Top-down sequencing of proteins combines information from measurements of intact protein molecular weight with fragmentation from a variety of dissociation techniques. For known proteins, fragments can be compared against the putative sequence to localize modifications. The difficulty of this approach increases if the nature of the modification is either unknown or occurs in the presence of other modifications or mutations.

Sequence verification of either the N or C termini of proteins is quite useful for this type of analysis and can concurrently verify the protein identification and localize modifications to a specific terminus. Without chemical modification, intact protein dissociation methods generally cannot localize fragments to either side of the protein without using sequence information.

Our experiments test the effectiveness of a new approach that combines CAD in the ion funnel source region of a hybrid Qq-FTICR followed by fragment isolation in the Q-region and ECD in the ICR cell. Since ‘b' type ions are known to lose a CO group from the C terminus during ECD, we can quickly generate domain assignments based on a 28 Da neutral loss tag without using sequence information. Observation of the 28 Da shift is used as a tag to indicate a particular fragment originates from the N terminus of the intact protein. A statistical treatment of this methodology will be reported as well as several examples relevant to biological interest.