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Top-Down Sequencing (TDS) was used in this work for the first time to establish a complete protein sequence de novo. The sample was made available for participation in a blind study in the course of the Martinsried Conference 2009 on “Micromethods in Protein Chemistry”. A pure ~14 kDa protein was received, which was not present in any database to prevent sequencing by simple annotation to sequences that can be searched. Samples were received as 120 pmol aliquots. About 10-20 pmol were prepared using sDHB or DAN matrix and run on a MALDI-TOF by in-source decay in reflector mode (reISD). They were directly analyzed by de novo TDS software and the entire protein sequence was manually assembled from 5 reISD and Tз-sequencing spectra. ReISD spectra called N- and C-terminal 70mer sequences up to ~8 kDa based on isotopically resolved fragments and 10-20 ppm mass accuracy. This provided an overlap of the N- and C-terminal calls and safe sequence determination also in the core part of the protein. The three N-terminal Leu residues were unequivocally assigned by heCID-TOF/TOF analysis of the tryptic N-terminal peptide after the sequence framework was established. The protein was identified based on the established sequence and a protein-BLAST search as a camelid variable heavy chain antibody (VHH domain “affibody”). The molecular weight was experimentally confirmed with a mass accuracy of 43 ppb, which permitted the exclusion of any artificial or in vivo protein deamidation and the assignment of the single disulfide bond as 100 % oxidized. It was demonstrated for the first time, that mass spectrometric top-down de novo sequencing of an entire protein in the 14 kDa range is possible. In the conclusion of the study, the top-down sequence assignment turned out being 100 % correct.