Intact mass measurement and sequence tag analysis

Electrospray ionisation generates multiple charge states of large molecules that are detectable in the lower m/z region of the MS-spectrum. A single charge state (up to ca. 20+) can be trapped in the linear ion trap (using an isolation width of 5-10) and transferred to the orbitrap for an accurate mass measurement. At a resolution of 100,000 the orbitrap can resolve the isotope envelope for the determination of the charge state. If the sequence of the protein is known the isotope composition of the most intense peak of the isotope cluster can be calculated and used for accurate mass determination. The error is significantly less than 1 Da for proteins up to 50 kDa. However, only purified proteins or low complexity samples are suitable for this approach.

Top-down approach: In a top-down approach proteins are identification by the mass determination of intact proteins and sequence tag analysis. Therefore a charge state of an intact protein is trapped in the linear trap and fragmented by CID (collision induced dissociation). A high resolution measurement of the fragment ions in the orbitrap allows the identification of doubly or triply charged daughter ions that can be used as precursors for an additional CID fragmentation in the linear trap. This MS/MS/MS analysis of intact proteins requires a reasonable amount of material but allows the identification of peaks of interest in more complex samples.

Electron transfer dissociation (ETD) is a more efficient fragmentation for larger molecules and higher charge states and is probably the best method for top-down protein analysis. Unfortunately ETD is yet not available at the Centre for Protein Research.