Quantification with LC/MSBiologics
To quantify proteins from biological matrixes, larger proteins (>10-20 kDa) are analysed using digestion and surrogate peptide approach, whereas smaller proteins and peptides can be quantified as intact with UV with or without MS-detection, although the method of choice depends also on the sample matrix.
Protein quantification using surrogate peptide approach with LC/MS starts with bioinformatics, instead of antibody sourcing or development like in traditional immunoassays (e.g. ELISA). Therefore, the method development time can be significantly shorter compared to the antibody-binding based analytical techniques. The target protein sequence is first compared with the sample matrix proteome and the predicted peptides, unique for the target protein, are identified. Next, the target protein is enriched or the background proteome depleted using, for example, selective precipitation or solid phase extraction. This is followed by protein digestion, using sequence specific proteases, to liberate the predicted peptides from the target protein. The target protein quantification is then based on monitoring LC/MS signal of the unique target peptides.