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Automated de novo sequencing of proteins using the differential scanning technique
Author(s) -
UttenweilerJoseph Sandrine,
Neubauer Gitte,
Christoforidis Savvas,
Zerial Marino,
Wilm Matthias
Publication year - 2001
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200104)1:5<668::aid-prot668>3.0.co;2-s
Subject(s) - peptide , dna sequencing , tandem mass spectrometry , computational biology , peptide sequence , biology , genome , mass spectrometry , chemistry , genetics , gene , biochemistry , chromatography
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 18 O labelled at their C ‐terminus and that two fragment spectra are acquired for each peptide, one selecting the 16 O/ 18 O isotopic cluster and a second fragmenting only the 18 O 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.