DTT protein equalization and Tryptophan protein quantification as a powerful tool in analytical proteomics.

Authors

  • Inês F. Domingos BIOSCOPE Research Group, LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal. PROTEOMASS Scientific Society, 2825-466 Costa da Caparica, Portugal.
  • Luís B. Carvalho BIOSCOPE Research Group, LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal. PROTEOMASS Scientific Society, 2825-466 Costa da Caparica, Portugal.
  • José L. Capelo BIOSCOPE Research Group, LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal. PROTEOMASS Scientific Society, 2825-466 Costa da Caparica, Portugal.
  • Carlos Lodeiro BIOSCOPE Research Group, LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal. PROTEOMASS Scientific Society, 2825-466 Costa da Caparica, Portugal.
  • Hugo M. Santos BIOSCOPE Research Group, LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal. PROTEOMASS Scientific Society, 2825-466 Costa da Caparica, Portugal.

DOI:

https://doi.org/10.5584/jiomics.v14i1.229

Keywords:

DTT equalization, tryptophan, proteomics, serum, multiple myeloma

Abstract

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.

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Published

2024-04-15