Bunk David M, Lowenthal Mark S
Biomolecular Measurement Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA.
Methods Mol Biol. 2019;2030:143-151. doi: 10.1007/978-1-4939-9639-1_12.
The role of amino acid analysis in bioanalysis has changed from a qualitative to a quantitative technique. With the discovery of both electrospray ionization and matrix-assisted laser desorption ionization in the early 1990s, the use of amino acid analysis for qualitative analysis of proteins and peptides has been replaced by mass spectrometry. Accurate measurement of the relative molecular masses of proteins and peptides, peptide mapping, and sequencing by tandem mass spectrometry provide significantly better qualitative information than can be achieved from amino acid analysis. At NIST, amino acid analysis is used to assign concentration values to protein and peptide standard reference materials (SRMs) which, subsequently, will be used in the calibration of a wide variety of protein and peptide assays, such as those used in clinical diagnostics. It is critical that the amino acid analysis method used at NIST for assigning concentration values in SRM deliver the highest accuracy and precision possible. Therefore, we have developed an amino acid analysis method that uses isotope dilution LC-MS/MS-the analytical technique routinely used at NIST to certify analyte concentrations in SRMs for a wide variety of analytes. We present here our most recent method for the quantification of amino acids using isotope dilution LC-MS/MS.
氨基酸分析在生物分析中的作用已从一种定性技术转变为定量技术。随着20世纪90年代初电喷雾电离和基质辅助激光解吸电离的发现，氨基酸分析用于蛋白质和肽定性分析的用途已被质谱法所取代。通过串联质谱法精确测量蛋白质和肽的相对分子质量、肽图谱分析和测序，能提供比氨基酸分析显著更好的定性信息。在美国国家标准与技术研究院（NIST），氨基酸分析用于为蛋白质和肽标准参考物质（SRM）确定浓度值，这些标准参考物质随后将用于校准各种蛋白质和肽分析方法，例如临床诊断中使用的那些方法。至关重要的是，NIST用于在SRM中确定浓度值的氨基酸分析方法要尽可能提供最高的准确度和精密度。因此，我们开发了一种使用同位素稀释液相色谱 - 串联质谱法的氨基酸分析方法——这是NIST常规用于为各种分析物在SRM中认证分析物浓度的分析技术。我们在此展示我们使用同位素稀释液相色谱 - 串联质谱法定量氨基酸的最新方法。
