MALDI Top-Down Sequencing: Calling N- and C-Terminal Protein Sequences with High Confidence and Speed

Abstract

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.

FIGURE 1.

Scheme of MALDI-TDS. Intense c-ions in MALDI-TDS spectra provide for direct N-terminal sequence assignment. Additional y (and z+2) ions call the C-terminal sequence at the same time. Spectrum acquisition takes seconds, and analysis may take only minutes.

MATERIALS AND METHODS

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.

FIGURE 2.

MALDI-TDS spectrum of the intact protein in ABRF-ESRG 2009 Sample 2. G3P_RABIT sequence assigned 68 N-terminal, 46 C-terminal calls. Database search using m/z 6086 as virtual parent ion provided a peptide score of 169.

RESULTS

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.

FIGURE 3.

Based on the “pure” MALDI-TDS spectrum, the purity of the protein in Sample 2 was assessed. No other signals than those of a, c, y, and z + 2 ions matching G3P_RABIT were present.

FIGURE 4.

MALDI-TDS spectrum from Sample 2 assigned to the C-terminal G3P_BOVIN sequence. The match indicates a sequence error in the G3P_RABIT sequence (A286D), which does not allow assignment of y47 and higher (see Figure 7).

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).

FIGURE 5.

MALDI-TDS analysis of Sample 1. N-terminal His6-β-Gal fusion vector and C-terminal ADH1_YEAST IDed by two Mascot searches in the NCBI database. De novo sequencing in FlexAnalysis (Bruker Daltonics) allowed reading through the fusion site.

FIGURE 6.

MALDI-TDS analysis of Sample 1. The fusion protein contains a point mutation, assuming ADH1_YEAST (His21Tyr) permitted the call of 70 aa residues from the N-terminus.

CONCLUSIONS

• Standard Mascot searches were used to call sequences from MALDI-TDS spectra of undigested proteins (Figure 7).

• 50–70 N-terminal sequence calls from undigested, 40 kDa proteins

• 30–50 C-terminal sequence calls from the same datasets as additional benefit from this technology

• For routine protein quality control work, MALDI-TDS is largely a push-button analysis that takes minutes.

FIGURE 7.

MALDI-TDS analysis summary. Sequences and covered calls (red) by MALDI-TDS from ABRF-ESRG 2009 Samples 1 (upper) and 2 (lower); mutations/DB errors are in circles. Approximately 70 sequence calls from N-terminal and 50 residues from C-terminal were obtained from either sample.