-mediated antibody immobilization to beaded support. Protein A/G (or Protein A or Protein G) binds to the Fc region of an IP antibody to immobilize it in the correct orientation to immunoprecipitate the target antigen.
Specificity of antibody-binding proteins.  Proteins used to immobilize antibodies to beaded support show specificity to different antibody domains. Protein A and G bind to the heavy chains of the antibody Fc region, while Protein L specifically binds the light chains of the two Fab regions of the F(ab')2antibody fragment.

Protein A/G supports are not compatible with certain IP experimental situations, such as when an incompatible antibody species or subclass is used or when immunoprecipitating from serum, which contains nonspecific immunoglobulins that would compete with the IP antibody for binding to the support.

Streptavidin beads with biotinylated antibodies

Many primary antibodies are commercially available in biotinylated form, or they can be easily biotin-labeled by the researcher in the laboratory using readily available reagents or kits, such as the Pierce Antibody Biotinylation Kit for IP. When a biotinylated antibody is used in IP, streptavidin beads are the obvious choice for capturing (immobilizing) it. Furthermore, because many other proteins or nucleic acids can be biotinylated, the avidin-biotin affinity system is a versatile and powerful strategy for all kinds of pull-down reactions.
Diagram for using biotinylated antibodies and streptavidin-coupled beads to perform immunoprecipitation. This strategy can be used for classes of IP antibodies that do not bind well to Protein A or G. The strategy is also effective for pull-down reactions with any sort of biotinylated molecule
Covalent antibody immobilization

Covalent immobilization strategies chemically bind the antibody to the beaded support and remove the requirement for Protein A/G-dependent antibody immobilization. Commercial products are available that provide beaded supports that react with primary amines (-NH2) on the antibody to permanently bind the antibody to the support. Although this method couples antibodies in random orientation (based on whichever surface amines contact the reactive groups on the beads), this usually has only slight effects on the antigen-binding function and capacity of the IP antibody. Besides eliminating the dependence on Protein A/G, the direct immunoprecipitation method prevents the IP antibody from co-eluting with the antigen (when a non-reducing elution buffer is used) and interfering in SDS-PAGE analysis. Additionally, because the antibody theoretically remains intact and permanently bound to the support, it may be possible to reuse the antibody-coated support many times.
Schematic representation of a direct method to covalently immobilize antibody to IP support. This diagram features aldehyde-activated agarose beads (AminoLink Resin), but a similar immobilization mechanism applies to epoxy-activated Dynabeads and other magnetic beads.
Crosslinking antibody immobilization

It is also possible to covalently attach antibodies to Protein A/G-bound supports using a crosslinker. Examples of such crosslinkers include DSS and BS3, which are short carbon chains with reactive N-Hydroxysuccinimidyl (NHS) ester groups at each end. NHS esters react with primary amines (side chain of lysine residues in proteins) to form covalent amide bonds. If an antibody is first bound to a Protein A/G support and then mixed with a crosslinker solution, the crosslinker molecules can react to covalently link adjacent amines of the antibody and Protein A/G.
Schematic representation of covalently crosslinking the antibody to Protein A/G-coated beaded support. This diagram features agarose beads, but the same immobilization mechanism applies to Dynabeads and other magnetic beads.

As with the direct immobilization method, the crosslink method eliminates co-elution of antibody fragments and potentially enables the antibody support to be reused several times. For obvious reasons, this method can only be used for antibodies that successfully bind to Protein A or G. Because antibodies contain multiple amine groups that are not exclusively limited to the Fc region, it is important to optimize the dosage of crosslinker. If too little or too much crosslinker is used, the antibody may not become successfully linked to the Protein A/G agarose or too many of the amine groups in the antibody binding site may become modified, rendering the antibody unable to bind antigen.

IP buffers and optimization

Lysis Buffers

Although IP methods are logically and procedurally simple, the variables affecting the success of any specific experiment are as numerous and peculiar as the specific differences between individual proteins and different primary antibodies. Empirical testing is nearly always required to optimize IP conditions to obtain the desired yield and purity of target proteins. Nevertheless, consideration of the main factors involved can help to identify the components that are most likely to affect particular experiments. e control for an IP or co-IP experiment; any products obtained with these control conditions can be attributed to nonspecific (off-target) interactions. One advantage of the direct immobilization strategy described above is the lack of Protein A/G as a component, which is a potential source of nonspecific binding interactions in the assay system.

The quality of the sample that is used for IP applications critically depends on the right lysis buffer, which stabilizes native protein conformation, inhibits enzymatic activity, minimizes antibody binding site denaturation and maximizes the release of proteins from the cells or tissue. The lysis buffer used for a particular application depends on the target proteins that will be immunoprecipitated, because the location of the protein in the cell (e.g., membrane, cytosol, nucleus) affects the ease of release during lysis.

Non-denaturing buffers are used when the IP antigen is detergent-soluble and when the antibody can recognize the native form of the protein. These buffers contain non-ionic detergents, such as NP-40 or Triton X-100. Denaturing buffers, such as radio-immunoprecipitation assay (RIPA) buffer, are more stringent than non-denaturing buffers because of the addition of ionic detergents like SDS or sodium deoxycholate. While these buffers do not maintain native protein conformation, proteins that are difficult to release with non-denaturing buffers, such as nuclear proteins, can be released with denaturing buffers. Both buffers contain NaCl and Tris-HCl and have a slightly basic pH (7.4 to 8). Detergent-free buffers can also be used if the target protein can be released from cells using only physical disruption, such as mechanical homogenization or heat. These simple buffers usually consist of just EDTA in phosphate buffered saline (PBS). See the table below for the ranges of each component to aid in protocol optimization.

Because cell lysates also contain proteases and phosphatases that can modify or degrade the target protein, most IP protocols are performed at 4°C. Proteasomal inhibitors, such as PMSF, aprotinin and leupeptin are commonly added to the lysis buffer just prior to use, along with sodium orthovanadate or sodium fluoride as a phosphatase inhibitor. While these components can be added individually, commercial inhibitor cocktails are available that are higher quality and easier to use.

Sample preclearing (optional for magnetic beads)

Because lysates are complex mixtures of proteins, lipids, carbohydrates and nucleic acids, one must generally assume that some amount of nonspecific binding to the IP antibody, Protein A/G, or the beaded support might occur and negatively affect the detection of the immunoprecipitated target(s).

Preclearing is designed to remove potentially reactive, non-specific components from a lysate sample prior to the actual immunoprecipitation procedure. Preclearing is frequently necessary when using agarose beads, but it is seldom required when using magnetic beads (see videos below).

The basic approach to preclear a lysate is to incubate the sample with exactly the same components that will be used for the immunoprecipitation, except use a nonspecific antibody from the same host species as the IP antibody. Any nonspecific immune complexes will form and be immobilized to the beaded support. Additionally, if Protein A/G or agarose beads are used, this approach will allow nonspecific binding to these IP components, which along with the nonspecific immune complexes are removed from the lysate. If successful, the nonspecific lysate products will be removed by this preclearing step so that they will not co-purify with the target antigen in the actual IP experiment.

These nonspecific immune complexes may be used as a negative control for an IP or co-IP experiment; any products obtained with these control conditions can be attributed to nonspecific (off-target) interactions. One advantage of the direct immobilization strategy described above is that it is devoid of Protein A/G as a component, thereby eliminating one potential source of nonspecific binding interactions in the assay system.

Binding and wash buffers

During the immunoprecipitation, assembling the immune complexes and maintaining complex stability depend on the compatibility of the binding buffer with all of the component binding interactions. In most cases, antibody-antigen interactions are fairly robust and will occur in any standard ionic strength buffer of near-neutral pH, such as PBS. By contrast, bait-prey interactions range in strength and time from irreversible and long-lived to labile and transient, which will be influenced by both the binding conditions and the temporal completion of protocol steps.

Even after lysate preclearing, IP components will still pull down nonspecific cellular components that must be removed by gentle washes prior to sample elution. Multiple washes with simple wash buffers, such as PBS either alone or with low detergent concentrations or by moderate adjustments to salt concentration, can be used to remove these contaminants.

Elution buffers

Traditional IP for downstream analysis by reducing SDS-PAGE and western blot detection typically involves elution directly in reducing SDS-PAGE sample buffer. This buffer, which is designed to denature and reduce proteins for electrophoresis, is very effective in dissociating the affinity interactions upon which IP is based. Other downstream applications for IP products are not compatible with this buffer system, nor is it possible to take advantage of certain IP methods, such as antigen elution, without antibody fragment contamination in the direct or crosslink IP methods, when this elution buffer condition is used.

The most generally effective, nondenaturing elution buffer for protein affinity purification methods is 0.1 M glycine at pH 2.5 to 3. The low pH condition dissociates most antibody-antigen interactions, as well as the antibody-Protein A/G interaction, assuming that it has not been crosslinked. However, low-pH glycine is not universally effective; some antibody-antigen interactions do not dissociate with this buffer, and conversely, some antibodies and target antigens denature or become inactive in this buffer. Several alternative types of elution buffers are described in the following documents.

The following videos feature Dynabeads magnetic beads and collectively summarize much of the information about immunoprecipitation described above. They also demonstrate several key advantages of using magnetic beads instead of agarose resin for immunoprecipitation.

References

1. Bjorck L. and Kronvall G. (1984) Purification and some properties of streptococcal protein g, a novel IgG-binding reagent. J Immunol. 133, 969-74.

2. Harlow, Ed, and Lane, David. (1999) Antibodies: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

3. Wikstrom M. et al. (1995) Mapping of the immunoglobulin light chain-binding site of protein l. J Mol Biol. 250, 128-33.

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Overview of the Immunoprecipitation (IP) Technique

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Immunoprecipitation (IP) is the small-scale affinity purification of antigens using a specific antibody that is immobilized to a solid support such as magnetic particles or agarose resin. Immunoprecipitation is one of the most widely used methods for isolation of proteins and other biomolecules from cell or tissue lysates for the purpose of subsequent detection by western blotting and other assay techniques.

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genomics in endocrinology: dna microarray analysis in endocrine health and disease (contemporary endocrinology)

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science / life sciences / molecular biology

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science / life sciences / molecular biology

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Genomics in Endocrinology: DNA Microarray Analysis in Endocrine Health and Disease (Contemporary Endocrinology)

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Buy Genomics in Endocrinology: DNA Microarray Analysis in Endocrine Health and Disease (Contemporary Endocrinology) 2008 by Stuart Handwerger, Bruce Aronow (ISBN: 9781588296511) from Amazon's Book Store. Everyday low prices and free delivery on eligible orders.