Equipment

Q Exactive Plus

Mass range: 50 to 6,000 m/z (mass over charge)

Resolution: up to 140,000 at 200 m/z , maximum 280,000

Ion fragmentation: HCD (Higher-Energy Collisional Dissociation)

Mass accuracy: < 3 ppm RMS (root mean square) external calibration, < 1 ppm RMS internal calibration

High scan speed (12 Hz, 17,500 resolution)

Multiplexing (up to 10 precursors/scan) and Targeted Proteomics (SIM/PRM)

The Q Exactive Plus is a benchtop instrument coupled with the Ultimate 3000 RSLC system for LC/MS applications. The Proxeon source with a steel emitter is used for electrospray ionization. Ionized peptides enter the Q Exactive Plus through a heated transfer capillary and an S-lens, focusing the ion stream. The ion stream is then directed to an advanced quadrupole mass filter and selected ions are collected and stored in a C-trap. The C-trap transfers ions to either the Orbitrap mass analyzer or the HCD cell for ion fragmentation, followed by mass analysis in the Orbitrap. The Q Exactive allows monitoring of selected ions (SIM) and parallel reactions (PRM). The instrument is used within the TRR130 proteomics platform to provide state-of-the-art LC/MS technology for research projects within the TRR130

Orbitrap XL

Mass range: 50 to 4.000 m/z (mass over charge)

Resolution: up to 60,000 at 400 m/z , maximum 100,000

Ion fragmentation: CID (collision induced dissociation)

Mass accuracy: < 3 ppm RMS (root mean square) external calibration, < 1 ppm RMS internal calibration.

The Orbitrap XL is used for routine applications, method optimization, and low complexity sample analysis. The instrument features a linear ion trap and an Orbitrap mass analyzer and is coupled to the Ultmiate3000 RSLC system for LC/MS applications. The eluting peptides are transferred via a fused silica emitter into a thermo nano LC source for electrospray ionization. Charged ions enter the mass spectrometer through a heated transfer capillary and are transferred to the linear ion trap through three multipoles for ion storage, selection, and fragmentation by CID. Mass analysis of precursor ions is performed in the Orbitrap.

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Synapt G1 HDMS Q-TOF system for native MS and ion mobility separation (IMS) of intact proteins and non-covalent protein complexes.

Instrument features:

• equipped with nano-electrospray ion source
• suitable for high mass detection and intact non-covalent protein complexes
• isolation up to m/z 32,000 for MS/MS
• collision-induced dissociation with argon or xenon gas
• Ion mobility separation in a drift tube filled with nitrogen.

The system uses electrospray to transfer proteins intact and in their folded state, as well as non-covalent protein complexes, from an aqueous buffer solution to the gas phase without dissociation or fragmentation. This mass spectrometric technology is called "Native MS" and allows the study of the binding affinity and stoichiometry of protein-protein or protein-ligand complexes in addition to the determination of the exact molecular masses of the individual components of a given complex. Native MS has recently become an important complementary method of existing structural biology tools. In addition, native MS has been successfully combined with IMS in the gas phase. In this method, ions are passed in packets through a drift tube filled with inert gas. Since the resulting collisions slow down larger ions more than small ones, a drift time spectrum can be recorded. Collision cross sections (CCS) are estimated by comparison with drift times of proteins of known size. This allows us to uncover subtle conformational changes induced by ligand binding or mutations, or to follow protein unfolding upon stepwise collision activation. A particular focus is currently on developing methods to analyze membrane protein complexes in the presence of detergent micelles.

Dionex/Thermo Ultimate 3000 systems

We have several differently configured Dionex/Thermo Ultimate 3000 HPLC systems in our laboratory. The HPLC systems that are used offline (i.e. not directly coupled to a mass spectrometer) have milliliter to microliter flow rates. In combination with various analytical columns, they are used for reversed-phase (RP) or size-exclusion chromatography of intact proteins or peptides after enzymatic digestion. The LC systems also include a sampler module that allows collection of chromatographic fractions. The on-line HPLC systems deliver flow rates in the nanolitre range required for stable electrospray ionization. They are equipped with a dual valve system that allows parallel loading of the sample onto one of the two precolumns while the other precolumn is washed and conditioned.