Mass accuracy and sequence requirements for protein database searching.
To elucidate the role of high mass accuracy in mass spectrometric peptide mapping and database searching, selected proteins were subjected to tryptic digestion and the resulting mixtures were analyzed by electrospray ionization on a 7 Tesla Fourier transform mass spectrometer with a mass accuracy of 1 ppm. Two extreme cases were examined in detail: equine apomyoglobin, which digested easily and gave very few spurious masses, and bovine alpha-lactalbumin, which under the conditions used, gave many spurious masses. The effectiveness of accurate mass measurements in minimizing false protein matches was examined by varying the mass error allowed in the search over a wide range (2-500 ppm). For the "clean" data obtained from apomyoglobin, very few masses were needed to return valid protein matches, and the mass error allowed in the search had little effect up to 500 ppm. However, in the case of alpha-lactalbumin more mass values were needed, and low mass errors increased the search specificity. Mass errors below 30 ppm were particularly useful in eliminating false protein matches when few mass values were used in the search. Collision-induced dissociation of an unassigned peak in the alpha-lactalbumin digest provided sufficient data to unambiguously identify the peak as a fragment from alpha-lactalbumin and eliminate a large number of spurious proteins found in the peptide mass search. The results show that even with a relatively high mass error (0.8 Da for mass differences between singly charged product ions), collision-induced dissociation can help identify proteins in cases where unfavorable digest conditions or modifications render digest peaks unidentifiable by a simple mass mapping search.
Green
M K
MK
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Johnston
M V
MV
Larsen
B S
BS
D000818:Animals; D001059:Apoproteins / Q000032:analysis*; D002417:Cattle; D016208:Databases, Factual; D005583:Fourier Analysis; D006736:Horses; D016247:Information Storage and Retrieval; D007768:Lactalbumin / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D009211:Myoglobin / Q000032:analysis*; D011786:Quality Control
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D007768:Lactalbumin