Lecture

MaxQuant Summer School 2012: Lecture Richard Scheltema

Mass spectrometry

The identification and quantification of the complement of all proteins present in a biological sample is a long-standing goal of modern biology. Mass spectrometry-based proteomics has matured as the method of choice to achieve this and address a great variety of biological questions.

In shotgun proteomics, analysis starts with several steps of sample preparation such as cell lysis, digestion of the proteins and optional fractionation of the peptide mixture. Peptides are then separated by reverse phase chromatography using an UHPLC, 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 an MS scan followed by several MS/MS scans (topN). Data analysis is carried out using our in-house developed MaxQuant software environment

  

Within the Prospects EU Framework our group has established a close collaboration with Thermo Fisher Scientific. This covers the development of novel instrumentation and software for proteomics applications. Our instrument park currently consists of 3 LTQ Orbitrap (XL), 1 Orbitrap Elite and 8 QExactive instruments coupled to Easy nLC 1000 UHPLCs.

 

 

Focus: Orbitrap Instrumentation

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.

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. 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

The Orbitrap analyser was built into a benchtop configuration called ‘Exactive’ in 2008 that was lacking an isolation device. However, the all ion fragmentation 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. 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 Elite. The instrument can be equipped with an ETD device.

 
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