Equipment

The AutoflexIII (Bruker Daltonics) is an orthogonal MALDI-TOF-TOF-system that offers high mass accuracy and resolution in both positive and negative ionization modes. The mass accuracy for metabolites and peptides can reach 5 ppm which is equal to +/- 0,005 Da at m/z = 1000. The instrument also offers MS/MS capabilities that can be used to obtain structural information by fragmentation of biomolecules. Using MS/MS analysis identifications of metabolites or Peptide Mass Fingerprint (PMF) identifications of proteins can be verified.

Applications:

• Identification of proteins from 1D-, 2D- or 2D DIGE-gels (PMF, Peptide Mass Fingerprint)
• Mass determination of peptides, proteins, oligosaccharides, oligonucleotides etc. for quality control or identification
• Top-Down mass spectrometry of intact proteins using in-source-decay (ISD). MALDI-ISD of intact proteins enables N-terminal sequencing of proteins.

The 5500 QTRAP ESI-MS (AB SCIEX) system is mainly used for the targeted quantification of metabolites. For the analysis of peptides and proteins a nano-spray ion source is available.  Metabolites or peptides with known m/z ratio can be detected and quantified with high sensitivity even in very complex mixtures by SRM (Selected Reaction Monitoring) or MRM (Multiple Reaction Monitoring). In a SRM experiment combinations of a precursor ion and one or more characteristic fragment ions of the metabolite of interest are analyzed. Since for both precursor ion and fragment ions only very narrow m/z ranges are selected by the quadrupoles (usually 1 Da) the chemical background is low and therefore the metabolite of interest can be detected with high sensitivity.

In contrast to Triple-Quadrupole mass spectrometers the 5500 QTRAP is a hybrid system including a linear ion trap (LIT) that can be used as quadrupole or LIT. Due to the high acquisition speed of the LIT MS/MS or MS3 scans can be acquired during an SRM quantification experiment to confirm of the metabolite identity. Furthermore the 5500 QTRAP can be used for precursor ion or neutral loss scan experiments to screen for the presence of certain substance classes or modifications (for example lipids, glycosylation, phosphorylation, etc.) within a sample. The LIT can also used within these screening experiments to record data dependent MS/MS spectra for metabolite or peptide identification.

For the analysis of proteins the 5500 QTRAP is coupled to a nano-UHPLC-system whereas the QTRAP is coupled to a 1290 Infinity UHPLC system (Agilent) for the analysis of metabolites. The 1290 Infinity UHPLC can be operated at flow rates of  ≤ 2 ml/min and up to 1200 bar backpressure. This allows the application of UHPLC columns with < 2 µm particle size, which leads to higher peak capacities and higher MS signal intensities compared to a standard HPLC.

Applications:

• Targeted quantification of metabolites and proteins by SRM/MRM experiments.
• Absolute quantification of metabolites and proteins by SRM/MRM experiments with internal standard.
• Precursor ion or neutral loss scans for the identification of certain substances classes or modifications.

The GenePix 4000B Microarray Scanner (Molecular Devices) allows the acquisition and analysis of fluorescent DNA- and protein arrays on standard glass microscopy slides (25 mm × 75 mm). The scanner has two lasers with excitation wavelengths of 532 nm and 635 nm and can acquire data simultaneously, which dramatically reduces scan times (approximately 14 min at maximum resolution of 5 µm) and provides real-time access to data as they are acquired. The lasers and filters are optimized for Cy3 and Cy5 dyes, but dyes with spectra similar to Cy3 and Cy5 will also give good results. For more information you can visit the manufacturers’ webpage (Molecular Devices). Two software packages are available for data analysis: GenePixPro for data acquisition and first stages of analysis (localization of features, normalization). Advanced statistical analysis and visualization of the data including comparison with data obtained on other platforms (Affimetrix) can be carried out with the Acuity Software. Hybridization chambers for sample preparation are available at the CFH on request.

For 2D-electrophoresis experiments an isoelectric focussing system (IPGphor3 (GE Healthcare) and a vertical electrophoresis-unit Ettan Daltsix (GE Healthcare) for large gels (26 × 20 cm) are available. In the first dimension (IEF) up to 12 IPG strips of different lengths (7-24 cm) can be processed simultaneously. In the second dimension (SDS-PAGE) up to six large format gels (26 × 20 cm) can be run in 4-5 h using the Ettan Daltsix electrophoresis unit.

The Typhoon Trio+ Imaging System (GE Healthcare) is a versatile imaging platform that can be used for a variety of applications:

Autoradiography of different radio-isotopes: ³H, ¹⁴C, ³²P, ³³P, ³⁵S, ¹²⁵I (with storage phosphor screen plates).
Chemiluminescence: direct, sensitive detection of chemiluminescence without applying a film or screen (Western Blots).
Fluorescence: the system has 3 lasers with excitation wavelengths of 488 nm (blue), 532 nm (green) and 633 nm (red) and a wide set of emission filters. This allows the application of a large number of commercially available fluorophores. In addition the analysis of up to four fluorophores in one analysis (»Multiplexing«) is possible, for example in differential protein expression analysis using 2D DIGE.
The instrument handles agarose and polyacrylamide gels, membranes and microplates. Data analysis can be carried out with ImageQuantTL Software. A software package for statistical evaluation of 2D DIGE gels is also available (Progenesis SameSpots 4.1.4).