Mass spectrometry

With the beginning of 2013, the mass spectrometers and nano-HPLCs of the IMP/IMBA/GMI protein chemistry facility and the MFPL present at the campus were integrated into the Campus Science Support Facility (CSF). Since then, the CSF provides a pool of mass spectrometers and nano-HPLCs. This equipment pool is overseen, managed and maintained by Karl Mechtler.

From October 2016 onwards, the following instruments are available for the facility:

Q Exactive HF (Thermo Scientific)

In October 2015, we upgraded 4 of our mass spectrometers to the newest generation of the Q-Exactive instrument series, which is called QExactive HF. This mass spectrometer contains an ultra-high-field Orbitrap analyzer which almost doubles its resolution at the same scan speed as compared to the preceeding Q Exactive Plus. The QExactive HF employs a quadrupole for the selection of precursor ions with a HCD cell for fragmentation and an Orbitrap for detection of ions. We use this instrument for all kinds of peptide identifications and quantifications, except for those, where other fragmentation modes than HCD have to be employed.

Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Scientific)

The Orbitrap Fusion Lumos Tribrid mass spectrometer belongs to the newest generation of the Orbitrap instrument family and was installed at our facility in Q4 of 2016. It includes 3 mass analysers, i.e. a quadrupole mass filter, a linear ion trap and an Orbitrap mass analyzer and 2 detectors. The instrument allows different and partially complementary fragmentation techniques, which are collision induced dissociation (CID), higher-energy collisional dissociation (HCD), electron transfer dissociation (ETD) and a combination of ETD and HCD called EThcD. One major application of this new instrument will be multiplex quantitation of TMT-labelled samples as this instrument offers a novel method called MultiNotch MS3 (McAlister et al., 2014), which provides a higher accuracy of TMT quantification. Additionally, we use the instrument for the analysis of peptides with labile post-translational modifications such as glycopeptides and for the analysis of chemically crosslinked peptides.

QTRAP 5500 (AB Sciex)

The QTRAP 5500 (AB Sciex) is a hybrid triple quadrupole/linear ion trap mass spectrometer. This instrument is characterized by a high scan speed, selectivity, sensitivity and a high dynamic range. We use this instrument mainly for the absolute quantification of proteins of interest either from affinity-purified samples or from total cell extracts using multiple reaction monitoring (MRM).

Q-TOF Synapt G2 (Waters)

We use this mass spectrometer for the characterization of intact proteins. The Q-TOF Synapt G2 employs a quadrupole for the selection of precursor ions, a second quadrupole for fragmention and a time-of-flight tube for the mass analysis. The instrument is coupled to a chromatography system using a microscale C4 column, which allows chromatographic separation of proteins before being ionized in a microspray source and entering the MS. The instrument is equipped with a high-efficiency T-Wave ion mobility technology, which gives access to an additional dimension of separation, based on molecular size and shape. As Ion mobility experiments are time consuming and difficult they are not offered as a service but can be done project based. The acquisition and the operation of this instrument is based on a joint initiative with the MFPL mass spectrometry facility.

ABI 4800 MALDI TOF/TOF (AB Sciex)

The ABSCIEX 4800 MALDI TOF/TOF is used for controlling the quality of synthetic peptides generated in house, for the mass measurement of intact proteins and for glycan analysis.

Nano reversed phase HPLC

All state of the art mass spectrometers described above (except the MALDI-TOF/TOF) are coupled on-line to nano-LCs (Dionex UltiMate 3000 Rapid Separation LCs; Thermo Scientific). For peptide separation prior to MS reversed phase C18 columns are used. For separation of intact proteins prior to MS reversed phase C4 columns or size exclusion columns are used.

HPLC for off-line sample fractionation

In cases were two-dimensional separations are required we employ off-line chromatography, mainly but not exclusively, strong cation exchange chromatography. This instrument is connected to a fraction collector.