De novo sequencing of tryptic peptides

De novo sequencing has evolved to become a very useful tool for the complete elucidation of protein primary structures - especially in case of an unknown proteome. In a recent contest, MALDI-TOF/TOF MS has shown its huge potential for this task.

Mass spectrometry (MS) is the most important analytical technique for the assignment of protein sequences. New developments in this field now provide additional methods for those cases where protein identification fails since the protein comes from an organism with insufficient genomic reference information. Here, de novo sequencing combined with amino acid sequence similarity searches (BLAST) is a very promising approach.

The 2002 'Martinsried Contest'

Since de novo sequencing is expected to play a pivotal role, it was a focus of the European workshop 'Micro methods in Protein Chemistry' held at the Max-Planck-Institute for Biochemistry in Martinsried in Germany, June 2002. This meeting has become an established event, which reliably provides insight into the 'real-life' performance of new techniques in protein analysis: all manufacturers of mass spectrometers are invited to take part in an analytical contest, with the intention to objectively update the biotech community.

The task

The sequences of two unknown peptides were to be determined by MS/MS analysis from a 10 pmol sample. Bruker Daltonics participated with its two major proteomics systems, the ultraflex MALDI-TOF/TOF MS and the esquire3000 plus ESI-Ion Trap MS. The results achieved with the ultraflex TOF/TOF are reported in this article; the esquire results are reported elsewhere.

The equipment

In the standard TOF/TOF mode, fragment ions are generated during the MALDI ionisation step by applying high laser intensity (LID, laser-induced decomposition). The resulting LIDLIFT MS/MS spectra are dominated by 'backbone' b and y fragments and easily interpreted (Figure 1). Additionally, a-ions, neutral loss ions of NH3 (-17 Da) or H2O (-18 Da), internal i-ions and (yb)-ions are present. C-, x- and z-ions are not observed in LIFT MS/MS spectra.

Alternatively, the ultraflex allows the use of collision gas for fragmentation (CID). CID-LIFT spectra provide complementary structural information, as side chain fragmentation occurs here (N-terminal d- and C-terminal w-ions).

Data interpretation using the BioTools software package provides interactive tools for results visualisation. A dedicated software module supports de novo sequencing, which iterates, evaluates and provides ranking for possible sequence candidates.

Results

Peptide 1 gave a clear MS/MS spectrum with a distinguished base peak. Software assisted interpretation of this spectrum is shown in Figure 2, Figure 3, Figure 4 and Figure 5.

1. Collecting first sequence hints: The low-mass fragment ions allowed to predict the presence and absence of certain amino acid residues and identified R as C-terminus by the intensive y1-ion at m/z 175.
2. Filtered search: The acquired information is used as input for the de novo sequencing module, which calculates and displays a list of possible sequences.
3. Evaluating the proposed sequences: The proposed sequences are annotated to the MS/MS spectrum for visual comparison.
4. Differentiating Leucine and Isoleucine: As a last step, a CID-LIFT-MS/MS analysis of the peptide distinguishes Leucine from Isoleucine, based on a characteristic side chain fragment.

Conclusion

The submitted sequence proposal for peptide 1 was TDFEAAINTR. Peptide 2 was analysed in the same way, unambiguously yielding the sequence ALHSYPPPVMPR. Both sequences were found to be 100% correct. The results were obtained in a short period of time with a high degree of automation. The workflow was:

• Do the sequencing work first using simple LID-LIFT MS/MS data
• Interrogate additional CID-LIFT spectra afterwards, only to differentiate between L and I residues.

High energy CID spectra contain a lot more fragment ions, which are not simple backbone cleavage products and therefore do not provide straight-forward sequence information for de novo work, while LID-LIFT spectra show higher intensities of the significant b- and y-ions. However, CID-LIFT adds valuable information on specific questions as a powerful second step analysis tool.