Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples
Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples
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直接输注-SIM 作为复杂样品中绝对蛋白质定量的快速而稳健的方法
DOI：
10.1016/j.euprot.2015.03.001
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发表时间：
2015
影响因子：
--
通讯作者：
Katrin Marcus
中科院分区：
文献类型：
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作者：
Christina Looße;Sara Galozzi;Linde Debor;Mattijs K. Julsing;Bruno Bühler;Andreas Schmid;Katalin Barkovits;Thorsten Müller;Katrin Marcus
生物和临床样品中蛋白质的相对和绝对定量是蛋白质组学中常用的方法。到目前为止，靶向蛋白质定量主要使用基于HPLC的肽分离和三重四极杆质谱仪上的选定反应监测的组合进行。在这里，我们第一次展示了在Q Exactive质谱仪上使用直接注入策略结合单离子监测（SIM）进行绝对定量的潜力。利用大肠杆菌复合膜组分，对重组表达的异源人细胞色素P450单加氧酶3A 4（CYP 3A 4）进行了绝对定量，并与传统的HPLC-SIM进行了比较。直接输注SIM仅显示14.7%（±4.1（s.e.m.））与HPLC-SIM相比，平均偏差降低，并且与LC-MS方法的65 min相比，单个样品的处理和分析时间减少4.5 min（可进一步减少至30 s）。总之，我们使用直接输注-SIM的简化工作流程提供了一种用于定量复杂蛋白质混合物中蛋白质的快速且稳健的方法。
Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM) on a Q Exactive mass spectrometer. By using complex membrane fractions ofEscherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4) comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m.)) deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s) for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.