Fred Hutchinson Cancer Center, Seattle, WA, USA.
Methods Mol Biol. 2024;2823:253-267. doi: 10.1007/978-1-0716-3922-1_16.
Targeted proteomics enables sensitive and specific quantification of proteins and post-translational modifications. By coupling peptide immunoaffinity enrichment with targeted mass spectrometry, we have developed the methodology for multiplexed quantification of proteins and phosphosites involved in the RAS/MAPK signaling network. The method uses anti-peptide antibodies to enrich analytes and heavy stable isotope-labeled internal standards, spiked in at known concentrations. The enriched peptides are directly measured by multiple-reaction monitoring (MRM), a well-characterized quantitative mass spectrometry-based method. The analyte (light) peptide response is measured relative to the heavy standard. The method described provides quantitative measurements of phospho-signaling and is generally applicable to other phosphopeptides and sample types.
靶向蛋白质组学能够实现蛋白质和翻译后修饰的灵敏和特异性定量。通过将肽免疫亲和富集与靶向质谱相结合，我们开发了用于定量分析 RAS/MAPK 信号网络中涉及的蛋白质和磷酸化位点的方法。该方法使用抗肽抗体富集分析物和以已知浓度掺入的重稳定同位素标记的内标。通过多重反应监测 (MRM) 直接测量富集的肽，这是一种经过充分验证的基于质谱的定量方法。分析物（轻）肽响应相对于重标准进行测量。所描述的方法提供了磷酸化信号的定量测量，并且通常适用于其他磷酸肽和样品类型。
