Photocrosslinking and click chemistry enable the specific detection of proteins interacting with phospholipids at the membrane interface.
New lipid analogs mimicking the abundant membrane phospholipid phosphatidylcholine were developed to photocrosslink proteins interacting with phospholipid headgroups at the membrane interface. In addition to either a phenylazide or benzophenone photoactivatable moiety attached to the headgroup, the lipid analogs contained azides attached as baits to the acyl chains. After photocrosslinking in situ in the biomembrane, these baits were used for the attachment of a fluorescent tetramethylrhodamine-alkyne conjugate or a biotin-alkyne conjugate using click chemistry, allowing for the selective detection and purification of crosslink products, respectively. Proteins crosslinked to the lipid analogs in inner mitochondrial membranes from Saccharomyces cerevisiae were detected and subsequently identified by mass spectrometry. Established interaction partners of phosphatidylcholine were found, as well as known integral and peripheral inner membrane proteins, and proteins that were not previously considered mitochondrial inner membrane proteins.
Gubbens
Jacob
J
Chemical Biology and Organic Chemistry, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.
Ruijter
Eelco
E
de Fays
Laurence E V
LE
Damen
J Mirjam A
JM
de Kruijff
Ben
B
Slijper
Monique
M
Rijkers
Dirk T S
DT
Liskamp
Rob M J
RM
de Kroon
Anton I P M
AI
D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000528:radiation effects; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D051336:Mitochondrial Membranes / Q000737:chemistry; D010713:Phosphatidylcholines / Q000138:chemical synthesis / Q000737:chemistry; D010743:Phospholipids / Q000138:chemical synthesis / Q000737:chemistry* / Q000528:radiation effects; D040901:Proteomics; D012235:Rhodamines / Q000138:chemical synthesis / Q000737:chemistry
D001386:Azides; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D010743:Phospholipids; D012235:Rhodamines; C014747:phenylazide; C005358:tetramethylrhodamine