fluorescent derivatisation of peptides

[andrewna]

Does anyone know if there are any agents that can be used to derivatise peptides and hence make them detectable by fluorescence? If such agents exist, what sort of column could I use to do the separation?

thanks
nick

[danko]

Is there any reason for not utilizing an ordinary absorbance detector?

Best Regards

[andrewna]

I think sensitivity might be the issue with a standard UV detector since I will be in the pg/ml range for my samples. For amino acids i have found pre-column derivatisation and fluorescence detection to be a sensitive way to measure low levels of analyte and I thought a similar approach (if possible) might enhance sensitivity here as well. Maybe there is a way to enhance sensitivity up front prior to UV or UP detection?

nick

[zokitano]

I have consulted one of my colleagues who works with HPLC/FLD.
She said that it is possible some proteins to have native fluorescence, but for peptides most convenient regents for derivatization are:

FMOC - fluorenylmethyl chloroformate (excit. wavelength = 266 nm)
OPA - o-phthalaldehyde
Rhodamine Green (excit. wavelength = 488 nm)
FITC - fluorescein isothiocyanate (excit. wavelength = 488 nm)

Hope this helps and good luck

Regards

[mbicking]

Pre-column fluorescence derivatization is a standard tool for analyzing amino acids. There are many reagents available, as noted above. You can also search www.sigmaaldrich.com for more information, or look in one of the books on derivatization.

Depending on the equipment you have, there is an excellent automated, pre-column derivatization method available from Agilent. The link is:

http://www.chem.agilent.com/temp/radDD7E1/00000385.PDF

Sometimes people have trouble accessing this link directly, so you may have to search the agilent web site. (search: amino acid derivatization. It will be one of the first few entries.)

[rick1112]

hi zokitano,

we were also thinking of doing same although we are trying to lable specific amino acids ina protein......could you tell info about derivatization reagents that could lable cystine,methionine,asparagine and tryptophane???
any info regarding this will be very helpful....
thanks a lot

[zokitano]

we were also thinking of doing same although we are trying to lable specific amino acids ina protein......could you tell info about derivatization reagents that could lable cystine,methionine,asparagine and tryptophane???

Dear Rick1112,

Personally I have no experience in fluorescence derivatization of amino acids, so maybe if there is an expert on this forum regarding this question I'll kindly let him/her to answer your question. My colleague who works on HPLC/FLD is on a conference at this moment so I couldn't get the answer you need.
But I tried to search the journals regarding your question and I find some interesting publications, which I think are worth to be read:

Derivatization and Fluorescence Detection of Amino Acids and Peptides with 9-Fluorenylmethyl Chloroformate on the Surface of a Solid Adsorbent, Dihua Shangguan, Yingxin Zhao, Huiwan Han, Rui Zhao, and Guoquan Liu* , Anal. Chem., 73 (9), 2054 -2057

HPLC-fluorescence determination of amino acids in pharmaceuticals after pre-column derivatization with phanquinone, R. Gatti a, M.G. Gioia a, P. Andreatta b, G. Pentassuglia, Journal of Pharmaceutical and Biomedical Analysis 35 (2004) 339–348

Phanquinone: a useful fluorescent pre-chromatographic derivatization reagent for liquid chromatographic analyses of amino acid dosage forms R. Gatti, M.G. Gioia, A.M. Di Pietra, Analytica Chimica Acta 474 (2002) 11–20

New reagents for determination of amino acids by liquid chromatography with pre-column fluorescence derivatization, Xinjun Fana, Jinmao Youa, Jingwu Kanga, Qingyu Oua, Qingcun Zhub, Analytica Chimica Acta 367 (1998) 81-91

High-performance liquid chromatography with fluorescence detection for quantitation of tryptophan and tyrosine in a shrimp waste protein concentrate, D.I. S´anchez-Machado, B. Chavira-Willys, J. L´opez-Cervantes, Journal of Chromatography B, 863 (2008) 88–93

Simple and rapid quantitative high-performance liquid chromatographic
analysis of plasma amino acids, Michael P. Frank a, Robert W. Powers, Journal of Chromatography B, 852 (2007) 646–649 {derivatization with OPA reagent}

Tryptophan determination in milk-based ingredients and dried sport supplements by liquid chromatography with fluorescence detection, Cristina Delgado-Andrade a, Jose A. Rufia -Henares,
Salvio Jimenez-Perez, Francisco J. Morales, Food Chemistry 98 (2006) 580–585

As I have briefly inspected those papers I saw different derivatizing reagents for amino acids like: OPA, FMOC, etc.

Good luck