Protein Analysis Services and quantitative Method Development
The mission of the Proteomics Core Facility at the Medical University of Vienna is to provide the researchers at the Medical University of Vienna with consulting, teaching, protein analysis services and quantitative method development.
Data Analysis and Protein Characterization
Molecular Weight Determination
We can perform molecular weight determination for a purified protein or a peptide by using LC/ESI/MS with a very fast and steep reversed-phase gradient and a QToF mass spectrometer. Due to the separation on the HPLC column, sample cleanup is performed during the workflow and your analyte is separated from interfering species that may be in the sample.
Identifications of proteins based on tandem mass spectrometry
Protein identifications are made based on automated database searching of tandem mass spectra of peptides.The Proteomics Core Facility at the MUW uses high resolution (R>10,000) tandem mass spectrometers (maXis, qToF Ultima, LTQ Velos) to provide accurate mass measurements of both peptide molecular weight (MS data) and peptide fragments (MS/MS) needed for database search and identification. The use of high-end mass spectrometers such as maXis yields high quality data providing for highly confident identifications of proteins based on peptide masses.
In case of qualitative identifications from 2D PAGE gel spots are performed using either a short (30 min) nanoscale capillary LC gradient coupled to the LTQ Velos ion-trap mass spectrometer. Currently, we cannot provide Data dependent acquisition and a MASCOT database search is used to return peptide, and thus protein, identifications.
Qualitative Identifications from gel bands or more complex samples such as cell lysates are performed using higher-capacity separations (60 min or up to 10 hours on 50 cm long nano separation columns) coupled to Q-Tof Mass Spectrometers. The higher capacity separation allows for analysis of more complex samples and more confident identification of larger numbers of proteins. Again, data dependent acquisition and a MASCOT database search is used to identify peptides and proteins.
For extremely complex samples such as tissue or organ lysis, multi-dimensional separations can be performed prior to MS/MS analysisThe last separation dimension of this approach is always reversed-phase nanoscale LC, but the first dimensional separation can involve several mechanisms: strong cation exchange (SCX), strong anion exchange (SAX), hydrophilic interaction chromatography (HILIC), size exclusion chromatography (SEC), or ERLIC LC. All multidimensional separations are applied as off-line separation methods. That means that the fractions from one separation approach e.g. SCX are collected and reinjected in the following separation dimension.
Methoden & Expertise zur Forschungsinfrastruktur
The Proteomics Core Facility provides a full proteomics infrastructure for the identification and characterization of proteins. The key technology of the Proteomics Core Facility is mass spectrometry for the qualitative and quantitative analysis and characterization of proteins. We primarily use the “bottom-up” proteomics approach, where all proteins are proteolytically (in most cases with tryptic, but other enzymes are also used) digested, producing peptide surrogates (signature peptides) of the original proteins.
Protein identifications are made using state-of-the-art search engines such as Mascot or X!Tandem, with the capability of searching standard or custom protein databases.