Maize seeds make researchers start from scratch
When mass spectrometry seems to fail
In the omics era, only high throughput protocols and technologies where data are analysed by sophisticated computer software make sense for protein identification. But even protein scientists have to start from the beginning sometimes. Faced with the absence of results when trying to identify maize seed proteins by mass spectrometry, researchers from the Plant Biochemistry Laboratory (and co-workers from Luxembourg) show that the typical protocol and software analysis may have to be re-designed in particular cases. The work is described in the Journal for Proteome Research.
Involved in a project aiming to relate maize seed content with the broa (a type of cornbread) quality of different maize varieties, researchers looked into the storage protein patterns. But this initial, apparently simple task turned out to be troublesome: despite the high number of proteins available in databases few proteins could be identified in the maize seeds by mass spectrometry.
Mass spectrometry-based proteomics uses trypsin to convert protein into analyzable peptide populations. The peptide fragmentation spectra are then searched against sequence databases, looking for matching peptide sequences. As it turns out, maize seed storage proteins have no or few trypsin cleavage sites and this made the typical trypsin-based method pretty much useless.
A different enzyme – chymotrypsin – gave clear and reproducible patterns but pose the difficulties of any atypical protocol. Surprisingly, researchers also found that keeping trypsin and manually analyzing the peptides was a good option when chymotrysin fails. While the canonical trypsin-generated peptides were absent, a set of reproducible peptides, usually disregarded as background noise, were produced. The required one-by-one analysis was to tedious to be effective but researchers were able to overcome this by adjusting of the peptide analysis software. Sometimes you really have to go back to basics.
Taking Advantage of Nonspecific Trypsin Cleavages for the Identification of Seed Storage Proteins in Cereals
Kjell Sergeant*, Carla Pinheiro*, Jean-François Hausman, Cândido Pinto Ricardo and Jenny Renaut
* Both authors contributed equally to this work