Peptide de Novo Sequencing Using 157 nm Photodissociation in a Tandem Time-of-Flight Mass Spectrometer

摘要

It has previously been shown that photodissociation ofntryptic peptide ions with 157 nm light in a matrix-assistednlaser desorption/ionization (MALDI) tandem time-of-flightn(TOF) mass spectrometer generates an abundance ofnx-type ions. A peptide de novo sequencing algorithm hasnnow been developed to interpret these data. By combinationnof photodissociation and postsource decay (PSD)nspectra, the algorithm identifies x-type ions and derivesnpeptide sequences. The confidence of amino acid assignmentsnis evaluated by observing complementary y-, v-, andnw-type ions that provide additional constraints to sequencenidentification. In the analysis of 31 tryptic peptidesnfrom 4 model proteins, the algorithm identified 322n(or 90.7%) of the 355 amino acids and made only 3nincorrect assignments. The other 30 amino acids were notnidentified because specific needed x-type ions were notndetected. Based on the observation of v- and w-type ions,n45 of 50 detected leucine and isoleucine residues werensuccessfully distinguished and there was only one mistake.nThe remaining four residues were not distinguishednbecause the corresponding v- and w-type ions were notndetected. These de novo sequencing results translated intonsuccessful identification of proteins through homologynsearches. To evaluate the robustness of the presentnsequencing approach, a collection of 266 tryptic peptidesnfrom 23 model proteins were analyzed and then sequenced.nA total of 167 peptides yielded sequence tagsnof 5 or more residues. In 5 peptides, 1 or 2 residues werenincorrectly assigned.