Hi, everyone. We are trying to extract a small peptide: cyclic Glycine-Proline (cGP) from brain tissues for HPLC-MS analysis. Since the HPLC machine is very sensitive to detergents or ions, we tried to use only milli-Q water for extraction, and then use the 3k filter to filter out the large molecules before adding to HPLC columns. However, the extraction strength of water is very low comparing to other buffers like RIPA buffer. When we used RIPA buffer for extraction, the total protein of the extract was around 30mg/ml, while we can only get 10mg/ml total protein concentration when extracting with water. As a result, the cGP level detected was super low with a lot of noise that interfere with the cGP spike, which is impossible or not credible to read.
Now it left two options for us: either increase the extraction strength to extract enough amount of cGP that can rule out the noise, or to purify the sample even after the 3k filtration. I would prefer the first option because we have no idea what molecules are inside the filtrate other than cGP molecules: the answer could be numerous. So I'm asking if we can use some other non-ionic, non-detergent extraction buffer that can be applied for HPLC-MS analysis?
Alternatively, do you have any other ideas of how to quantify the small peptide cyclic Glycine-Proline from brain tissues? As far as we know, there is currently no antibody in the market that can bind such molecule.
Thank you very much!
Please look at the following below links which may help you in your analysis:
http://www.bioanalysis.dicp.ac.cn/publication/2007/Development%20of%20Efficient%20Protein%20Extraction%20Methods%20for%20Shotgun.pdf
https://academic.oup.com/endo/article/157/8/3130/2422389
https://experiments.springernature.com/articles/10.1007/978-1-4939-7119-0_9
https://www.nature.com/articles/ncomms11436
Thanks
You are definitely seeing a matrix effect here. Are you performing any homogenization of the tissue sample to increase extraction efficiency? Beside I will do liquid-liquid extraction to separate small molecule lipids and other interfering element present in the tissue matrices. MWCO of 3k will not remove lipids and other small molecule metabolites. Water extraction should provide a good yield of your peptides. Finally, if possible I will optimize MS parameters and will use a targeted analysis to reduce noise/background interference.
Using water to extract peptides does not sound optimal to me. Try to use a low-ionic strength buffer with volatile compounds like ammonium acetate. This is suitable for buffering around pH 5 for basic peptides or around pH 9 for acid peptides.
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