Protein Sequencing: Reverse Phase C18

Mass Spectrometry and Protein Sequencing

Most mass spectrometers that are used for protein sequencing are equipped with collision cells that are ideal for sequencing peptides that are under 4 KDa range. Mainly for this reason proteins need to be cut using proteases that will result in peptide fragments that on average are less than 4KDa. Once these peptides are generated they need to be fractionated before ionization into mass spectrometer. This is mainly to allow mass spectrometers time to analyze different peptides that are present in a mixture. The fractionation schemes can be carried out online or offline. The advantage of online schemes are that they require very little sample material which ultimately increases sensitivity of the system. For MALDI-TOF peptide sequencing, peptides will need to be fractionated offline for complex protein mixtures.

Reverse Phase (RP) C18 Resin Liquid Chromatography

The simplest fractionation strategy for low protein complexity samples is the reverse phase (RP) C18 resin liquid chromatography. In this separation scheme peptides bind the C18 resin based on their hydrophobicity. They are then eluted off the C18 resin using an organic reagent.