Our Research Support and Equipment

The proteins are typically cleaved by trypsin into peptides (in-gel, in-solution or bead-based SP3 prep) 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 or NCBI 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 can be submitted either in-solution, as a protein pellet or a protein band 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, please use an MS compatible silver staining method (e.g. Pierce Silver Staining for Mass Spectrometry Kit, Thermo #24600). 

Isotopically modified peptides with known concentrations are added during the sample preparation and are quantified in parallel with its non-modified counterparts to derive absolute peptide concentration. This targeted LCMS methods are called Single or Parallel Reaction Monitoring (SRM/PRM), highly sensitive and selective mass spectrometry methods.

In addition, the ProteomEdge technology allows absolute quantification of up to hundreds of selected proteins from biological and clinical samples. Available ProteomeEdge panels, as ApoEdge18, LiverEdge39, ThromboEdge11, ComplementEdge81, and DiscoveryEdge176, are ideal for biomarker validation and translational studies. 

During sample preparation, several recombinant human protein fragments containing heavy isotopes are introduced. These labeled fragments act as internal protein standards (qRePS), which are quantified in parallel to their non-labeled endogenous counterparts using Parallel Reaction Monitoring (PRM) LC–MS. 

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). 

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 relative proteomics analysis, TMT). The number of quantified peptides depends mainly on the cellular response and tissue type, but can cover 10,000-12,000 different phosphopeptides. 

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 global 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 of the isobaric tags created during fragmentation in the mass spectrometer.