Proteomics by Bottom-up

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.).

The proteins are digested by an enzyme, usually trypsin, to obtain a mixture of peptides. The peptides are separated by a nano-liquid chromatography (nanoLC) system and an ion mobility module before being analysed online by a high-resolution tandem mass spectrometer (nanoLC-MS/MS). Typically, we use DDA (Data Dependent Acquisition) methods. The aim is to measure the mass of peptides and then select them sequentially to fragment them in order to obtain structural information (sequence, location and nature of modifications). These data are then compared with databases (e.g. UniprotKB, NCBInr) to identify proteins and characterise their post-translational modifications. Data independent acquisition (DIA) methods can also be applied to models of interest. In this particular case, several precursors are fragmented together and the data is interpreted on the scale of the fragmentation spectra using dedicated tools.

When proteins are digested in-solution and analysed by nanoLC-MS/MS, this is referred to as a shotgun strategy. This strategy is generally preferred. However, proteins can also be digested in-gel (1D or 2D SDS-PAGE) and peptide extracts analysed by nanoLC-MS/MS, a strategy known as GeLC-MS/MS. This strategy has the advantage of reducing the complexity of protein samples.

PIXANIM offers bottom-up proteomics approaches:

Global approaches :

• Deep proteome: Reference map, the most exhaustive inventory possible of a given proteome or sub-proteome (sub-cellular fractionation) from protein/peptide extracts: Identification of proteins from complex mixtures by one-dimensional liquid nanochromatography coupled with ion mobility and a high-resolution tandem mass spectrometer (LC-1D-MS/MS, RP columns).
• Search for specific biomarkers using differential and quantitative analyses (different physiological states, healthy vs pathological, pharmacological effects, etc.). Differential analyses are carried out essentially without labelling (Label free) using quantitative methods such as XIC (Extracted Ion Chromatogram)
• µproteomics and single cell proteomics (SCP): the aim is to apply proteomics to rare and precious biological models with limited quantities of material (≤ qq pg or ng of peptides/injection) for fine cellular phenotyping, on an individual scale, in order to limit variability (inter-individual) and gain statistical power during comparative analyses.

Targeted approaches :

• 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.