Wiśniewski Jacek R, Gaugaz Fabienne Z
†Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Anal Chem. 2015 Apr 21;87(8):4110-6. doi: 10.1021/ac504689z. Epub 2015 Apr 9.
The determination of total protein content is one of the most frequent analytical tasks in biochemistry and molecular biology. Here we evaluate measurements of tryptophan fluorescence (WF) for total protein determination in whole tissue lysates and for peptide quantification in protein digests. We demonstrate that the fluorescence spectrometry of tryptophan offers a simple, sensitive, and direct method for protein and peptide assays. The WF assay is fully compatible with SDS and other solutes that are commonly used for lysis of tissue and cells. We found that the content of tryptophan varies only a little between mouse tissues (1.16 ± 0.08% of total amino acids) and is similar in human cells (1.19 ± 0.06%). Therefore, free tryptophan can be used as a universal standard. We show that the assay can be carried out on a standard fluorescence spectrometer with cuvettes as well as in a 96-well format using a plate reader. The method is particularly suitable for determination of peptide content in diluted samples. Notably, the whole sample can be recovered after the measurement.
总蛋白含量的测定是生物化学和分子生物学中最常见的分析任务之一。在此，我们评估了利用色氨酸荧光（WF）对全组织裂解物中的总蛋白进行测定以及对蛋白质消化产物中的肽进行定量的方法。我们证明，色氨酸荧光光谱法为蛋白质和肽的测定提供了一种简单、灵敏且直接的方法。WF 测定法与 SDS 以及其他常用于组织和细胞裂解的溶质完全兼容。我们发现，小鼠组织中色氨酸的含量仅略有差异（占总氨基酸的 1.16±0.08%），在人类细胞中也相似（1.19±0.06%）。因此，游离色氨酸可作为通用标准。我们表明，该测定法既可以在配备比色皿的标准荧光光谱仪上进行，也可以使用酶标仪以 96 孔板的形式进行。该方法特别适用于测定稀释样品中的肽含量。值得注意的是，测量后整个样品可以回收。
