Besides gel electrophoresis, it is also possible to analyze samples in a gel-free manner. This is preferred when no immunoreactivity experiments are necessary and identification of the sample as a whole is the experimental aim. In more complex samples, an extra separation step (strong cation exchange, high pH) can be included in order to obtain different fractions (= multidimensional chromatography). Each fraction is then analyzed by LC-MS/MS. This setup will lead to more identifications in complex protein mixtures.
Advantages of gel-free proteomics
- Little sample loss
- Deeper protein coverage with multidimensional chromatography
- Gold standard for fishing experiment
Workflow
You provide us with your protein(mixture) of interest, preferable with a known amount of proteins. The composition of the buffer from your protein mixture should be mentioned. Detrimental for LC-MS/MS is the presence of salt in your protein mixture. The proteins are first digested into peptides with an enzyme such as trypsin. Other enzymes can be used as well. The customer can decide whether only one ion of multiple dimensions is preferred, such as high pH reversed phase (RP) or strong cation exchange (SCX).