Mass spectrometry (MS) is the most comprehensive and versatile tool in large-scale proteomics. In this review, we dissect the overall framework of the MS experiment into its key components. We discuss the fundamentals of proteomic analyses as well as recent developments in the areas of separation methods, instrumentation, and overall experimental design. We highlight both the inherent strengths and limitations of protein MS and offer a rough guide for selecting an experimental design based on the goals of the analysis. We emphasize the versatility of the Orbitrap, a novel mass analyzer that features high resolution (up to 150,000), high mass accuracy (2-5 ppm), a mass-to-charge range of 6000, and a dynamic range greater than 10(3). High mass accuracy of the Orbitrap expands the arsenal of the data acquisition and analysis approaches compared with a low-resolution instrument. We discuss various chromatographic techniques, including multidimensional separation and ultra-performance liquid chromatography. Multidimensional protein identification technology (MudPIT) involves a continuum sample preparation, orthogonal separations, and MS and software solutions. We discuss several aspects of MudPIT applications to quantitative phosphoproteomics. MudPIT application to large-scale analysis of phosphoproteins includes (a) a fractionation procedure for motif-specific enrichment of phosphopeptides, (b) development of informatics tools for interrogation and validation of shotgun phosphopeptide data, and (c) in-depth data analysis for simultaneous determination of protein expression and phosphorylation levels, analog to western blot measurements. We illustrate MudPIT application to quantitative phosphoproteomics of the beta adrenergic pathway. We discuss several biological discoveries made via mass spectrometry pipelines with a focus on cell signaling proteomics.

中文翻译：

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质谱蛋白质组学：方法，进展和应用。

质谱（MS）是大规模蛋白质组学中最全面，用途最广泛的工具。在这篇综述中，我们将MS实验的总体框架分解为其关键组成部分。我们讨论了蛋白质组学分析的基础知识，以及分离方法，仪器和整体实验设计领域的最新进展。我们着重介绍了蛋白质MS的固有优势和局限性，并为根据分析目标选择实验设计提供了粗略的指导。我们强调Orbitrap的多功能性，这是一种新型的质量分析仪，具有高分辨率（高达150,000），高质量精度（2-5 ppm），质荷范围为6000和动态范围大于10（ 3）。与低分辨率仪器相比，Orbitrap的高质量精确度扩展了数据采集和分析方法的范围。我们讨论了各种色谱技术，包括多维分离和超高效液相色谱。多维蛋白质识别技术（MudPIT）涉及连续样品制备，正交分离以及MS和软件解决方案。我们讨论了MudPIT在定量磷酸化蛋白质组学中的应用的几个方面。MudPIT在磷蛋白的大规模分析中的应用包括（a）用于磷酸肽基序特异性富集的分馏程序，（b）开发用于查询和验证shot弹枪磷肽数据的信息学工具，（c）深度数据分析，可同时测定蛋白质表达和磷酸化水平，类似于蛋白质印迹测量。我们说明了MudPIT在β肾上腺素途径定量磷酸化蛋白质组学中的应用。我们讨论了通过质谱管线取得的一些生物学发现，重点是细胞信号蛋白质组学。