Has anyone done LCMS on condition media for protein quantification?
I have this media (after the exposure of different flow conditions) that i would like to use LCMS for protein quantification because I want to test for any potential proteins that were exocytotically released during the conditioning. Any potential papers or suggestions would be greatly appreciated.
If you don't use serum-free media as Alexey indicates, serum proteins and serum-derived exosomes overwhelm the amount of protein coming from your cells. Rather than using acetone precipitation, we concentrated protein by filter centrifugation using low MWCO Amicon concentrators. Using either method, you should calculate protein concentration in the media prior to concentrating, and in the case of filter centrifugation, after concentrating as well. If you're comparing across conditions or samples, you should compare based on the protein from equivalent initial volume of media, not equivalent amount of protein when running MS analysis.
Keep in mind that the amount of protein you obtain will be extremely low. In our studies, 48 hrs of cell culture in serum-free conditions yielded low ng per mL concentrations of protein (15-40 ng/mL, depending on experimental condition), a range that clearly requires concentration prior to MS analysis.
I would like to express my opinion. MS is not a quantitative detector at all, but an HPLC-photometric detector uniquely gives a true result.
Protein separation by reversed-phase ODS-column should be performed by our high-recovery acidic-phosphate buffer (pH 2.0)/2-propanol/ethyleneglycol system (please see file; Lysozyme by RP-HPLC).
Separated proteins by ODS-column and/or SEC-column (please see file; SEC column 300A silica) are fractioned and quantitatively identified by our new proteomics of PDMD method, which is based upon the famous Edman degradation (please see file; HepG2 fucoidan).
Therefore, you can avoid the use of non-reliable and high cost LC-MS method for the proteomics.
We have done quantitative proteomic analysis of proteins in cell growth media before. You should use serum-free medium, and up-scale your cellllab experiment to collect enough material. We have used aceton precipitation to extract proteins, followed by standard shotgun proteomics workflow.
If you don't use serum-free media as Alexey indicates, serum proteins and serum-derived exosomes overwhelm the amount of protein coming from your cells. Rather than using acetone precipitation, we concentrated protein by filter centrifugation using low MWCO Amicon concentrators. Using either method, you should calculate protein concentration in the media prior to concentrating, and in the case of filter centrifugation, after concentrating as well. If you're comparing across conditions or samples, you should compare based on the protein from equivalent initial volume of media, not equivalent amount of protein when running MS analysis.
Keep in mind that the amount of protein you obtain will be extremely low. In our studies, 48 hrs of cell culture in serum-free conditions yielded low ng per mL concentrations of protein (15-40 ng/mL, depending on experimental condition), a range that clearly requires concentration prior to MS analysis.
Hello Sana,
in addition to Dr. Alexey and Dr. Kent answers above, have a look to this chapter
Article Analysis of Secreted Proteins
and this article
Article Investigating the Secretome Lessons About the Cells That Com...
Hope it helps.
Hi Alexey, Could you please provide me with some of your published work in this area? Thanks - Sana
Dear Sana,
We've done this analysis as a service at our core facility, not sure if they already published the results.
Sample prep included filtration (3kDa cutoff filter), acetone precipitation, and then standard proteomics workflow - redissolving in 50mM AmBic + ProteaseMax, protein concentration measurement (BCA assay), reduction, alkylation and digestion with trypsin.
Cheers
Kou Hayakawa is correct, MS is not a quantitative tool, but can be used as a ratiometric tool. If you are looking for the relative abundance differences between two different cultures, MS can work, but only if well-designed. If you are trying to quantify each of the exogenously-expressed proteins in a single culture don't even bother with MS, do straight to those methods cited by Hayakawa. Don't do anything until you fully understand that fundamental difference.
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