MALDI-ISD is a useful technique for N- and C-terminal sequencing of intact proteins. MALDI is commonly classified as a soft ionisation technique where laser powers close to threshold values generate intact protonated peptide and protein Ions. In the case of peptides, increasing the laser fluence results in unimolecular decomposition in the time-of-flight region – post-source decay (PSD). However, in the case of proteins, fragmentation can occur in the MALDI plume prior to ion extraction from the ion source i.e. in-source decay. Typically, protein identification is achieved by subjecting the protein(s) of interest to enzymatic digestion followed by MS/MS sequencing of the resulting peptides in a bottom-up approach. In contrast, MALDI-ISD is referred to as a top-down approach as sequencing is performed on the intact protein, thereby eliminating the lengthy digestion step.

Further benefits of the top-down approach are found in the analysis of post-translational modifications (PTMs) as even labile PTMs (that may be lost in a bottom-up approach) are retained thereby facilitating localisation during sequencing. MALDI-ISD does however have a limitation in the lower mass region where ISD fragment ions begin to overlap with chemical noise and matrix-related ions. A solution to this issue is to perform pseudo MS³ through MS/MS fragmentation of precursor ions created by ISD. However, using conventional MALDI-TOF-TOF instrumentation, this approach is limited to pseudo MS³. In this application, we report ISDⁿ: the extension of top-down sequencing to maximise sequence coverage of intact modified and unmodified proteins by using a unique MALDI-QIT-TOF for MALDI-ISD and pseudo MS⁵.

The advantages of ISDⁿ over a conventional ISD analysis of proteins are demonstrated using recombinant Tau protein. Tau has been reported to be involved in Alzheimer’s disease and is a good in vitro model for protein modification analysis. The unmodified protein along with the corresponding oxidised form, were used to demonstrate the ability of the described technique for the characterisation of modified proteins.

The ISDⁿ spectrum obtained for unmodified Tau contained both N-terminal cn- and bn-ions and C-terminal yn-ions. ISDⁿ analysis of oxidised Tau did not show significant losses of sulfenic acid (64 Da) indicating the modification remained intact during analysis. In addition to N-terminal cn-ions, c-ions showing both a mass shift of +16 Da: oxidised Met10 (sulfoxide form) and +32 Da: oxidised Met10 (sulfone form) were also detected. C-terminal yn-ions and y-ions showing a mass shift of +16 Da: oxidation of Met418 were also observed.