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能，使蛋白质的蛋白质组谱分析。这些方法不仅可以测量蛋白质的上调或下调，还可以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。这使得整个蛋白质组的全局足迹分析和比较生物体或组织的不同生理状态成为可能。我们的研究重点是蛋白质的各种翻译后修饰的阐明和相互作用，如植物中的ac（et）酰化，磷酸化和糖基化。对于特定肽修饰的某些鉴定，Orbitrap技术获得的高分辨率质谱分析是绝对必要的。此外，为了鉴定和定量化学修饰，多路复用和交联肽样品，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离。电喷雾电离后，前体离子必须通过四极杆选择，在不同的离子阱中进行数据采集，使用不同的方法，如CID， HCD， ETD，以及这些方法的组合。进一步的碎片化（MS3）有利于从多路复用样品中使用报告离子进行准确的蛋白质定量，也有利于从交联样品中对二肽进行一定的鉴定。此外，大肽、完整蛋白和复杂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.