Mass-spectrometry based Proteomics
In shotgun proteomics an LC-MS analysis follows several steps of sample preparation such as cell lyses, digestion of the proteins with trypsin or other proteases and potential fractionation of the peptide mixture (FASP). Peptides are separated on C18 columns during two or four hour gradients that are applied though an UltraHPLC (Easy nLC 1000, ThermoScientific) on-line coupled to the mass spectrometer with a nano electrospray source. The majority of our samples are measured in a data-dependent manner with cycles of one MS scan followed by ten to twenty MS/MS scans (dd topN) using the Xcalibur software (ThermoScientific). Data analysis is carried out using our in-house developed MaxQuant software environment (Cox et al., Nature Biotechnology, 2008). Further details on data analysis can be found on our website MaxQuant.
Within the Prospects EU Framework our group closely collaborates with ThermoScientific on the development of novel instrumentation and software for proteomics applications even before these are introduced to the market. Our instrument park consists of 2 LTQ Orbitrap (XL), 2 Orbitrap Elite and 7 QExactive instruments.
The first commercially available Orbitrap analyser (ThermoScientific) was introduced in 2005 in a hybrid format combined with a linear ion trap providing low resolution CID fragmentation. As with the previous LTQ FT ICR instrument, the LTQ Orbitrap was very well suited for proteomics. Major advantages were the high resolution of the Orbitrap analyser and its superior sensitivity, while the maintenance requirements were greatly reduced in comparison to the FT ICR technology (Makarov et al., JASMS, 2006). The LTQ Orbitrap was later on equipped with further fragmentation capabilities such as HCD and ETD in addition to the by then exclusively used CID fragmentation mechanism (Olsen et al., MCP, 2005, Mikesh et al., Biochem Biophysica Acta, 2006). A significant improvement to the LTQ part of the instrument was realized by dividing the ion trap into two segments each with different pressure for improved fragmentation and detection. Together with an S-lens, this provided the ‘LTQ Orbitrap Velos’ with higher sensitivity and speed (Olsen et al., MCP, 2009). The Orbitrap analyser was built into a benchtop configuration called ‘Exactive’ in 2008 that was lacking an isolation device. However, the all ion fragmentation (Geiger et al., MCP, 2010) mode was developed to circumvent this shortcoming and apply it to proteomics studies. Recently, the ‘Q Exactive’ was introduced to the market overcoming the disadvantage of not being able to filter for precursor masses by connecting a quadrupole to the Orbitrap analyser (Michalski et al., MCP, 2011). This particular combination allows special features such as multiplexing of several mass ranges. Latest developments in the LTQ Orbitrap hybrid series include a compact, high-field Orbitrap analyser providing ultra high resolution and a next generation LTQ part that is more robust and allows higher scan speed in the Orbitrap Velos Pro and Orbitrap Elite (Michalski et al., MCP, 2011). Both instruments can be equipped with an ETD device.
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