Dear all,
I am currently working with cell lines that need to grow on DMEM medium with usually 10 %FCS. A lot of the proteins inside the FCS are BSA or similar. After the induction of the cells with cytokines and a reduced 1%FCS medium, I need to screen the supernatant with MALDI-TOF to see some proteins.
I am usually working in a 3-15 kDa range. BSA is usually 66 kDa and I need to work with α-CHCA matrix. Before testing with Sinapinic acid, I wonder if someone knows if BSA can interact with this quantity of FCS in the Medium.
I have read in some publications that maybe working with a Serum-free medium can greatly enhance detection with MALDI-TOF. Did someone already worked with similar conditions and know if I need to change for a FCS-free medium for the analysis with the MALDI.
I will definitely try to change the conditions in a couple of weeks, but before that if someone as a brilliant idea, I'll be grateful.
Yours sincerely,
Mathieu Rebeaud
Mathieu: You are facing a number of biological issues/challenges as well as technical ones. First of all, 3-15 kDa range proteins show up on reducing SDS-PAGE analysis, but may NOT exist by themselves, ie, monomers in your cells secretion. Second, you must do at least one or multiple steps of purification from the condition medium to enrich your protein of interest, as MALDI-TOF is sensitive yet easily overshadowed by contaminating proteins. Third, find a way to culture your cells in serum-free medium. Most cells can be cultured for less than 24 hrs with traditional medium supplemented with protein-free (but peptide-rich) additives. Please check our papers (PMID: 22949401 and PMID: 25900303) for details. Good luck.
@Mathieu
I agree with Ke-Wei's suggestions above, especially because you will be studying the secretome. The level of interference from media components in MALDI-TOF analysis may also vary according to cell health and treatment conditions. Below are some additional resources for optimizing your experimental conditions to reduce interference from FCS and other media components. Feel free to contact the corresponding authors for additional advice.
• From Technical U Munich in Germany (open-access publication)
"Enrichment and Detection of Molecules Secreted by Tumor Cells Using Magnetic Reversed-Phase Particles and LC-MALDI-TOF-MS"
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2392993/
• From CNRS/CEA/BBSI in Grenoble, France (open-access publication):
"Toward a better analysis of secreted proteins: the example of the myeloid cells secretome"
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2386146/
• From Medical U Vienna in Austria (not open-access, but your school's library may already have a subscription to Electrophoresis journal, or the authors may be willing to share a copy)
"A novel technique to specifically analyze the secretome of cells and tissues"
http://onlinelibrary.wiley.com/doi/10.1002/elps.200410387/full
• From Johns Hopkins U in Baltimore, USA (open-access review article with helpful references for various analytical techniques including mass spectrometry. NOTE: It is focused on cardiac cells, which may not be what you are studying.)
"Investigating the Secretome: Lessons About the Cells that Comprise the Heart"
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282018/
Hope this helps!
Lael gave a very useful list of papers for guidance. I just want add one more point to alert people in dealing with serum-free culture: wherever there is a DMEM, use alpha-MEM plus nucleosides and non-essential amino acids instead for better nutrition. Collect secretion medium from 1-5 10-cm dishes cultured for ~6 hr is more than enough for purification and eventual MS.
@Ke-Wei Zhao, @Lael L Cheung
Thank you both of you, this is more than I can ask for. I'll try these methods and contact the authors of these different paper.
Regards,
Mathieu
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