Retention Time Prediction and Protein Identification

In bottom-up proteomics, proteins are typically identified by enzymatic digestion into peptides, tandem mass spectrometry and comparison of the tandem mass spectra with those predicted from a sequence database for peptides within measurement uncertainty from the experimentally obtained mass. Although now decreasingly common, isolated proteins or simple protein mixtures can also be identified by measuring only the masses of the peptides resulting from the enzymatic digest, without any further fragmentation. Separation methods such as liquid chromatography and electrophoresis are often used to fractionate complex protein or peptide mixtures prior to analysis by mass spectrometry. Although the primary reason for this is to avoid ion suppression and improve data quality, these separations are based on physical and chemical properties of the peptides or proteins and therefore also provide information about them. Depending on the separation method, this could be protein molecular weight (SDS-PAGE), isoelectric point (IEF), charge at a known pH (ion exchange chromatography), or hydrophobicity (reversed phase chromatography). These separations produce approximate measurements on properties that to some extent can be predicted from amino acid sequences. In the case of molecular weight of proteins without posttranslational modifications this is straightforward: simply add the molecular weights of the amino acid residues in the protein. For IEF, charge and hydrophobicity, the order of t...
Source: Springer protocols feed by Protein Science - Category: Biochemistry Source Type: news