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The ability to match Top-Down protein sequencing (TDS) results by MALDI-TOF to protein sequences by classical protein database searching was evaluated in this work. Resulting from these analyses were the protein identity, the simultaneous assignment of the N- and C-termini and protein sequences of up to 70 residues from either terminus. In combination with de novo sequencing using the MALDI-TDS data, even fusion proteins were assigned and the detailed sequence around the fusion site was elucidated. MALDI-TDS allowed to efficiently match protein sequences quickly and to validate recombinant protein structures—in particular, protein termini—on the level of undigested proteins.
After Edman sequencers disappeared from the commercial instrument market in 2008, there was great interest in finding new methods for intact protein sequencing. MALDI Top-Down Sequencing (MALDI-TDS) is one such method (Figure 1) and was applied to this year’s Association of Biomolecular Resource Facilities-Edman Sequencing Research Group (ABRF-ESRG) 2009 research study. MALDI-TDS provided up to ~70 N-terminal and C-terminal sequence calls from a single MALDI-TDS spectrum of an intact protein. MALDI-TDS worked for N-terminally blocked proteins as well.
The ABRF-ESRG 2009 study samples—two proteins in the 35-kDa MW range—were analyzed. Protein (50 pmol) was prepared with super 2,5-dihydroxybenzoic acid on the MALDI target without any digestion steps. An Ultraflex III MALDI-TOF/TOF (Bruker Daltonics, Bremen, Germany) with a smart beam laser was used to acquire MALDI-TDS spectra by in-source decay analysis in reflector mode.
Proteins were identified from MALDI-TDS spectra with Mascot using “virtual precursor ions”. A new “MALDI-TDS” instrument was defined on Mascot 2.2 server (Matrix Science, Boston, MA, USA): comprising 1+, a, c, y, and z + 2 fragment ions for most specific scoring.
Retrieval of the IDed protein from Sample 2 into the BioTools 3.2 software (Bruker Daltonics) provided full assignment of G3P_RABIT (Figure 2). Sample 1, representing a fusion protein, required additional de novo sequencing using the dominant c-ions to read through the fusion site into the native ADH1_YEAST sequence.
Sample 2 was characterized as being pure (Figure 3). The G3P_RABIT sequence in SPROT appeared special with regard to Ala286, which is uncommon in other mammal sequences. Careful evaluation of the spectrum in Figure 4 provided evidence for a Ala286Asp error in the G3P_RABIT sequence.
ADH1_YEAST was identified as the native C-terminal of Sample 1, a His6-tag fusion vector as its N-terminal (Figure 5). Manual work with the spectrum established the sequence of the fusion site, plus it detected a point mutation ADH1_YEAST (His21Tyr) at sequence call 55 (Figure 6).