Proteomics Unit, Viikki

Protein digestion in-gel or in-solution using a proteolytic enzyme, usually trypsin. Analysis is performed in data-dependent acquisition mode. 

For protein identification and quantification FragPipe is used as the default search engine. Databases can be customized to user´s needs. 

When proteins interact with other proteins, they form protein-protein interactions (PPI). PPIs are fundamental for the function of cells and consequently any living organism. Gene expression, cell growth, proliferation, apoptosis, and intercellular communication are among the many biological processes that are facilitated by proteins.

Samples are lysed, affinity-purified and digested into peptides using trypsin, followed by C18-desalting. Purified samples are subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.

For protein identification and quantification from data-dependent acquisition we use FragPipe as the default search engine. Databases can be customised to user´s needs.

For interactomics we recommend to have appropriate controls and considering adding biological and technical replicates for the analysis. 

Proteomes are sets of proteins produced in an organism, system, or biological context. For example, one can refer to the proteome of a species, e.g. the human proteome, or the proteome of an organ, e.g. the liver.

Besides overall understanding of human biology, proteomics has created opportunities to study proteins in diseases. Disease proteomics aim to understand how altered protein expression, structure and function can cause illness.

Depending on the specific needs of the project, the starting point for global proteomics analysis can be a piece of tissue, cell pellet or a lysate of known protein concentration. The sample is proteolytically digested to peptides, desalted and analyzed on a LC-MS/MS. 

Analysis can be performed in data-dependent or data-independent acquisition mode. For both approaches specific data processing workflows will be used. 

In addition, the analysis can be performed label-free or labelled. Label-free quantification determines the relative amount of proteins in two or more biological samples. Label-free quantification don´t use stable isotopes compounds to bind and label the protein. Labelled methods are used to compare protein or peptide abundance between samples. Spiking unlabeled samples with isotopically labeled peptides we can yield absolute quantitation for target peptides. Labelled protein quantification approach is more costly and time-consuming. Quantification methods for labeled analysis include ICAT, iTRAQ, ICPL, IDBEST, TMT, MeCAT, SILAC and TAILS.

Depending on the specific needs of the project, the starting point for phosphoproteomics analysis can be a piece of tissue, cell pellet or a lysate of known protein concentration. The sample is phosphoenriched with our in-house enrichment protocol, proteolytically digested to peptides, desalted and analyzed on a LC-MS/MS. The recommended acquisition mode is data-inpedenpent (DIA). For data analysis we use DIA-NN software.

We recommend to have appropriate controls and considering biological and technical replicates.