New methods for qualitative and quantitative proteome analysis
Abstract
To achieve the deep-coverage proteome profiling, we proposed an ionic liquid based FASP protocol (i-FASP), by which with 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl), both soluable and insoluable proteins could be efficiently extracted from cells, and denatured simultaneously at 95°C with DTT added, followed by on-filter alkylation, digestion and desalting. Different from the typical FASP protocol, most C12Im-Cl could be easily removed by ammonium bicarbonate. buffer, the identified protein and membrane protein number in HeLa cells was obviously improved within reduced time. Besides, to improve the throughput for proteome sample preparation, we developed a hollow fiber membrane-aided fully automated sample treatment (FAST) method, by which samples could be denatured, reduced, desalted and digested in 6 min via “one-stop” service. Through the on-line combination of FAST with nano-LC-ESI-MS/MS, we further established a fully integrated platform for high-throughput proteome quantification, and demonstrated its capacity to analyze sub-nanogram starting materials. Furthermore, mass defect-based pseudo-isobaric dimethyl labeling (pIDL) method based on the subtle mass defect between 12C/13C and 1H/2H was introduced. Lys-C protein digests were labeled with CD2O/13CD2O and reduced with NaCNBD3/NaCNBH3 as heavy and light isotopologues, respectively. The fragment ion pairs with mass differences of 5.84 mDa were resolved by high-resolution MS/MS and used for quantification. The pIDL method described resulted in approximately 100-fold dynamic range and highly accurate results with relative error less than 2%. Morever, this method can be extended to 6-plex proteome quantification. All the above-metioned new methods were succesfully applied to perform the comparative proteome quantification of hepatocellular carcinoma cells with high and low metastatic potential.
Functional Proteomics for Learning and Memory Disorders
Abstract
The AMPA receptor (AMPA-R) is a major excitatory neurotransmitter receptor in the brain. The regulation of activity and trafficking of AMPA-R is a potent way of modulating synaptic plasticity, which underlies many neurobiological functions including learning and memory. Through proteomic analyses, we discovered several novel components of AMPA-R protein complex. Our function studies of the novel AMPA-R associated proteins revealed new molecular mechanisms underlying AMPA-R trafficking, functional and structural synaptic plasticity, and thus learning and memory.
Cyclin Y inhibits AMPA receptor exocytosis during long-term potentiation
Abstract
Cyclin Y (CCNY) is a member of the cyclin protein family, known to regulate cell division in proliferating cells. Interestingly, CCNY is expressed in neurons that do not undergo cell division. Here, we report that CCNY negatively regulates long-term potentiation (LTP) of synaptic strength through inhibition of AMPA receptor trafficking. CCNY is enriched in postsynaptic fractions from rat forebrain and is localized adjacent to postsynaptic sites in dendritic spines in rat hippocampal neurons. Using live-cell imaging of a pH-sensitive AMPA receptor, we found that during LTP-inducing stimulation, CCNY inhibits AMPA receptor exocytosis in dendritic spines. Furthermore, CCNY abolishes LTP in hippocampal slices. Taken together, our findings demonstrate that CCNY inhibits plasticity-induced AMPA receptor delivery to synapses and thereby blocks LTP, identifying a novel function for CCNY in post-mitotic cells.
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