most widely used in published BioID experiments but digestion of coupled streptavidin, which is in large excess to the
captured biotinylated proteins, yields a large amount of Streptavidin peptides that might affect the sensitivity of the MS
analysis. On the other hand, elution of biotinylated proteins
requires very harsh denaturing conditions that induce significant release of streptavidin from the beads. We address this by
running the eluted sample on a gel and cutting the lane above
the streptavidin bands (running at 17 kDa). Whereas this procedure allows minimizing streptavidin contamination it has the
disadvantage of excluding small molecular weight proteins
(MW < approximately 20 kDa) from the MS analysis.
StrepTactin-coupled beads were described to allow efficient
capture of biotinylated proteins and elution in mild conditions
using an excess of free biotin [9, 10]. This procedure may thus
be the method of choice to minimize streptavidin contamination and allow analyzing the full range of captured biotinylated
proteins.
26. In our hands this procedure that has been previously described
by Dyballa and Metzger [11] consistently produces very sensitive staining with a fast and easy protocol. Other sensitive and
MS-compatible staining procedures can also be applied.
27. Standard MS analysis protocols similar to those used to analyze
sample from AP approaches are fine. More elaborate protocols
that also aim at enriching and identifying biotinylated peptides
were also described [9, 12–14].
Acknowledgments
This work was supported by the excellence initiative by the German
federal and state governments: DFG-EXC81, cluster of excellence
CellNetworks.
References
1. Scott DE, Bayly AR, Abell C et al (2016) Small
molecules, big targets: drug discovery faces the
protein-protein interaction challenge. Nat Rev
Drug Discov 15(8):533–550. https://doi.
org/10.1038/nrd.2016.29
2. Lam SS, Martell JD, Kamer KJ et al (2015)
Directed evolution of APEX2 for electron
microscopy and proximity labeling. Nat Methods 12(1):51–54. https://doi.org/10.1038/
nmeth.3179
3. Roux KJ, Kim DI, Raida M et al (2012) A
promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in
mammalian cells. J Cell Biol 196(6):801–810.
https://doi.org/10.1083/jcb.201112098
4. Kim DI, Birendra KC, Zhu W et al (2014)
Probing nuclear pore complex architecture
with proximity-dependent biotinylation. Proc
Natl Acad Sci U S A 111(24):E2453–E2461.
https://doi.org/10.1073/pnas.1406459111
5. Lambert JP, Tucholska M, Go C et al (2015)
Proximity biotinylation and affinity purification
are complementary approaches for the interactome mapping of chromatin-associated protein
complexes. J Proteome 118:81–94. https://
doi.org/10.1016/j.jprot.2014.09.011
Context-Specific Proximity-Dependent Proteomics with Split-BioID
317
captured biotinylated proteins, yields a large amount of Streptavidin peptides that might affect the sensitivity of the MS
analysis. On the other hand, elution of biotinylated proteins
requires very harsh denaturing conditions that induce significant release of streptavidin from the beads. We address this by
running the eluted sample on a gel and cutting the lane above
the streptavidin bands (running at 17 kDa). Whereas this procedure allows minimizing streptavidin contamination it has the
disadvantage of excluding small molecular weight proteins
(MW < approximately 20 kDa) from the MS analysis.
StrepTactin-coupled beads were described to allow efficient
capture of biotinylated proteins and elution in mild conditions
using an excess of free biotin [9, 10]. This procedure may thus
be the method of choice to minimize streptavidin contamination and allow analyzing the full range of captured biotinylated
proteins.
26. In our hands this procedure that has been previously described
by Dyballa and Metzger [11] consistently produces very sensitive staining with a fast and easy protocol. Other sensitive and
MS-compatible staining procedures can also be applied.
27. Standard MS analysis protocols similar to those used to analyze
sample from AP approaches are fine. More elaborate protocols
that also aim at enriching and identifying biotinylated peptides
were also described [9, 12–14].
Acknowledgments
This work was supported by the excellence initiative by the German
federal and state governments: DFG-EXC81, cluster of excellence
CellNetworks.
References
1. Scott DE, Bayly AR, Abell C et al (2016) Small
molecules, big targets: drug discovery faces the
protein-protein interaction challenge. Nat Rev
Drug Discov 15(8):533–550. https://doi.
org/10.1038/nrd.2016.29
2. Lam SS, Martell JD, Kamer KJ et al (2015)
Directed evolution of APEX2 for electron
microscopy and proximity labeling. Nat Methods 12(1):51–54. https://doi.org/10.1038/
nmeth.3179
3. Roux KJ, Kim DI, Raida M et al (2012) A
promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in
mammalian cells. J Cell Biol 196(6):801–810.
https://doi.org/10.1083/jcb.201112098
4. Kim DI, Birendra KC, Zhu W et al (2014)
Probing nuclear pore complex architecture
with proximity-dependent biotinylation. Proc
Natl Acad Sci U S A 111(24):E2453–E2461.
https://doi.org/10.1073/pnas.1406459111
5. Lambert JP, Tucholska M, Go C et al (2015)
Proximity biotinylation and affinity purification
are complementary approaches for the interactome mapping of chromatin-associated protein
complexes. J Proteome 118:81–94. https://
doi.org/10.1016/j.jprot.2014.09.011
Context-Specific Proximity-Dependent Proteomics with Split-BioID
317