Our Research Support and Equipment
We offer research support including study design, protein identification, characterization of post-translational modifications, and protein interaction analysis. Global quantitative analyses of differentially expressed proteins reflect the majority of our projects.
The Proteomics Core Facility has experience regarding preparation of a huge range of samples originating from tissues, cells, isolated cellular compartments, pull-downs, other biological samples, or expressed proteins. The facility is equipped with the latest high-resolution mass spectrometers.
We provide comprehensive data analysis through a variety of database search engines (Mascot, Sequest) and software packages (Proteome Discoverer, Byonics and MaxQuant).
Protein identification
Identification and determination of sequence coverage of a highly purified protein or identification of proteins in a complex mixture is a service provided by the Core Facility.
The proteins are typically cleaved by trypsin into peptides (in-gel, in-solution or filter-aided sample preparation (FASP)) and analyzed by nano LC MS-MS. The detected peptides are matched against the sequence of the purified protein or against a publicly available protein database as SwissProt using Proteome Discoverer.
For complex samples an optional off-line fractionation step as High pH liquid chromatography can be included in the workflow to facilitate identification of low abundant proteins and to improve the number of protein identifications.
Samples are either in-solution, as a protein pellet or a protein bands from a gel separation. Avoid the use of detergents as NP40, Triton X100 and Tween in any buffers since their presence significantly interferes with the downstream MS analysis.
Gel cuts are submitted in a tube and covered by 3% acetic acid in ultra-pure water. We prefer Coomassie blue-visible protein bands, in case of silver staining use an MS compatible silver staining method (e.g. Pierce Silver Staining for Mass Spectrometry Kit, Thermo #24600)
Quantitative Mass Spectrometry
Quantitative proteomics includes powerful global discovery or targeted methods to analyze and understand protein changes in cell, tissue or other biological material.
Discovery proteomics can provide critical insights for our understanding of the global protein expression as well as modification profiles (e.g. phosphorylation) underlying the molecular mechanisms of biological processes and disease states. Technological advances in instrumentation have increased the number of proteins that can be covered in a single sample up to several thousands.
Changes of protein expression, phosphorylation patterns, or interaction partners can be identified using relative quantification. Quantitative proteomic studies often follow a sequential workflow where methods such as off-line fractionation by high pH liquid chromatography is performed to reduce sample complexity prior to nano LC-MS. Reduced sample complexity extremely important, since identification and quantification rates are directly proportional to sample complexity. Normalisation and relative quantification of raw data originating from nano LC-MS are performed using Proteome Discoverer or MaxQuant.
Post-translational modifications
Protein post-translational modifications (PTMs) are chemical modifications that regulate cellular activity and influence the molecular mechanisms of biological processes and disease states. The main challenge studying PTMs is that they are low abundant. PTMs of purified proteins or a highly expressed proteins can be characterised by mass spectrometry directly, while PTMs in complex samples have to be enriched for prior to the MS-analysis.
Glycosylation
Protein glycosylation is one of the most common and the most complex post-translational modification.
Glycobiosynthesis is a non-template driven process, where complex structures (branched and linear) are produced from monosaccharide building blocks by multiple and competitive enzymatic actions. This complexity of the glycobiosynthesis presents an analytical challenge for studies of protein glycosylation.
Due to the structural complexity and the diversity of glycans, protein glycosylation studies are traditionally carried out on the released glycans. The analysis of released glycans (glycomics) provides detailed quantitative and structural glycan data, however, it lacks their protein site-specific information and glycoproteomics should be applied for studies that require characterization of the glycans on the proteome level.
Analogous to proteomics, glycoproteomics is based on advanced Mass Spectrometry technologies optimized for either global or directed analysis of glycoproteins in biological samples. Depending on the selected methodologies, glycoproteomics is capable to provide:
- identification and quantification of the proteins carrying glycosylation
- identification of the glycosylation sites and site occupancies
- site-specific characterization of the glycan structures (monosaccharide compositions and micro heterogeneity)
Protein glycosylation studies often demand a complex design and addressing even one of the above listed questions would frequently require application of multiple advanced technologies for sample preparation and analysis as well as an in-depth knowledge of systems biology and medicine. Therefore, we encourage you to come and discuss with us about your glycoproteomic projects to ensure optimal design in respect to the available material and your biological question.
Glycoproteomics has been identified as an important research field and is currently supported by the national BioMS infrastructure (www.bioms.se) as well as SciLifeLab (www.scilifelab.se).
Phosphorylation
Phosphorylation is one of the most common post-translational modification. Cells tightly control phosphorylation of proteins by an interplay beweeen kinases and phosphatases, creating a fast, powerful, and transient mechanism adapting cellular processes in response to stimuli.
Phosphoproteomics is the study of changes in the protein phosphorylation pattern due to different treatments or conditions. Quantification of phosphorylation sites among different conditions is often accomplished using labeling with isobaric tags (see quantitative MS).
Especially the treatment and harvest of samples for phosphoproteomics experiments require attention; therefore please contact the Proteomics Core Facility already during study design.
Phosphoprotein samples are lysed in the presence of phosphatase inhibitors and digested with trypsin. The resulting peptides are labeled with different versions of isobaric tags, and combined into one sample. A small aliquot is used for quantitative proteomics in order to check the protein level background, whereas the majority is subjected to phosphopeptide enrichment. Typically, (phospho)peptides originating from whole cell lysates are pre-fractionated to reduce complexity. Phosphorylation sites are detected by their additional mass of 80 Da on the modified amino acid. Quantification is performed on TMT reporter ions created during fragmentation in the mass spectrometer.
Other modifications
Any modification of proteins can be studied as long as the exact composition of the modification is known. The mass shift of the modification is entered manually into the database and used in the database matching. Common post-translational modifications are ubiquitination, methylation and acetylation.
Interaction or network analyses
Proteins rarely act alone, and their functions are regulated by interacting partners. Protein-protein interaction (PPIs) databases provide known and predicted protein-protein interaction networks.
Immunoprecipitations are performed using antibodies for protein-of-interest isolation; whereas affinity purifications and pull-down experiments are done using a tagged bait like RNA or peptides.
There are several critical sample preparation steps that affect the downstream MS-analysis and they must be considered before performing the experiment. Please contact the Core Facility to discuss the project prior to sample preparation.
The sample will be concentrated and digested using filter aided sample preparation (FASP). Tryptic digested samples will then be analyzed with nanoLC MS/MS followed by matching against a publicly available protein database as SwissProt using Proteome Discoverer for protein identification.
Optimization of protocol
Detailed protocols and guidance for setting up an IP/pull down experiment are provided by the manufacturer of the beads. In order to optimize experimental conditions, work initially on a small scale. For the final MS-analysis it is important to do a large scale preparation.
We recommend that you optimize your IP by monitoring the efficiency with Western blotting (WB) against your protein. To determine the quality of IP/pull-down and amount of protein in the sample (protein of interest, background proteins and antibody) SDS-PAGE and Coomassie staining are required. Image of WB and Coomassie-stained gel are send together with the sample submission form, when you hand-in the samples to the Proteomics Core Facility.
To reduce the background elute using mild conditions. SDS in elution buffer will give massive background, since non-specifically bound proteins will be eluted together with target proteins. In IP experiments, it is important to cross-link the antibody to the beads. Both background proteins and antibody in the eluate will obstruct identification of the proteins in the complex. PEG-containing detergents as NP40, TritonX100, and Tween should be avoided or used in very low concentrations.
The researcher is responsible for the IP/pull-down optimization and procedure and that it has been performed in a MS-compatible way.
Equipment
Mass spectrometers
- Bruker timsTOF SCP
- Bruker timsTOF HT
- Thermo Orbitrap Eclipse Tribrid
- Thermo Orbitrap Fusion Lumos Tribrid
- Thermo Orbitrap Fusion Tribrid
- Thermo Orbitrap Exploris 480
- Thermo QExactiveHF
- Thermo Orbitrap LTQ
Chromatographic systems
- online: Thermo Easy nLC1200 systems
- online: Evosep One nLC systems
- offline: high pH UPLC fractionation: Thermo Dionex Ultimate 3000