NEW YORK (GenomeWeb) – Researchers at the Broad Institute and Stanford University have developed an antibody-based workflow for the pulldown of biotinylated proteins.
The method, described in a paper published last week in Nature Methods, offers a simpler and more effective approach than conventional streptavidin-based techniques, said Steven Carr, senior director of proteomics at the Broad and senior author on the paper. He added that it will allow researchers to better identify the location of biotinylation sites on specific peptides, which could improve the quality of data generated by certain experiments that rely on biotin labeling of target proteins.
Biotin labeling is commonly used in protein analysis. Biotin molecules can be chemically or enzymatically bound to protein chemical groups or amino acids. Bound proteins can then be pulled down using a separate biotin-binding molecule.
Most commonly, researchers use streptavidin-based systems to pull down biotin-labeled molecules. Streptavidin binds to biotin with great strength, making it an effective approach to purifying biotin-bound targets. However, as Carr and his co-authors noted, the strength of the biotin-streptavidin bond presents a challenge to recovering the bound targets after pulldown.
This is particularly an issue for downstream mass spec analyses because the harsh reagents required to elute targets from the streptavidin used for pulldown are poorly compatible with mass spec workflows.
The biotin-streptavidin approach is particularly limiting when it comes to recovery of specific peptides, as opposed to whole proteins, added Namrata Udeshi, a Broad proteomics researcher and first author on the study.
This is especially relevant to work Carr and colleagues are pursuing using engineered ascorbic acid peroxidase (APEX) for the study of protein-protein interactions and other phenomena.
Developed by Stanford University researcher Alice Ting, a co-author on the Nature Methods paper, APEX labeling uses this peroxidase to biotinylate proteins in a particular cellular region, making it possible to pull them down and analyze them via mass spec.
Researchers insert the genetic tag for this peroxidase into a protein of interest. Upon stimulation with hydrogen peroxide, this tag releases biotin-phenoxyl radicals that tag nearby proteins in the cell, and these tagged proteins can then be pulled out of the sample.
The method allows researchers to analyze the protein components of specific subcellular regions by, for instance, adding an APEX tag to a protein known to localize to a given organelle and then pulling down all the proteins biotinylated upon addition of hydrogen peroxide. In a study published this year in Cell, Ting and colleagues used the approach to study the interactome of G-protein coupled receptors, which have been largely intractable to conventional methods of protein-interaction analysis.
Udeshi said she and her colleagues realized they might be able to further refine the cellular information they were collecting using this technique if they could better identify the specific sites where the peroxidase was labeling the proteins they pulled down. This could provide them with information not just on the proximity in the cell of a given protein to the APEX-tagged molecule but also, for instance, data on its orientation to this molecule.
Additionally, she noted, it would provide definitive evidence that this protein had, in fact, been biotinylated as opposed to pulled down via a non-specific interaction with streptavidin.
"It became clear that that would really add another layer of information," she said. "But the only way to look at those sites is, of course, to look at the modified peptides."
Antibodies against protein post-translational modifications like phosphorylation are commonly used to enrich modified peptides.
"So we thought, 'Why don't we try [anti-biotin] antibodies?'" Udeshi said. "We knew that we shouldn't have trouble eluting modified peptides from these antibodies and that we would be able to use reagents compatible with mass spec."
As an initial test of the idea, the researchers labeled peptides from a whole-cell lysate with biotin and then spiked these biotinylated peptides into a mixture of unlabeled peptides from the same lysate. Without antibody enrichment, they identified 129 biotinylated peptides. Using enrichment with an anti-biotin antibody, they were able to identify 4,810 biotinylated peptides.
They did a similar spike-in experiment comparing antibody-based enrichment to enrichment using NeutrAvidin, a form of avidin modified to have a lower affinity for biotin and so ease the challenges of eluting targets pulled down by a biotin-streptavidin system. In these experiments they identified two to three times as many biotinylated peptides using the antibody approach.
They then applied the antibody method to an APEX-labeling study of mitochondrial matrix proteins. Using streptavidin-based enrichment, they identified 511 putative mitochondrial proteins across three replicates, but identified only 185 biotinylation sites and only 38 that were detected in two or more replicates.
Using the antibody-based approach, they identified 526 proteins across two replicates and 1,695 biotinylation sites, 1,122 of which they identified in at least two replicates.
"Defining the exact sequence locations of biotinylation could provide direct insight into the topological organization of proteins in membranes," the authors wrote, and with the biotinylation site data generated using their antibody approach they looked at 42 sites on mitochondrial transmembrane proteins with known membrane topologies finding that this site location data information was able to corroborate the topological information.
Because the antibody-based approach enriches at the peptide level, it also makes for a more straightforward mass spec analysis, Udeshi said.
Enriching at the protein level and then digesting leaves a mixture not only of the biotinylated peptides but also the other peptides comprising these proteins, which, she noted, requires fractionation upfront of the mass spec analysis.
Using antibody enrichment after digestion, on the other hand, pulls out just the biotinylated peptides, a considerably less complex mixture, Udeshi said. "Once we had the [digested] peptides, we just did the antibody enrichment and ran the sample."