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Proteomics by Bottom-up

nanoUPLC
The PIXANIM platform offers different analytical strategies to identify, quantify and structurally characterize proteins through the analysis of peptides obtained after proteolytic digestion.

A bottom-up proteomic approach consists of analysing proteolytic peptides from purified proteins or complex mixtures by mass spectrometry. This approach is the most widely used in proteomics to identify proteins but also to quantify them or to characterise their post-translational modifications (phosphorylation, methylation, acetylation, etc.).
Proteins are digested by an enzyme, most often trypsin, to obtain a mixture of peptides. The latter are separated by a liquid nano-chromatography system and then analysed online by a tandem mass spectrometer (nanoLC-MS/MS). The objective is to measure the mass of the peptides and then to select them sequentially to fragment them in order to obtain structural information (sequence, location and nature of the modifications). These data are then compared with databases (e.g. UniprotKB, NCBInr) in order to identify the proteins and characterise their post-translational modifications.
When the proteins are digested in-solution and analysed by nanoLC-MS/MS, this is called a shotgun-type strategy.
When proteins are digested in-gel (SDS-PAGE 1D or 2D) and analysed by nanoLC-MS/MS, it is called a GeLC-MS/MS type strategy. This strategy has many advantages both on the preparation of the peptide sample (peptide extract devoid of all reduction/alkylation reagents and peptides of a size adapted to MS/MS) and on the complexity of the analysis (reduction of heterogeneity through prior separation in SDS-PAGE 1D or 2D gel).
PIXANIM offers you bottom-up proteomics for approaches :

Global approach :   

  • Reference cards (inventory) of a given proteome or sub-proteome from protein/peptide extracts: Identification of proteins from semi-complex mixtures by monodimensional liquid nanochromatography coupled to a tandem mass spectrometer (LC-1D-MS/MS, RP columns).
  • Identification by de novo sequencing (partial or total peptide sequences by MS/MS) of endogenous peptides.
  • Search for specific biomarkers through differential and quantitative analyses (different physiological states, healthy vs pathological, pharmacological effects...). Differential analyses can be carried out with iTRAQ labelling or without Label free type labelling using 2 complementary quantitative methods: by "spectral counting" and by XIC (Extracted Ion Chromatogram).

Targeted approach :

  • Study of protein polymorphism with the characterisation and localisation of post-translational modifications (phosphorylation, methylation, acetylation, pyroglutamic acid, glycosylation site, prenylation, etc.). In this context, we can proceed to an enrichment of peptides carrying in particular a post-translational modification (e.g. HILIC, IMAC, TiO2 for phosphopeptides), use more adapted MS/MS methods (Neutral Loss- MS3, MSA (Multistage Activation) or use different fragmentation modes (CID, HCD), characterization of N-glycosylated sites after deglycosylation by PNGase.
  • Control of sample purity (recombinant proteins, synthetic peptides, purification monitoring, etc.).

Analyses by mass spectrometry are accompanied by the exploitation and interpretation of results, advice and assistance with publication.