Halogenated Peptides as Internal Standards (H-PINS) INTRODUCTION OF AN MS-BASED INTERNAL STANDARD SET FOR LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
Halogenated Peptides as Internal Standards (H-PINS) INTRODUCTION OF AN MS-BASED INTERNAL STANDARD SET FOR LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
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DOI：
10.1074/mcp.m800569-mcp200
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发表时间：
2009-08-01
影响因子：
7
通讯作者：
Aebersold, Ruedi
中科院分区：
文献类型：
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作者：
Mirzaei, Hamid;Brusniak, Mi-Youn;Aebersold, Ruedi
近年来，随着定量蛋白质组学在生命科学中的应用不断增长，对质量控制和校准的更强大和更普遍适用的方法的需求也越来越大。定量蛋白质组学的可靠性与分析平台的重现性和稳定性密切相关，分析平台通常是多组分的(例如，样品制备、多步骤分离和质谱分析)，单个组分对整个系统的重复性的贡献并不相等。因此，定量准确度的变化是不可避免的，质量控制和校准成为评估分析本身质量的关键。为此，使用内部标准不仅有助于检测和消除由不可复制的系统(质量控制)获取的离群值数据，而且还可以用于检测仪器的变化，以便随后进行性能和校准。在这里，我们介绍了一套卤化肽作为内标。这些多肽是定制设计的，具有适合各种质量控制评估、数据校准和归一化过程的特性。卤化肽独特的同位素分布使其在加入到复杂的多肽混合物中时，其质谱学检测变得容易和明确。此外，它们被设计成在整个水到有机LC梯度上顺序洗脱，并具有在通常扫描的质量范围(300-1800 Da)内的m/z值。在一系列实验中，这些多肽被添加到从人血浆中分离的浓缩N-糖基多肽部分(即从以前脱糖形式的N-糖基化完整蛋白中)，我们展示了这些卤化多肽在样品制备和LC注射质量控制以及保留时间和质量校准方面的效用和性能。此外，还展示了用于选定反应监测实验的信号强度归一化和保留时间同步的进一步使用。《分子与细胞蛋白质组学8：1934-1946》，2009。
As the application for quantitative proteomics in the life sciences has grown in recent years, so has the need for more robust and generally applicable methods for quality control and calibration. The reliability of quantitative proteomics is tightly linked to the reproducibility and stability of the analytical platforms, which are typically multicomponent (e.g. sample preparation, multistep separations, and mass spectrometry) with individual components contributing unequally to the overall system reproducibility. Variations in quantitative accuracy are thus inevitable, and quality control and calibration become essential for the assessment of the quality of the analyses themselves. Toward this end, the use of internal standards cannot only assist in the detection and removal of outlier data acquired by an irreproducible system (quality control) but can also be used for detection of changes in instruments for their subsequent performance and calibration. Here we introduce a set of halogenated peptides as internal standards. The peptides are custom designed to have properties suitable for various quality control assessments, data calibration, and normalization processes. The unique isotope distribution of halogenated peptides makes their mass spectral detection easy and unambiguous when spiked into complex peptide mixtures. In addition, they were designed to elute sequentially over an entire aqueous to organic LC gradient and to have m/z values within the commonly scanned mass range (300-1800 Da). In a series of experiments in which these peptides were spiked into an enriched N-glycosite peptide fraction (i.e. from formerly N-glycosylated intact proteins in their deglycosylated form) isolated from human plasma, we show the utility and performance of these halogenated peptides for sample preparation and LC injection quality control as well as for retention time and mass calibration. Further use of the peptides for signal intensity normalization and retention time synchronization for selected reaction monitoring experiments is also demonstrated. Molecular & Cellular Proteomics 8: 1934-1946, 2009.