Sequencing is performed by High Resolution Electron Spray Tandem Mass Spectrometry (HR-ESI-MSn) and allows for the sequential determination of amino acids present in a peptide. However, sequencing is not limited to consecutive amino acid residues. Other moieties such as protecting groups, aliphatic modifications, cyclic structures, active site labels, and most other groups correlated with peptide chemistry are also covered during sequence determination.
The analyte is dissolved in an appropriate solvent containing a volatile acidic additive (e.g. formic acid). This mixture is infused into the electron spray ion source of the mass spectrometer. Here, droplets are formed which contain charged, quasi molecular ions. These droplets are further dried by a heated stream of nitrogen. The remaining, fine mist of particulates is transferred into the ion optics of the mass spectrometer and accelerated into a collision chamber. Here, the kinetic energy of the incoming analyte ions is dissipated by colliding with a collision gas, leading to collision induced decay (CID).
CID generates a myriad of peptide fragment ions which are subsequently transferred to the time-of-flight (TOF) analyser of the mass spectrometer. As a result, the weight of CID fragments is determined and correlated with the peptide sequence.
Typically, two abundant ion series are produced during CID. If the charge is retained on the N-terminal fragment of the peptide, the ion is classed a, b or c, depending on the exact location of the charge. If the charge is retained on the C-terminus, a series of x, y and z ions is detected. Usually, b and y ion series are used for peptide sequence determination, as those are of highest intensity and yield the best sequence coverage.
Usually a complete sequence determination is possible for peptides with a length of <20 amino acids. For longer peptides, the sequence determination of the N- and/or C-terminus might not be achievable.
Cyclic peptides need to be converted into their linear form prior to sequencing. For peptides containing one or multiple disulphide bonds, this is performed by selective reduction by either DTT or TCEP. In this case, the molecular weight of the peptide is determined prior and after reductive cleavage in order to confirm the cyclic structure of the analyte by a mass increase of 2 Da (per disulphide bridge). A chromatographic separation of linear and cyclic analytes could be performed by HPLC-MS prior to fragmentation.
Reversed phase HPLC-MSn is offered as a support service for protein sequence analysis, determination of molecular weight of peptides and identification of side products. In this hyphenated approach, the analyte mixture is subjected to chromatographic separation on high resolution column materials such as state of the art Core-Shell technology. Here, analytes are detected via diode-array-detector (DAD) and are subsequently subjected to HR-ESI-MSn analysis.