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Top-Down de Novo Protein Sequencing of a 13.6 kDa Camelid Single Heavy Chain Antibody by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight/Time-of-Flight Mass Spectrometry
| Type: | Application |
Scientific Paper |
Expression Proteomics |
| Number: | Technology |
10.1021/ac1000515 |
MALDI-TOF, UHR-TOF |
| Year | Products |
2010 |
ultrafleXtreme, Compass 1.3, Bruker Easy-nLC, maXis, solariX |
| Author | |
Anja Resemann†, Dirk Wunderlich†, Ulrich Rothbauer‡, Bettina Warscheid§, Heinrich Leonhardt‡, Jens Fuchser†, Katja Kuhlmann§ and Detlev Suckau*† Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany, Department of Biology and Center for Integrated Protein Science, Ludwig Maximilians University Munich, Grosshaderner Strasse 2, 82152 Planegg-Martinsried, Germany, Medizinisches Proteom-Center, Ruhr-Universitaet Bochum, Universitaetsstrasse 150, 44780 Bochum, Germany, and Clinical & Cellular Proteomics, Medical Faculty and Center for Medical Biotechnology, Duisburg-Essen University, 45117 Essen, Germany |
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| Reference | |
Anal. Chem., Article ASAP DOI: 10.1021/ac1000515 |
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Abstract |
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The primary structure of a 13.6 kDa single heavy chain camelid antibody (VHH) was determined by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) top-down sequence analysis. The majority of the sequence was obtained by mass spectrometric de novo sequencing, with the N-terminal 14 amino acid residues being determined using T3-sequencing and database interrogation. The determined sequence was confirmed by liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis of a tryptic digest, which also provided high-energy collisionally induced dissociation (CID) data permitting the clear assignment of 3 of the 14 isobaric Leu/Ile residues. Five of the 11 Leu/Ile ambiguities could be resolved by homology comparisons with known VHH sequences. The monoisotopic molecular weight of the VHH was determined by ultrahigh-resolution orthogonal electrospray (ESI)-TOF analysis and found to be 13
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610.6066 Da, in excellent agreement with the established sequence. To our knowledge, this is the first time that the entire primary structure of a protein with a molecular weight >13 kDa has been established by mass spectrometric top-down sequencing.
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