title: Database-independent Protein Sequencing (DiPS) Enables Full-lengthde NovoProtein and Antibody Sequence Determination.
abstract: Traditional "bottom-up" proteomic approaches use proteolytic digestion, LC-MS/MS, and database searching to elucidate peptide identities and their parent proteins. Protein sequences absent from the database cannot be identified, and even if present in the database, complete sequence coverage is rarely achieved even for the most abundant proteins in the sample. Thus, sequencing of unknown proteins such as antibodies or constituents of metaproteomes remains a challenging problem. To date, there is no available method for full-length protein sequencing, independent of a reference database, in high throughput. Here, we present Database-independent Protein Sequencing, a method for unambiguous, rapid, database-independent, full-length protein sequencing. The method is a novel combination of non-enzymatic, semi-random cleavage of the protein, LC-MS/MS analysis, peptidede novosequencing, extraction of peptide tags, and their assembly into a consensus sequence using an algorithm named "Peptide Tag Assembler." As proof-of-concept, the method was applied to samples of three known proteins representing three size classes and to a previously un-sequenced, clinically relevant monoclonal antibody. Excluding leucine/isoleucine and glutamic acid/deamidated glutamine ambiguities, end-to-end full-lengthde novosequencing was achieved with 99-100% accuracy for all benchmarking proteins and the antibody light chain. Accuracy of the sequenced antibody heavy chain, including the entire variable region, was also 100%, but there was a 23-residue gap in the constant region sequence.
authors.affiliation: From ‡The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot.
authors.forename: Alon
authors.initials: A
authors.lastname: Savidor
authors.affiliation: From ‡The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot.
authors.forename: Rotem
authors.initials: R
authors.lastname: Barzilay
authors.affiliation: From ‡The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot.
authors.forename: Dalia
authors.initials: D
authors.lastname: Elinger
authors.affiliation: the §Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel 76100.
authors.forename: Yosef
authors.initials: Y
authors.lastname: Yarden
authors.affiliation: the §Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel 76100.
authors.forename: Moshit
authors.initials: M
authors.lastname: Lindzen
authors.affiliation: From ‡The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Gabashvili
authors.affiliation: From ‡The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot.
authors.forename: Ophir
authors.initials: O
authors.lastname: Adiv Tal
authors.affiliation: From ‡The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot; yishai.levin@weizmann.ac.il.
authors.forename: Yishai
authors.initials: Y
authors.lastname: Levin
mesh_terms: D000906:Antibodies / Q000235:genetics; D002853:Chromatography, Liquid; D030562:Databases, Protein; D009211:Myoglobin / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D017421:Sequence Analysis; D020539:Sequence Analysis, Protein / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000235:genetics; D053719:Tandem Mass Spectrometry; D060749:alpha-2-HS-Glycoprotein / Q000235:genetics
chemical_list: D000906:Antibodies; D009211:Myoglobin; D060749:alpha-2-HS-Glycoprotein; D012710:Serum Albumin, Bovine