Cell surface protein labeling
As 66% of drugs in the DrugBank target the surfaceome, these proteins are a key source for potential diagnostic and therapeutic agents. ^[Current Opinion in Chemical Biology, 2019, 48, 26]^
Biotinylation
NHS-biotin
2004. Yingming Zhao*. AC.
Proteomic Analysis of Integral Plasma Membrane Proteins.
10.1021/ac0354037
Sulfo-NHS-SS-Biotin (0.1 mg/mL in PBS), RT, 15 min
Half of 10 cm H1299 cells:
Reported:
898 unique proteins,
781 were annotated with regard to their plasma membrane localization.
Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins.
In-gel digestion
49 gel bands.
Use Gao’s database:
Uniprot reviewed: 484
Membrane proteins: 207
Plasma membrane proteins: 111
2005. Toshiaki Isobe*. MCP. 10.1074/mcp.M500216-MCP200
sulfo-NHS-LC-biotin (1 mg/ml EZ-Link) in PBS+ for 20 min at 4 °C
2008. Yinsheng Wang*. JPR. 10.1021/pr700651b
sulfo-NHS-LC-biotin (0.5 mg/mL, Pierce) in PBS, 37 °C, for 10 min
2019. Mingliang Ye*. J Proteomics
Sensitive Profiling of Cell Surface Proteome by Using an Optimized Biotinylation Method.
Aminooxy-biotin
2009. James C. Paulson*. Nat Methods
High-Efficiency Labeling of Sialylated Glycoproteins on Living Cells.
10.1038/nmeth.1305
Hydrazide-biotin
2009. Bernd Wollscheid*. Nat Biotechnol
Mass-Spectrometric Identification and Relative Quantification of N-Linked Cell Surface Glycoproteins.
10.1038/nbt.1532
Alkyne-biotin
2017. Bertozzi*. Angew
Bioorthogonal Labeling of Human Prostate Cancer Tissue Slice Cultures for Glycoproteomics.
10.1002/anie.201701424
2019. Ronghu Wu. AC, 91, 6934−6942
Peptide level enrichment.
Deglycosylation in heavy-oxygen water
Three fractions, 80-minute gradients, Orbitrap Elite
HeLa:
321 membrane proteins (use Gao’s list)
306 plasma membrane proteins
Amine-biotin
2018. Nurhan Özlü*. JPR.
10.1021/acs.jproteome.7b00825
Labeling Carboxyl Groups of Surface-Exposed Proteins Provides an Orthogonal Approach for Cell Surface Isolation.
低引
EDC in PBS, pH 5.7
Shaving / cell surface trypsinization
2010. María Luisa Hernáez*. J Proteomics.
10.1016/j.jprot.2010.02.008
Identification of Candida Albicans Exposed Surface Proteins in Vivo by a Rapid Proteomic Approach.
cells were resuspended in 0.8 mL of 25 mM ABC (pH 8.0) and incubated at 37 °C with different treatments. A total amount of 10 µg of trypsin (Rec. Sequencing grade, Roche) was added to 108 cells.
Since unfolded proteins are more accessible to tryptic digestion, the efficiency of surface digestion was assayed in the absence and presence of 5 mM DTT. After incubation at different conditions, samples were centrifuged at 3500×g for 5 min and the supernatants were collected. The peptide supernatants were filtered through 0.22-μm pore-size filters (Millipore). Proteolytic reactions were stopped by adding 0.1% TFA (v/v).
Techniques Q&A
biotin-PEG4-azide可以穿过细胞膜 [Chengzhi Cai, Chem Sci. 2017 10.1039/c6sc02297a]
Enzymatic →
