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 Vienna Biocenter Core Facilities (VBCF). Since then, the VBCF provides a pool of mass spectrometers and nano-HPLCs. This equipment pool is overseen, managed and maintained by the protein chemistry facility.

The following instruments are available for the facility:

  • 4 Q-Exactive HF-X (Thermo Scientific)
  • 1 Orbitrap Fusion Lumos (Thermo Scientific)
  • 1 Orbitrap Exploris 480 (Thermo Scientific)
  • 1 TimsTOF Pro (Bruker)
  • 1 Q-TOF Synapt G2 (Waters)
  • 1 MALDI-TOF/TOF 4800 (Sciex, used for characterization of synthetic peptides)
  • HPLCs

Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer

The facility operates 4 Q Exactive HF-X instruments, which are used to analyse the major part of the submitted samples. This mass spectrometer uses a high-capacity transfer tube for maximum ion loading, an electrodynamic ion funnel that accommodates and transmits ions over a broader mass range, and a high-field Orbitrap mass analyser with a resolution of 240000 (FWHM) at m/z 200. The main advantage is the higher sensitivity and scan rate. The Advanced Peak Determination (APD) algorithm significantly increases the number of precursors available for data-dependent analysis, which in turn results in more MS/MS spectra, and peptide identifications.

The Q Exactive HF-X employs a quadrupole for the selection of precursor ions with an 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 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 application of this instrument is 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 arginine phosphorylation and for the analysis of chemically crosslinked peptides.

Orbitrap Exploris 480 (Thermo Scientific)

Similar to the HF-X the Exploris contains the hybrid quadrupole-Orbitrap design, but the layout of the instrument was reconfigured to a more compact and robust platform. It has the same ion source, atmosphere-to-vacuum interface and user-facing software as the Orbitrap Fusion Lumos mass spectrometer and can be interfaced on the front end with a FAIMS device (high-Field Asymmetric waveform Ion Mobility Spectrometry). The instrument has a new pumping concept, instead of a multi-stage pumping system it has a single six-stage turbo pump that evacuates the entire path from the ion funnel to the orbitrap. Additionally, the ion optics and instrument software were refined to reduce spectral noise and ion optics contamination. The most prominent improvement compared to the HFX is the option to interface it with the FAIMS device, which adds a new dimension of selectivity. The application of FAIMS leads to a reduction of chemical noise, leading to better signal to noise ratios. 

TimsTOF Pro (Bruker)

The timsTOF Pro is a time-of -flight instrument coupled to a dual Trapped Ion Mobility Spectrometry (TIMS) analyser. Here, ions are separated in the gas phase based on their ion mobility which adds a 3rd dimension of separation in addition to retention time and m/z. The design of the dual TIMS device allows for ions to be accumulated in the first part of the analyser, while they are sequentially released dependent on their ion mobility from the second section (PASEF; Parallel Accumulation and Serial Fragmentation). Thereby a duty cycle of nearly 100% is achieved resulting in nearly no ion loss. The design of the instrument offers a high sequencing speed (up to 100Hz) by synchronizing the quadrupole isolation mass window with the elution time of the specific peptide packages from the TIMS funnel. This design allows to reach new depths in shotgun proteomics and phosphoproteomics, using low sample amounts.

MS instruments used for shotgun proteomics require cleaning when run 24 hours a day on large sample numbers, and performance degradation can be noticeable over short time periods. The superior robustness of the timsTOF Pro means that the instrument can be run 24/7 over many days or weeks without noticeable loss of sensitivity or other performance metrics.

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 with a pre-column set-up to allow for online desalting and pre-concentration. 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.