Free Cysteine in blood plasma

[Gene44]

Hello all,

I am currently working on the method for 20 essential amino acids. We purchased EasyFaast kit from Phenomenex, but it does not have cystein as one of the targets. Does anyone know if you can measure free cystein in blood plasma?

Any method references?

Thank you very much for any info!

[MSCHemist]

Are you using GC/MS or LC/MS?

For GC and GC/MS the kit uses a SCX resin packed tip for cleanup, propyl chloroformate for derivitization, and a proprietary column probably a 1701 variant for analysis. On the GC it can do all the amino acids except arginene. Arginine is impervious to derivitization because of its gaunidino group. I've heard the sample can be treated with arginase to convert arginine to ornithine and quantitiate arginine as ornithine.

http://www.ncbi.nlm.nih.gov/pubmed/14570314
http://www.ncbi.nlm.nih.gov/pubmed/22349320
http://epub.uni-regensburg.de/12318/1/H ... 090719.pdf

[Gene44]

Thanks for the links, we are actually using LC-MS/MS for analysis. It looks like 90% of cystein in plasma is in the form of cystine, that is why no one is measuring free cystein unless a reducing agent is used, like DTT, to treat plasma first.

[Andy Alpert]

Robert Croes presented a poster at ASMS 1999 in which he measured the content of various amino acids in individual corn and soybean seeds. He ground each seed in 10-15 mm ammonium formate, pH ~ 3, containing 60% ACN. The supernatant was injected directly onto a PolyHYDROXYETHYL A column being eluted isocratically with the same solvent. This effected a separation in the HILIC mode. Most amino acids eluted in about 4-5 minutes and were analyzed via MS/MS. At ASMS 2000 he presented an improved version of this process. You could implement this by adding 3 vol. ACN to 1 vol. plasma, cooling, and centrifuging the mixture. The supernatant would contain only peptides smaller than about 5 KDa and other small molecules and could probably be injected directly for the same kind of analysis. There's no reason this wouldn't work for cystine. Contact me offlist (aalpert@polylc.com) and I'll send you the posters.

Incidentally, I believe that the most abundant sulfhydryl-containing small molecule in plasma is the tripeptide glutathione (GSH). That means that your cysteine is more likely to be circulating in plasma in the form of a GSH-Cys mixed disulfide than it is as cystine. Be prepared to analyze for both of those compounds.

[Gene44]

Thanks Andy, we'll check it out.

[carls]

Cysteine, cystine and arginine can all be analyzed using EZFaast with LC-MS.

https://phenomenex.blob.core.windows.ne ... c86cb0.pdf

[Andy Alpert]

That's nice, but most of the cysteine in plasma is going to be in a mixed disulfide with glutathione, and I don't see glutathione listed in the literature for EZFaast.

[carls]

The post states "free" cysteine, right?

Besides, glutathione should be derivatized as well so it should be detectable.