Instrumentation

Mass Spectrometry

LTQ-Orbitrap hybrid mass spectrometer

The LTQ-Orbitrap is a hybrid mass spectrometer combining the strengths of two fundamentally different mass analysers. The front part is a robust very sensitive linear ion trap (dynamic electric field ion trap) mass spectrometer with high scan rates for high throughput analyses. The second mass analyser, the Orbitrap (static electric field ion trap), measures the m/z dependent frequency of a harmonic ion oscillation along a central electrode rode using non-destructive ion detection. With the Orbitrap analyser a mass resolution of 100,000 and mass accuracies of <3ppm (<1ppm after calibration) are achievable in routine analyses.

The LTQ-Orbitrap instrument is equipped with a nanospray ionisation source that is in-line coupled to nano-flow liquid chromatography. This setup allows high throughput and high accuracy mass spectrometric analyses of tryptic digests from very complex protein mixtures. Typically tryptic peptides are fractionated by nano-flow reverse phase liquid chromatography and directly injected through an emitter tip into the ion source of the mass spectrometer. While the Orbitrap determines the accurate mass of the tryptic peptides that elute from the LC column during a 1.5 sec scan the linear ion trap performs collision induced dissociation tandem mass spectrometry on the strongest 4-6 precursor signals to gain sequence information. The combination of high accuracy mass determination of tryptic peptides with sequence information from a very efficient CID MS/MS gives high confidence in protein identification.

ABI 4800 MALDI tandem time-of-flight mass spectrometer

The MALDI tandem time-of-flight mass spectrometer combines the high mass accuracy and resolution of a TOF-analyser with highly specific precursor ion selection for sensitive CID (collision induced dissociation) analysis. Matrix assisted laser desorption/ionization is a robust ionisation method that tolerates certain amounts of additives/contaminations. The 4800 MALDI TOF/TOF-analyzer is able to separate molecules of a wide mass range. Ions of up to 6-8 kDa can be detected in reflector mode and all ions of up to > 100 kDa in linear mode. However mass accuracy and resolution is low in linear mode whereas an accuracy of 5ppm and a resolution of 15000-25000 FWHM (full width at half maximum) are achievable in a mass range of up to 4000 m/z in reflector mode. The effective sensitivity for MS/MS analyses is in the low fmol range.

Offline coupling of MALDI TOF/TOF mass spectrometry with liquid chromatography via automated MALDI-plate spotting of the fractions enables high throughput protein analyses.

Protein/peptide separation/fractionation

Liquid Chromatography

The Centre for Protein Research is equipped with various LC-systems suitable for peptide and protein separation/fractionation. E.g. the Ultimate 3000 is a capillary HPLC-instrument which is set up for 1- and 2-dimensional peptide chromatography using strong cation exchange liquid chromatography (SCX-LC) connected upstream to nano-flow reversed phase liquid chromatography (RP-LC). The nano-flow RP-LC operates at flow rates between 200 and 400 nL/min. The LC-system can also be used for protein separation on C4 columns at higher flow rates.

The nano-flow LC-system is usually connected either in-line to the LTQ-Orbitrap mass spectrometer or the Probot fraction collector for coupling LC peptide fractionation with MALDI TOF/TOF MS.

2-D PAGE

We are routinely performing large scale 2-D gel electrophoresis (IEF coupled with SDS-PAGE) for protein separation and quantification of relative protein abundances.

2-D PAGE is particularly suitable for differentially displaying the protein complements (up to ca. 1500 protein spots per sample) of two or more related samples such as disease state vs healthy, time course experiments etc. Absolute or quantitative differences between samples are usually excised from the gel and processed for mass spectrometric protein identification.

We usually visualise the proteins by in-gel detection either with fluorescent dyes or colloidal coomassie. Both staining methods are compatible with downstream mass spectrometric protein identification.

Robotic workstations

Robotic workstation for automated protein digestion and MALDI plate spotting

In-gel enzymatic protein hydrolysis is the most crucial, and also the most labour intensive sample processing step for gel-based mass spectrometric protein identification. The liquid handling robotic workstation for protein digestion represents an efficient automation and standardisation of sample processing between gel electrophoretic protein separation and mass spectrometric protein identification. The automated protein digestion (i) allows a high sample throughput for large scale gel-based proteomics experiments, (ii) minimise hands-on work which avoids sample contamination that affects downstream mass spectrometry-based protein identification and (iii) standardise sample processing which gives more confidence for less experienced users.

Probot fraction collector

The Probot workstation is a fraction collector that interfaces liquid chromatography with MALDI mass spectrometry. The Probot workstation is in-line coupled to nano-flow liquid chromatography. It automatically mixes the LC-fractions with matrix and spots them onto a MALDI plate. Typically an RP-LC separation of tryptic peptides from a complex protein mixture is fractionated and spotted into 400-800 fractions of 75 nL each that are pre-mixed with 300 nL of matrix. In a 2-dimensional peptide separation (SCX and RP-LC) we routinely fractionate the peptite mixture into 1600 fractions onto two MALDI plates.