Peptide de novo sequencing using 157 nm photodissociation in a tandem time-of-flight mass spectrometer.
It has previously been shown that photodissociation of tryptic peptide ions with 157 nm light in a matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF) mass spectrometer generates an abundance of x-type ions. A peptide de novo sequencing algorithm has now been developed to interpret these data. By combination of photodissociation and postsource decay (PSD) spectra, the algorithm identifies x-type ions and derives peptide sequences. The confidence of amino acid assignments is evaluated by observing complementary y-, v-, and w-type ions that provide additional constraints to sequence identification. In the analysis of 31 tryptic peptides from 4 model proteins, the algorithm identified 322 (or 90.7%) of the 355 amino acids and made only 3 incorrect assignments. The other 30 amino acids were not identified because specific needed x-type ions were not detected. Based on the observation of v- and w-type ions, 45 of 50 detected leucine and isoleucine residues were successfully distinguished and there was only one mistake. The remaining four residues were not distinguished because the corresponding v- and w-type ions were not detected. These de novo sequencing results translated into successful identification of proteins through homology searches. To evaluate the robustness of the present sequencing approach, a collection of 266 tryptic peptides from 23 model proteins were analyzed and then sequenced. A total of 167 peptides yielded sequence tags of 5 or more residues. In 5 peptides, 1 or 2 residues were incorrectly assigned.
Zhang
Liangyi
L
Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405, USA.
Reilly
James P
JP
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D014357:Trypsin / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism
D009211:Myoglobin; D010455:Peptides; D025801:Ubiquitin; D007532:Isoleucine; D045304:Cytochromes c; D014357:Trypsin; D007930:Leucine