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DTT protein equalization and Tryptophan protein quantification as a powerful tool in analytical proteomics
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Resumo(s)
Assessing total protein levels in biological samples is a common procedure in biochemistry and molecular biology. In this study, we compare tryptophan fluorescence (WF) with Bradford and BCA assays to determine total protein in serum samples. Our results indicate that tryptophan fluorescence spectrometry is an efficient, sensitive, and straightforward technique for quantifying proteins in serum. We observed minimal variation between the three methods: BCA de one with the lowers LOD and LOQ. The tryptophan method offers the possibility of reusing the intact sample that does not need colourimetric reagents for quantification. Consequently, free tryptophan serves as a reliable universal standard. This assay can be performed using a conventional fluorescence spectrometer with cuvettes or in a 96-well plate format with a plate reader. The method was successfully used as proof of concept, using serum from patients diagnosed with myeloma and serum from healthy donors.
Descrição
PM003/ 2016). This work received support from Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia and Minist\u00E9rio da Ci\u00EAncia, Tecnologia e Ensino Superior (FCT/MCTES) through the projects LA/P/0008/2020 DOI 10.54499/LA/P/0008/2020, UIDP/50006/2020 DOI 10.54499/UIDP/50006/2020 and UIDB/50006/2020 DOI 10.54499/UIDB/50006/2020. H.M.S. acknowledges the Associate Laboratory for Green Chemistry-LAQV (LA/P/0008/2020) DOI 10.54499/LA/P/0008/2020 funded by FCT/ MCTES for his research contract. L.B.C. is supported by the FCT/ MCTES PhD grant 2019 (SFRH/BD/144222/2019).
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Palavras-chave
DTT equalization multiple myeloma proteomics serum tryptophan Biochemistry Molecular Biology Genetics