Extraction
All types of biological samples are analyzable by mass spectrometry provided that proteins can be extracted from them.
Our staff are experts in extracting proteins from a wide variety of samples (tissues, cells, biological fluids, culture media, etc.) and organisms (human, mammalian, plant, microorganisms, etc.).
We use mechanical extraction methods (grinding, sonication, bead beating, etc.) or chemical methods (detergents, solvents, etc.).
We adapt our protocols to your project.
After extraction, proteins are denatured, usually by heating, and we also proceed with reduction and alkylation of disulfide bridges to fully unfold the proteins.
Enzymatic Digestion
The type of analysis we perform is called “Bottom-Up Proteomics.” This strategy involves digesting the extracted proteins with an enzyme. The resulting peptides are then injected into the mass spectrometer. In most cases, we use trypsin, which cleaves after basic amino acids (lysine and arginine), thus generating peptides of suitable size and easily ionizable in the instrument’s source.
It is also possible to directly digest proteins present in an SDS-PAGE gel band or immobilized on beads (immunoprecipitation, pull-down).
Protein or Peptide Assay
To verify if your samples contain sufficient proteins for analysis, we can perform protein assays after extraction (Bradford method) or peptide assays after digestion (nanodrop assay at 205nm). This step also allows for analysis normalization by injecting identical amounts of different samples into the mass spectrometer.
Peptide Purification
The peptides must then be purified before injection. For this purpose, we use Solid Phase Extraction (SPE) method on C18 beads, which retain peptides based on hydrophobicity.
Other Preparations
Depending on the desired analysis type, it is also possible to perform other sample preparation steps:
- Concentration by ultrafiltration
- Post-translationnal modifications (PTMs) enrichment
- Deglycosylation
- TMT labeling
- High pH fractionation
- Etc.