Protein Neighbors and Proximity Proteomics.
Within cells, proteins can co-assemble into functionally integrated and spatially restricted multicomponent complexes. Often, the affinities between individual proteins are relatively weak, and proteins within such clusters may interact only indirectly with many of their other protein neighbors. This makes proteomic characterization difficult using methods such as immunoprecipitation or cross-linking. Recently, several groups have described the use of enzyme-catalyzed proximity labeling reagents that covalently tag the neighbors of a targeted protein with a small molecule such as fluorescein or biotin. The modified proteins can then be isolated by standard pulldown methods and identified by mass spectrometry. Here we will describe the techniques as well as their similarities and differences. We discuss their applications both to study protein assemblies and to provide a new way for characterizing organelle proteomes. We stress the importance of proteomic quantitation and independent target validation in such experiments. Furthermore, we suggest that there are biophysical and cell-biological principles that dictate the appropriateness of enzyme-catalyzed proximity labeling methods to address particular biological questions of interest.
From the ‡Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom CB2 1QW, the §Cambridge Centre for Proteomics, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom CB2 1QR, and jsr31@cam.ac.uk.
Johanna S
http://orcid.org/0000-0003-2066-8617
JS
Rees
the ‖National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Xue-Wen
XW
Li
the ‖National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China sarah.perrett@cantab.net.
Sarah
http://orcid.org/0000-0003-0137-0997
S
Perrett
the §Cambridge Centre for Proteomics, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom CB2 1QR, and k.s.lilley@bioc.cam.ac.uk.
Kathryn S
http://orcid.org/0000-0003-0594-6543
KS
Lilley
From the ‡Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom CB2 1QW, apj10@cam.ac.uk.
Antony P
http://orcid.org/0000-0002-2895-7387
AP
Jackson
D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D014439:Tyramine / Q000031:analogs & derivatives
D029968:Escherichia coli Proteins; D020543:Proteome; D012097:Repressor Proteins; D001710:Biotin; D006735:Horseradish Peroxidase; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D019793:Fluorescein; D014439:Tyramine