Automated de novo sequencing of proteins using the differential scanning technique.
Despite the progress in genomic DNA sequencing de novo sequencing of peptides is still required in a biological research environment since many experiments are done in organisms whose genomes are not sequenced. A way to unambiguously retrieve a peptide sequence from a tandem mass spectrum is to assign the correct ion type to the fragments. Here we describe a method which improves the specificity in y-ion assignment throughout the spectrum. The differential scanning technique requires that the peptides are partially 18O labelled at their C-terminus and that two fragment spectra are acquired for each peptide, one selecting the 16O/18O isotopic cluster and a second fragmenting only the 18O labelled ions. When the spectra are acquired with a quadrupole time of flight mass spectrometer y-ions can be very specifically filtered from the spectrum using a computer algorithm. Partial or complete peptide sequences can be assigned automatically simply by finding the most abundant series of fragments spaced by amino acid residue masses. This method was used extensively in a project investigating vesicular transport in bovine brain cells. Human or mouse homologues to the bovine proteins were found in EST databases facilitating rapid cloning of the human homologues.
Uttenweiler-Joseph
S
S
European Molecular Biology Laboratory, Heidelberg, Germany.
Neubauer
G
G
Christoforidis
S
S
Zerial
M
M
Wilm
M
M
D000465:Algorithms; D000595:Amino Acid Sequence; D004705:Endocytosis; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes; D010455:Peptides / Q000737:chemistry; D050600:SNARE Proteins; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins / Q000737:chemistry
D008565:Membrane Proteins; D010103:Oxygen Isotopes; D010455:Peptides; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins