Mass accuracy and sequence requirements for protein database searching.

The research article explores the role of high mass accuracy in mass spectrometric peptide mapping and database searching, with a focus on selected proteins, and how this can minimize false protein matches.

Research methods

• The proteins selected for this research were equine apomyoglobin and bovine alpha-lactalbumin. These were chosen because they present two extremes – apomyoglobin digests easily and produces few misleading masses, whereas alpha-lactalbumin generates many misleading masses under certain conditions.
• These proteins were put through a process of tryptic digestion and then analyzed via electrospray ionization on a high magnetic field (7 Tesla) Fourier transform mass spectrometer, which featured a mass accuracy of 1 part per million (ppm).
• The research explored the impact of accurate mass measurements in reducing false protein matches. This was tested by altering the mass error permitted in the search over a broad range (from 2 ppm to 500 ppm).
• Additionally, collision-induced dissociation was used to help identify an unassigned peak in the alpha-lactalbumin digest.

Key Findings

• For the easily digested apomyoglobin, very few masses were necessary to return valid protein matches. The mass error allowed in the search had little effect up to 500 ppm.
• In contrast, for alpha-lactalbumin, more mass values were needed, and low mass errors increased the search specificity.
• Lower mass errors, particularly below 30 ppm, were particularly useful for eliminating false protein matches when there were few mass values used in the search.
• Furthermore, even with a relatively high mass error, collision-induced dissociation was able to help in identifying proteins when digest conditions or modifications made digest peaks unidentifiable through a simple mass mapping search.
• An unassigned peak in the alpha lactalbumin digest provided sufficient data to identify the peak as a fragment from alpha-lactalbumin, thus eliminating numerous spurious proteins found in the peptide mass search.