is the sum of the amino acid residue masses (see Table II) that are contained within the fragment ion. is the mass of the C-terminus (e.g., 17.0027 Da for unmodified peptides and 16.0187 Da for amidated C-terminus). is the mass of the N-terminus (e.g., 1.0078 Da for unmodified peptides and 43.0184 Da for acetylated N-terminus). Table I Calculating the masses of positively charged fragment ions. In other words, a high quality MS/MS spectrum is one that is sequenceable. Computer programs that perform de novo sequencing can provide insight into the numbers of high quality unidentified MS/MS spectra for which a plausible peptide sequence can be deduced. Likewise, autosampler carryover and contamination can lead to the collection of numerous tandem mass spectra that are from a different species other than what was intended. One can learn about carbamylation, or other sample preparation issues that result in unexpected mass changes.
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Third, often the majority of high throughput LC-MS/MS tandem mass spectra are not matched in a database search, and it can be prudent to learn why. proteins identified on the basis of a single peptide identification from a database search). This could be particularly useful for “one-hit wonders” ( i.e. Second, given the significant differences between database searching and de novo sequencing approaches, any agreement between them should provide significant validation of the search result. For example, one of the authors of this manuscript (RJ) managed to show that a cell line was contaminated with a species of mycoplasma whose genome had not yet been sequenced (unpublished results, because no one would report on their contaminated cell lines). There are quite a few unsequenced genomes remaining ( i.e. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 1).
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This tutorial will present background important to understanding de novo sequencing, suggestions on how to do this manually, plus descriptions of computer algorithms used to automate this process and to subsequently carryout homology-based database searches. Homology searches are particularly useful for the study of organisms whose genomes have not been sequenced. However, these sequence tags can then be searched in a sequence database to identify the exact or a homologous peptide. Although powerful, de novo sequencing often can only determine partially correct sequence tags because of imperfect tandem mass spectra. Such analyses have traditionally been performed manually by human experts, and more recently by computer programs that have been developed because of the need for higher throughput. In proteomics, de novo sequencing is the process of deriving peptide sequences from tandem mass spectra without the assistance of a sequence database.