High-resolution hybrid mass spectrometer with nano liquid chromatography for proteomics analyses

近年来，基于质谱的方法已经成为可用的，可用于蛋白质的蛋白质组范围的分析。这些方法不仅测量蛋白质的上调或下调，而且测量由于翻译后修饰、复合物形成、重新定位和各种其他细胞过程而改变的其他物理化学性质。这使得能够对整个蛋白质组进行全局足迹样分析，并比较生物体或组织的不同生理状态。我们的研究重点是蛋白质的各种翻译后修饰，如乙酰化、磷酸化和糖基化的阐明和相互作用。对于特定肽修饰的确定性鉴定，绝对需要通过Orbitrap技术获得的高分辨率质谱。此外，为了鉴定和定量化学修饰的、多重的和交联的肽样品，需要通过在线反相纳米液相色谱和高场不对称波形离子迁移谱进行肽分级分离。在电喷雾电离后，必须通过四极杆选择前体离子，以在不同的离子阱中进行数据采集，在碎片化之前和之后使用不同的方法，如CID，HCD，ETD以及这些方法的组合。进一步的片段化（MS 3）有利于用来自多重样品的报告离子进行准确的蛋白质定量，以及用于从交联样品中确定二肽。对于大肽、完整蛋白质和复合体分析，还需要高质量范围。

In recent years, mass spectrometry-based methods have become available thatenable the proteome-wide profiling of proteins. These approaches do not only measureup- or down regulation of proteins, but also other physicochemical properties, whichchange due to posttranslational modifications, complex formation, re-localization andvarious other cellular processes. This enables a global footprint-like profiling of the entireproteome and to compare different physiological states of an organism or tissue. Aparticular focus of our research lies on the elucidation and interplay of variousposttranslational modifications of proteins, such as ac(et)ylations, phosphorylation, andglycosylations in plants. For the certain identification of a particular peptide modification,high resolution mass spectrometry, as obtained by the Orbitrap technology, is anabsolute requirement. In addition, to identify and quantify chemically modified,multiplexed, and cross-linked peptide samples, peptide fractionation via online reversedphase nano-liquid chromatography and high-field asymmetric waveform ion mobilityspectrometry is required. After electrospray ionization precursor ions have to be selectedvia a quadrupole for data acquisition in different ion traps, before and after fragmentationusing different methods such as CID, HCD, ETD, and a combination of those. Furtherfragmentation (MS3) is beneficial for accurate protein quantifications with reporter ionsfrom multiplexed samples as well as for the certain identification of dipeptides fromcrosslinked samples. A high mass range is additionally required for large peptides, intactproteins and complexome profilings.