Quantification of gel-separated proteins and their phosphorylation sites by LC-MS using unlabeled internal standards - Analysis of phosphoprotein dynamics in a B cell lymphoma cell line
Quantification of gel-separated proteins and their phosphorylation sites by LC-MS using unlabeled internal standards - Analysis of phosphoprotein dynamics in a B cell lymphoma cell line
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DOI：
10.1074/mcp.m500078-mcp200
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发表时间：
2005-08-01
影响因子：
7
通讯作者：
Waterfield, MD
中科院分区：
文献类型：
--
作者：
Cutillas, PR;Geering, B;Waterfield, MD
蛋白质磷酸化在正常细胞功能中起着至关重要的作用，但在疾病中常常被破坏。尽管最近在通过 MS 鉴定和定量蛋白质磷酸化位点方面取得了重大进展，但目前的方法学限制仍然阻碍了对蛋白质磷酸化的常规、易于使用和全面的定量分析。在这里，我们报告了一种简单的 LC-MS 方法来量化凝胶分离的蛋白质及其磷酸化位点；在这种方法中，所研究蛋白质的肽分析物的积分色谱峰面积被标准化为在凝胶内消化之前掺入切除的凝胶样品中的非同位素标记的内标蛋白质的色谱峰面积。内标强度可校正凝胶内消化和从凝胶碎片中提取肽的过程中可能发生的酶活性差异和样品损失。我们使用这种带有内标的峰面积测量方法来研究过钒酸盐对 WEHI-231 B 细胞淋巴瘤细胞系中蛋白质磷酸化的影响，并评估磷酸肌醇 3-激酶 (PI3K) 在这些磷酸化事件中的作用。使用 IMAC 或使用 Tyr(P) 抗体通过免疫沉淀从总细胞裂解物中分离出的磷蛋白，使用该方法进行分析，从而鉴定出超过 400 种蛋白质，其中一些在过钒酸盐处理后在磷蛋白组分中发现了较高水平。用 PI3K 抑制剂渥曼青霉素预处理细胞会降低某些蛋白质（例如 STAT1 和磷脂酶 C gamma 2）的磷酸化水平，同时增加其他几种蛋白质的磷酸化水平。还使用内标测量峰面积来跟踪已知和新型磷脂酶 C γ 2 磷酸化位点的翻译后修饰中 PI3K 依赖性和独立变化的动态。我们的结果说明了这种概念上简单的 LC-MS 方法在定量凝胶分离的蛋白质及其磷酸化位点以及对生物系统进行定量分析方面的能力。
Protein phosphorylation plays a critical role in normal cellular function and is often subverted in disease. Although major advances have recently been made in identification and quantitation of protein phosphorylation sites by MS, current methodological limitations still preclude routine, easily usable, and comprehensive quantitative analysis of protein phosphorylation. Here we report a simple LC-MS method to quantify gel-separated proteins and their sites of phosphorylation; in this approach, integrated chromatographic peak areas of peptide analytes from proteins under study are normalized to those of a nonisotopically labeled internal standard protein spiked into the excised gel samples just prior to in-gel digestion. The internal standard intensities correct for differences in enzymatic activities and sample losses that may occur during the processes of in-gel digestion and peptide extraction from the gel pieces. We used this method of peak area measurement with an internal standard to investigate the effects of pervanadate on protein phosphorylation in the WEHI-231 B cell lymphoma cell line and to assess the role of phosphoinositide 3-kinase (PI3K) in these phosphorylation events. Phosphoproteins, isolated from total cell lysates using IMAC or by immunoprecipitation using Tyr(P) antibodies, were analyzed using this method, leading to identification of > 400 proteins, several of which were found at higher levels in phosphoprotein fractions after pervanadate treatment. Pretreatment of cells with the PI3K inhibitor wortmannin reduced the phosphorylation level of certain proteins ( e. g. STAT1 and phospholipase C gamma 2) while increasing the phosphorylation of several others. Peak area measurement with an internal standard was also used to follow the dynamics of PI3K-dependent and - independent changes in the post-translational modification of both known and novel phospholipase C gamma 2 phosphorylation sites. Our results illustrate the capacity of this conceptually simple LC-MS method for quantification of gel-separated proteins and their phosphorylation sites and for quantitative profiling of biological systems.