Single-cell chemical proteomics with an activity-based probe: identification of low-copy membrane proteins on primary neurons.
We propose a novel single-cell chemical proteomics (SCCP) strategy to profile low-abundance membrane proteins in single cells. In this approach, the membrane protein GB1 and its splicing variants were targeted on cultured cell lines and primary neurons using a specifically designed activity-based probe. The functionally labeled single cells were encapsulated in individual buffer droplets on a PDMS microwell array, and were further picked up one at a time and loaded into a capillary electrophoresis system for cell lysis, separation, and laser-induced fluorescence detection of the targeted proteins. The results revealed the expression of GB1 splicing variants in HEK and MEF cells, which was previously only suggested at the transcriptional level. We further applied this method to investigate single primary cells and observed significant heterogeneity among individual mouse cerebellar granule neurons. Interference experiments with GB1 antagonist and agonist validated this observation.
Wuhan National Laboratory for Optoelectronics-, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074 (China).
Fei
F
Xu
Han
H
Zhao
Xiaojun
X
Feng
Linhai
L
Chen
Dongjuan
D
Chen
Yang
Y
Zhang
Fajun
F
Nan
Jianfeng
J
Liu
Bi-Feng
BF
Liu
D000818:Animals; D001896:Boron Compounds / Q000737:chemistry; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D019075:Electrophoresis, Capillary; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D015335:Molecular Probes / Q000737:chemistry; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D040901:Proteomics; D018080:Receptors, GABA-B / Q000737:chemistry / Q000378:metabolism*; D014466:Ultraviolet Rays
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D015335:Molecular Probes; D018080:Receptors, GABA-B
activity-based probes; membrane proteins; primary neurons; proteomics; receptors