The measurement of KRAS G12 mutants using multiplexed selected reaction monitoring and ion mobility mass spectrometry.
The measurement of KRAS G12 mutants using multiplexed selected reaction monitoring and ion mobility mass spectrometry.
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DOI：
10.1002/rcm.8657
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发表时间：
2020-09
期刊：
影响因子：
--
通讯作者：
Jones DJL
中科院分区：
文献类型：
--
作者：
Norman RL;Singh R;Langridge JI;Ng LL;Jones DJL
有相当大的临床需求来确定与癌症相关的基因中的关键突变，这需要部署高度特异性和稳健的分析方法。多重液相色谱与选择性反应监测（LC/SRM）分析提供了实现定量低至临床样品中预期水平的能力。离子迁移率质谱（IMS/MS）分析可以提供更高的峰容量，从而在极短的时间范围内实现分离，此外还可以提供有关分析物碰撞截面的物理化学数据，这些数据可以与离子的m/z值结合使用，以提高检测特异性。对于LC/SRM，将未标记的肽和相应的稳定同位素标记的标准品加标至消化的人血浆中，并使用与三重四极杆质谱仪偶联的高效液相色谱法（UHPLC）进行分析，以生成分析物特异性校准线。将合成的未标记的肽注入Synapt G2质谱仪中进行行波离子迁移率分离，并通过与先前生成的TWCCSN 2校准值进行比较得出TWCCSN 2值。建立了每种KRAS肽的线性校准线（0.125 - 25 fmol/μL）。UHPLC分离了肽，因此能够将其分为不同的保留时间函数/窗口。这种分离能够检测每种轻肽和重肽的三个或四个转换，每个峰至少10个点，以进行准确定量。使用IMS/MS分离所有六种KRAS G12肽，从而能够确定精确的TWCCSN 2值。尽管一些G12肽在色谱上共洗脱，但所有肽均通过m/z、保留时间和/或漂移时间进行区分。本研究主张LC/SRM和IMS/MS都可以用于鉴定KRAS中的单个氨基酸取代，作为循环肿瘤DNA分析等常用方法的替代方法。
There is a considerable clinical demand to determine key mutations in genes involved with cancer which necessitates the deployment of highly specific and robust analytical methods. Multiplex liquid chromatography with selected reaction monitoring (LC/SRM) assays offer the ability to achieve quantitation down to levels expected to be present in clinical samples. Ion mobility mass spectrometry (IMS/MS) assays can provide increased peak capacity and hence separation in an extremely short time frame, and in addition provide physicochemical data regarding the collision cross‐section of an analyte which can be used in conjunction with the m/z value of an ion to increase detection specificity. For LC/SRM, unlabelled peptides and corresponding stable‐isotope‐labelled standards were spiked into digested human plasma and analysed using ultrahigh‐performance liquid chromatography (UHPLC) coupled to a triple quadrupole mass spectrometer to enable the generation of analyte‐specific calibration lines. Synthetic unlabelled peptides were infused into a Synapt G2 mass spectrometer for travelling wave ion mobility separation and TWCCSN2 values were derived from comparison with previously generated TWCCSN2 calibration values. Linear calibration lines (0.125 to 25 fmol/μL) were established for each of the KRAS peptides. UHPLC separated the peptides and hence enabled them to be split into different retention time functions/windows. This separation enabled detection of three or four transitions for each light and heavy peptide with at least 10 points per peak for accurate quantitation. All six KRAS G12 peptides were separated using IMS/MS, enabling precise TWCCSN2 values to be determined. Although some of the G12 peptides chromatographically co‐eluted, all the peptides were distinguished by m/z, retention time and/or drift time. This study advocates that LC/SRM and IMS/MS could both be used to identify single amino acid substitutions in KRAS as an alternative to commonly used methods such as circulating tumour DNA analysis.
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影响因子：
8.8
作者：
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通讯作者：
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