Arsene Cristian G, Ohlendorf Rüdiger, Burkitt William, Pritchard Caroline, Henrion André, O'Connor Gavin, Bunk David M, Güttler Bernd
Department Metrology in Chemistry, Physikalisch-Technische Bundesanstalt, Braunschweig, D-38116, Germany.
Anal Chem. 2008 Jun 1;80(11):4154-60. doi: 10.1021/ac7024738. Epub 2008 Apr 30.
The practice of quantifying proteins by peptide fragments from enzymatic proteolysis (digestion) was assessed regarding accuracy, reliability, and uncertainty of the results attainable. Purified recombinant growth hormone (rhGH, 22 kDa isoform) was used as a model analyte. Two tryptic peptides from hGH, T6 and T12, were chosen to determine the amount of the protein in the original sample. Reference solutions of T6 and T12 (isotopically labeled forms), value assigned by quantitative amino acid analysis (AAA) after complete hydrolysis, were used as internal standards. The accuracy of protein quantification by fragments T6 and T12 was evaluated by comparison of peptide results to those obtained for the same rhGH sample by AAA. The rate of cleavage (and thus the experimental protocol used) turned out to be crucial to the quality of results in protein quantification using enzymatic fragments. Applying a protocol customarily found in (qualitative) bottom-up proteomics gave results significantly higher than the target value from AAA (+11% with T6 and +6% with T12). In contrast, using a modified protocol optimized for fast and complete hydrolysis, results were unbiased within the limits of uncertainty, while the time needed for completion of proteolysis was considerably reduced (30 min as compared to 1080-1200 min). The method assessed highlighted three important criteria deemed necessary for successful protein quantification using proteolysis-based mass spectrometry methods. These are the following: the requirement for both the selected peptides and labeled internal standard to be stable throughout digestion; the correct purity assignment to the selected peptide standards; the proof of equimolar release of the selected peptides. The combined (overall) uncertainty for protein quantification was established by combination of estimates obtained for individual components and found to be U = 4% for this example. This uncertainty is of the same order as that typically attainable in quantification of "small" organic molecules using liquid chromatography/isotope dilution mass spectrometry.
对通过酶促蛋白水解（消化）产生的肽片段对蛋白质进行定量的实践，在可获得结果的准确性、可靠性和不确定性方面进行了评估。纯化的重组生长激素（rhGH，22 kDa 异构体）用作模型分析物。选择来自 hGH 的两种胰蛋白酶肽 T6 和 T12，以确定原始样品中蛋白质的含量。T6 和 T12 的参考溶液（同位素标记形式），在完全水解后通过定量氨基酸分析（AAA）赋值，用作内标。通过将肽结果与通过 AAA 对相同 rhGH 样品获得的结果进行比较，评估了通过片段 T6 和 T12 进行蛋白质定量的准确性。裂解速率（以及因此使用的实验方案）对于使用酶片段进行蛋白质定量的结果质量至关重要。应用（定性）自下而上蛋白质组学中常见的方案得到的结果明显高于 AAA 的目标值（T6 时高 +11%，T12 时高 +6%）。相比之下，使用针对快速完全水解优化的改良方案，结果在不确定度范围内无偏差，同时蛋白质水解完成所需的时间大大减少（与 1080 - 1200 分钟相比为 30 分钟）。所评估的方法突出了使用基于蛋白水解的质谱方法成功进行蛋白质定量所需的三个重要标准。如下所述：所选肽和标记内标在整个消化过程中都需要稳定；所选肽标准品的正确纯度赋值；所选肽等摩尔释放的证明。通过组合各个组分的估计值确定了蛋白质定量的综合（总体）不确定度，此示例中发现为 U = 4%。该不确定度与使用液相色谱/同位素稀释质谱法对“小”有机分子进行定量时通常可达到的不确定度处于同一量级。
