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University of Gothenburg
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Our Research Support and Equipment

We offer research support including study design, protein identification, characterization of post-translational modifications e.g. phosphorylation and glycosylation, and protein-protein interaction analysis. Global quantitative analyses of differentially expressed proteins are available using Tandem Mass Tags (TMT).

The Proteomics Core Facility has experience of sample preparation of a range of species and material such as tissues, cells, isolated cellular compartments, pull-downs, other biological samples, or expressed proteins. High pH liquid chromatography fractionation prior to nLCMS is used for in-depth analysis and to study lower abundant proteins in complex biological samples.

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 nanoLC 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.

Relative quantitation with chemical labeling as Tandem Mass tag (TMT) is used to compare protein abundances between groups and is suitable for all biological samples. Metabolic labeling using Stable Isotope Labeling with Amino Acids in Cell culture (SILAC) is suitable for labeling of proteins in cell cultures.

Quantitative mass spectrometry studies often follow a sequential workflow where methods such as off-line fractionation by high pH liquid chromatography are performed to reduce sample complexity prior to nanoLC. Reduced sample complexity is of significant importance, as identification and quantification rates are directly proportional to sample complexity.

Relative quantification is performed using Proteome Discoverer or MaxQuant.

Tandem Mass Tags and Isobaric labeling

Relative quantitation with isobaric labeling is used to compare abundance of peptides and their corresponding proteins in multiple samples at MS/MS level. This methodology can also be applied to study post-translational modifications as phosphorylation between multiple samples.

The proteins are digested with trypsin, the resulting peptides in each sample are labeled with different versions of the isobaric tags and then combined into one sample. Identical peptides, derived from different samples, are indistinguishable in their intact form during analysis. However, upon fragmentation in the mass spectrometer, each peptide variant produces a unique reporter ion used for quantification. Only peptides unique for a specific protein are considered for quantification. This multiplexed quantification reduces the variation between samples in the experiment and allow fold-change as low as ± 10-20% to be considered as significant results.

Study design: A TMT (tandem mass) set contains up to 16 isobaric tags or labels (16-plex) allowing comparison of two to 16 samples in one experiment. Depending on the number of samples to compare, also 10-plex or 11-plex TMT sets can be used. Often, one of the samples is a reference pool to be able to do relative quantification between samples in many TMT sets (more than 16 samples). At least three biological replicates in each group are required for t-test comparisons. However, it is recommended to have as many replicates as possible to increase the power of the statistical analysis. Concerning study design, optimally use all labels within one TMT set (either 10, 11 or 16 samples or even 18, 20 or 30, and so forth).


Stable isotope labeling by amino acids in cell culture (SILAC) is a simple and straightforward approach for in vivo incorporation of 'light' or 'heavy' forms of amino acids into proteins for quantitative proteomics (Ong SE, et al, MCP, 2002).

In a SILAC experiment, differential labeling of two to three cell types can be performed where cells are grown with the natural amino acids,  13C6-lysine and 13C6-arginine or 15N213C6-lysine and 15N413C6-arginine. The samples are mixed, digested with trypsin and analyzed by nanoLC MS/MS. Since trypsin cleaves at the amino acids arginine and lysine all peptides will be labelled. The labeling does not affect the chemical properties of the peptides and they co-elute from the nanoLC column but they remain distinguishable by MS. The peptide peaks of the differentially labeled samples can be very accurately quantified relative to each other to determine the peptide and corresponding protein ratios. This methodology can also be applied to study post-translational modifications as phosphorylation between multiple samples.

Customers are required to perform the SILAC incorporation themselves. We offer consultation setting the experimental design. Prior to large-scale experiments, incorporation testing by nanoLC MS analysis must be performed to determine the incorporation efficiency.

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 and commonly require enrichment prior to mass spectrometric characterization.

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.


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 by the Swedish Research Council and is currently supported by the national BioMS infrastructure, where research project application can be submitted to receive partial financial support (


Phosphorylation is the addition of a phosphate group to serine, threonine, and tyrosine by a specialized protein family called protein kinases. The cell tightly controls the phosphorylation of proteins by an interplay with the de-phosphorylating enzymes (phosphatases), creating a fast, powerful, and transient mechanism to adapt to changes of the environment.

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 or SILAC.

Especially the treatment and harvest of samples for phosphoproteomics experiments requires attention. 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 the 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. Phosphorylation sites are detected by their additional mass of 80 Da on the modified amino acid. Quantification is performed on reporter ions created during fragmentation in the mass spectrometer. Only unique peptides for a protein are considered for quantification.

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 into the database and used in the database matching. Common post-translational modifications are ubiquitination, methylation and acetylation.

Immunoprecipitation and pull-down experiments

Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody. The protein of interest is isolated and enriched from a sample containing many thousands of different proteins.

Pull-down experiments or affinity purifications are similar to immunoprecipitation, but instead of an antibody a bait (e.g. protein, RNA, peptide) is used. The bait is tagged and captured on an immobilized affinity ligand specific for the tag. In protein-interaction analysis, the protein or protein-complexes that interact with the protein or the bait are identified.

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.


Mass spectrometers

  • Thermo Orbitrap Fusion Lumos Tribrid
  • Thermo Orbitrap Fusion Tribrid
  • Thermo QExactiveHF
  • Thermo QExactive
  • Thermo Orbitrap Elite

Chromatographic systems

  • online: Thermo Easy nLC1200 systems
  • offline high pH UPLC fractionation: Thermo Dionex Ultimate 3000