Automated de novo sequencing of proteins using the differential scanning technique

Despite the progress in genomic DNA sequencing de novo sequencing of peptid es is still required in a biological research environment since many experi ments are done in organisms whose genomes are not sequenced. A way to unamb iguously retrieve a peptide sequence from a tandem mass spectrum is to assi gn the correct ion type to the fragments. Here we describe a method which i mproves the specificity in y-ion assignment throughout the spectrum. The di fferential scanning technique requires that the peptides are partially O-18 labelled at their C-terminus and that two fragment spectra are acquired fo r each peptide, one selecting the O-16/O-18 isotopic cluster and a second f ragmenting only the O-18 labelled ions. When the spectra are acquired with a quadrupole time of flight mass spectrometer y-ions can be very specifical ly filtered from, the spectrum: using a computer algorithm. Partial or comp lete peptide sequences can be assigned: automatically simply by finding the most abundant series of fragments spaced by amino acid residue masses. Thi s method was used extensively in a project investigating vesicular transpor t in bovine brain cells. Human or mouse homologues to the bovine proteins w ere found in EST databases facilitating rapid cloning of the human homologu es.