Ryerson Analytical Biochemistry Laboratory (RABL) Department of Chemistry and Biology, Faculty of Science Ryerson University, Toronto M5B 2K3, Canada.
J Am Soc Mass Spectrom. 2021 Jan 6;32(1):301-306. doi: 10.1021/jasms.0c00323. Epub 2020 Nov 16.
The alkaline phosphatase-streptavidin enzyme amplification conjugate (APSA) was diluted and quantified to the equivalent of one enzyme molecule injected on column by monitoring the production of excess adenosine from adenosine monophosphate (AMP) using sensitive and selective enzyme-linked mass spectrometric assay. The APSA enzyme conjugate has a mass of about 195 kDa and catalyzed the production of millions of enzyme products over the course of incubation that may be sensitively quantified by liquid chromatography, electrospray ionization, and mass spectrometry. APSA enzyme conjugate from fg/mL to ag/mL alongside 0 g/mL (control) was incubated with the substrate 1 mM AMP for 2 h in free solution before collecting a 1 μL of sample of the enzyme product adenosine for injection and analysis by LC-MS. The enzyme product adenosine showed a Gaussian distribution after log transformation. The safe limit of detection and quantification was approximately 250 zg of APSA enzyme conjugate injected on column. A linear signal with acceptable error was observed at the mass of the enzyme product adenosine from 10 to 10000 zg of APSA enzyme conjugate injected, compared to controls without enzyme. It was possible to make a linear and Gaussian measurement to the single molecule range of the universal APSA enzyme amplification conjugate per micro liter injected with approximately 10% error. This study describes the first linear and Gaussian quantification of enzyme product from the equivalent of one enzyme conjugate molecule injected onto LC-MS for analysis.
碱性磷酸酶-链霉亲和素酶放大偶联物（APSA）经稀释和定量，相当于在柱上注射一个酶分子，通过监测腺苷单磷酸（AMP）产生的多余腺苷，使用灵敏和选择性的酶联质谱检测法进行监测。APSA 酶偶联物的质量约为 195 kDa，并在孵育过程中催化产生数百万个酶产物，这些产物可以通过液相色谱、电喷雾电离和质谱进行灵敏定量。APSA 酶偶联物从 fg/mL 到 ag/mL 以及 0 g/mL（对照）与 1 mM AMP 底物在游离溶液中孵育 2 小时，然后收集 1 μL 酶产物腺苷进行 LC-MS 分析。酶产物腺苷经对数转换后呈高斯分布。在柱上注射约 250 zg APSA 酶偶联物时，检测限和定量限的安全范围约为 250 zg。与不含酶的对照相比，在注射 10 至 10000 zg APSA 酶偶联物的范围内，观察到酶产物腺苷的质量呈线性信号，误差可接受。可以对每微升注入的通用 APSA 酶放大偶联物进行线性和高斯测量，达到单个分子的范围，误差约为 10%。本研究描述了首次对 LC-MS 分析中相当于一个酶偶联物分子的酶产物进行线性和高斯定量。
