Workflows

    Protein identification
  • Protein bands/spots of interest are excised from the gel and digested in-gel with a site specific protease such as trypsin. The tryptic peptides are eluted from the gel matrix and subjected to mass spectrometric analysis. Single protein bands/spots are usually analysed by MALDI tandem Time-of-Flight mass spectrometry. more »

  • The goal of high throughput proteomics is basically the identification of all proteins in a certain protein complement including their post-translational protein modifications and quantitative information (see below). It can be performed either as a gel-based or a gel-free approach. The gel-based approach includes the pre-fractionation and purification of proteins by 1-dimensional protein gel electrophoresis. more »

  • Targeted Proteomics
  • MRM (multiple reaction monitoring) also called SRM (selected reaction monitoring) is a targeted data acquisition where known precursor ion signatures such as precursor m/z, chromatographic retention time and specific CID-based fragment ion m/z values are used for compound detection and quantification. MRMs are usually performed on triple quadrupole (QqQ) instruments where the first quadrupole serves as a mass selective filter open for only the selected m/z value (often within one unit resolution), the second as a collision quadrupole to generate CID-based fragment ions of the selected precursor m/z and the third as a second mass filter to only select one fragment ion at a time. In the context of MRM fragment ions specific for the compound of interest are called transitions. more »

  • SWATH (sequential window acquisition of all theoretical fragment ion spectra) is a new strategy for high throughput, label-free protein quantification. It generates global, quantitative protein maps using data-independent acquisition of collision-induced dissociation (CID) spectra of all precursor ions. As a data-independent acquisition (also referred to as MS/MSall) SWATH-MS has a greater coverage of peptide identification compared to classical discovery approaches. more »

  • Protein quantification
  • In 2-D PAGE protein complements of two or more different samples are compared by their relative staining intensities after in-gel detection of proteins e. g. by fluorescent dyes or colloidal coomassie. more »

  • ICAT is a duplex protein labelling technique to differentially compare relative protein abundances of two samples directly with the mass spectrometer. The ICAT chemistry labels the cysteine residues of proteins. Each sample is labelled either with a light or a heavy label that differ in mass by 9 Da. more »

  • iTRAQ is a multiplex protein labelling for mass spectrometry-based protein quantification. It is available as 4-plex and 8-plex labels (we only use 4plex labelling at present). iTRAQ reagents label primary amines (N-terminal amino group and epsilon amino group of lysines) either of intact proteins or proteolytically digested proteins. more »

  • Protein characterisation
  • Not all organisms that are of significance for research are completely sequenced yet i. e. conventional mass spectrometry-based approaches for protein identification may fail. more »

  • Many post-translational protein modifications (PTMs) can be identified by mass spectrometry. Modern high accuracy mass measurements give more confidence in the identification of these modifications. The resolution and mass accuracy of an Orbitrap instrument enables us to distinguish between phosphorylation vs sufation (delta mass of 0. more »

  • Electrospray ionisation generates multiple charge states of large molecules that are detectable in the lower m/z region of the MS-spectrum. A single charge state (up to ca. 20+) can be trapped in the linear ion trap (using an isolation width of 5-10) and transferred to the orbitrap for an accurate mass measurement. more »