Proteomics and protein analysis

This facility offers service and expertise in gel-based proteomics by two-dimensional gel electrophoresis (2-DE). With 2-DE proteins are separated according to two different protein characteristics: 1.) protein charge (isoelectric point, pI) and 2.) molecular mass. Since hundreds to thousands of protein spots can be separated within one 2D gel it is especially suitable for the large-scale screening of complex protein extracts. Here, the separation of intact proteins allows not only the study of protein levels (spot intensity) but also the detection of modifications as well as specific isoforms (spot position).

In addition to 2-DE the facility offers also various native electrophoresis techniques to study protein interactions and complexes:

Protein patterns of different samples can be compared to investigate e.g.:

• disease mechanisms (including biomarker screening)
• influence of drugs, environmental factors, growth or developmental stages, regulators etc.
• quality of commercial protein extracts (quality control)

Our group has a long term expertise in the analysis of numerous proteomes and subproteomes from bacteria to man to investigate various research questions. Main emphasis is on clinical proteomics to study disease mechanisms with the own research focus on human lung diseases.

Equipment and main approaches

The charge-dependent separation in the first dimension (isoelectric focusing, IEF) is performed with an IPGphor 3 IEF system or a Multiphor II flatbed system (GE Healthcare). Following IEF the proteins are separated in the second dimension (SDS-PAGE) by their molecular mass in an Ettan DALT II system (GE Healthcare) which allows the separation of up to 12 gels.

The protein pattern is visualized by labelling the proteins with fluorescent dyes (e.g. Cy3, Cy5) prior separation (2-D DIGE) or staining the gels with silver nitrate or different fluorescent dyes. Several fluorescent dyes allow also the specific detection of phosphorylated or glycosylated proteins. Since 2D-DIGE offers high accuracy and sensitivity in spot detection and quantification it represents the current major 2-DE approach. The two major DIGE approaches are the minimal DIGE (50 µg protein) and the cysteine-specific saturation DIGE for the analysis of scarce samples (5 µg protein).

After the separation (2D-DIGE) or staining the gels are scanned with an ImageScanner III (GE Healthcare) for silver stained 2-D gels and Octoplus QPLEX (NH DyeAGNOSTICS) or MolecularImager FX Pro (Bio-Rad) for fluorescent 2-D gels. The protein pattern is analyzed with specific softwares, e.g. Delta 2D (Decodon) or Melanie (GeneBio). Interesting protein spots are identified and further characterized in our mass spectrometry facility.

Prices

Fluorescent dyes, DIGE kits or Refraction-2D kits will be charged extra.

Contact person:

Dr. Steffen Ohlmeier

Faculty of Biochemistry and Molecular Medicine

University of Oulu

Aapistie 7B (office F009, lab F004)

90230 University of Oulu, Finland

tel. +358-(0)294 481209

e-mail: Steffen.Ohlmeier (at) oulu.fi

Mass analysis is an indispensable step in the profound analysis of biomolecules, since nearly all modifications that happen to biomolecules are reflected in a change of their molecular weight, which can be measured with high sensitivity and accuracy with different mass spectrometers. In addition to “simple” molecular weight measurements, molecules can be fragmented within the mass spectrometer to provide structural information that allows identification and detailed characterization. Aim of the Biocenter Oulu core facility is to develop applications for mass spectrometry in biomedical research and to provide mass spectrometry service. Next to the routine mass spectrometry service, the facility is focused on identification and in depth characterization of proteins and their PTMs. The spectrum of potential samples covers purified proteins, complex proteomic samples, and proteins from 2D gel-based proteomics. General services include protein identification from protein mass finger prints or MSMS-spectra (sequencing), characterization of PTMS, accurate mass measurements of full length proteins, top down sequencing of proteins. Under development are methods to offer routine service for label free quantification of proteomes and targeted proteins.

The core facility is situated on the Kontinkangas campus in the Faculty of Biochemistry and Medical Biochemistry (F-block, Aapistie 7 room F041A).

The major instruments of the core facility are a Thermo Orbitrap Fusion Lumos Tribrid, a Waters Synapt G2 HDMS Q-Tof and a Bruker UltrafleXtreme MALDI Tof/Tof. These cutting edge instruments can cover most applications in biological mass spectrometry.

Contact person:

Dr. Ulrich Bergmann

Faculty of Biochemistry and Molecular Medicine

University of Oulu

PL 3000, 90014 University of Oulu, Finland

tel. +358-(0)294 481209

e-mail: Ulrich.Bergmann (at) oulu.fi

This facility offers expertise and service for the label-free and in solution study of biomolecular interaction, protein conformation and stability analysis, as well as amino acid analysis of proteins and other biological samples. It is equipped with two surface plasmon resonance instruments, one isothermal titration calorimeters, one BioLayer interferometry instrument, one circular dichroism spectropolarimeters, one nanoDSF device, one thermofluor instrument and one HPLC for amino acid analysis. The devices are located in room F040A and L041, Faculty of Biochemistry and Molecular Medicine.

The equipment and service can be booked in iLab reservation system.

Detailed information about the different services and equipment

Contact person:

Dr. Hongmin Tu

Faculty of Biochemistry and Molecular Medicine

University of Oulu

Aapistie 7B, 90014 Oulu, Finland

tel. +358-(0)294 485821

e-mail: hongmin.tu(at)oulu.fi