Peptide Immunoaffinity Enrichment with Targeted Mass Spectrometry: Application to Quantification of ATM Kinase Phospho-Signaling.
Peptide Immunoaffinity Enrichment with Targeted Mass Spectrometry: Application to Quantification of ATM Kinase Phospho-Signaling.
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使用靶向质谱法进行肽免疫亲和富集：应用于 ATM 激酶磷酸信号定量。
DOI：
10.1007/978-1-4939-6955-5_15
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Paulovich,AmandaG
中科院分区：
文献类型：
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作者：
Whiteaker,JeffreyR;Zhao,Lei;Schoenherr,RegineM;Kennedy,JacobJ;Ivey,RichardG;Paulovich,AmandaG
多肽免疫亲和富集法与靶向质谱联用是一种对多肽分析物进行可靠和重复性定量的定量方法。该方法能够对多肽进行多重量化，包括翻译后修饰，如磷酸化。抗肽抗体用于浓缩分析物和重稳定同位素标记的标准品。通过多反应监测(MRM)直接测量富集肽，这是一种基于定量质谱学的良好特征的方法。定量是通过测量分析物(轻)肽相对于重标准的反应来进行的，重标准是在已知浓度下添加的。在这里，我们描述了在细胞裂解产物中对ATM激酶及其未修饰的多肽类似物上的磷酸化多肽进行多路测量的方法。该方法提供了磷酸信号的定量测量，并可扩展到许多其他磷酸肽和样品类型。
Peptide immunoaffinity enrichment coupled with targeted mass spectrometry is a quantitative approach for the robust and reproducible quantification of peptide analytes. The approach is capable of multiplexed quantification of peptides, including posttranslational modifications such as phosphorylation. Anti-peptide antibodies are used to enrich analytes and heavy stable isotope-labeled standards. The enriched peptides are directly measured by multiple reaction monitoring (MRM), a well-characterized quantitative mass spectrometry-based method. Quantification is performed by measuring the analyte (light) peptide response relative to the heavy standard, which is spiked at a known concentration. Here, we describe the methodology for multiplexed measurement of phosphorylated peptides on the ATM kinase and their nonmodified peptide analogs in cellular lysates. The method provides quantitative measurements of phospho-signaling and can be extended to a number of other phosphopeptides and sample types.