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.
The primary structure of a 13.6 kDa single heavy chain camelid antibody (V(H)H) 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 T(3)-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 V(H)H sequences. The monoisotopic molecular weight of the V(H)H was determined by ultrahigh-resolution orthogonal electrospray (ESI)-TOF analysis and found to be 13 610.6066 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.
%0 Journal Article
%1 resemannTopdownNovoProtein2010
%A Resemann, Anja
%A Wunderlich, Dirk
%A Rothbauer, Ulrich
%A Warscheid, Bettina
%A Leonhardt, Heinrich
%A Fuchser, Jens
%A Kuhlmann, Katja
%A Suckau, Detlev
%C United States
%D 2010
%J Analytical chemistry
%K Analysis Antibodies/*chemistry,Spectrometry Chains/*chemistry,Sequence Desorption-Ionization/*methods,to_read Heavy Immunoglobulin Laser Mass Matrix-Assisted Protein,Single-Chain
%N 8
%P 3283--3292
%R 10.1021/ac1000515
%T 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.
%V 82
%X The primary structure of a 13.6 kDa single heavy chain camelid antibody (V(H)H) 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 T(3)-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 V(H)H sequences. The monoisotopic molecular weight of the V(H)H was determined by ultrahigh-resolution orthogonal electrospray (ESI)-TOF analysis and found to be 13 610.6066 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 $>$13 kDa has been established by mass spectrometric top-down sequencing.
@article{resemannTopdownNovoProtein2010,
abstract = {The primary structure of a 13.6 kDa single heavy chain camelid antibody (V(H)H) was determined by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) top-down sequence analysis. The majority of the sequence was obtained by mass spectrometric de novo sequencing, with the N-terminal 14 amino acid residues being determined using T(3)-sequencing and database interrogation. The determined sequence was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a tryptic digest, which also provided high-energy collisionally induced dissociation (CID) data permitting the clear assignment of 3 of the 14 isobaric Leu/Ile residues. Five of the 11 Leu/Ile ambiguities could be resolved by homology comparisons with known V(H)H sequences. The monoisotopic molecular weight of the V(H)H was determined by ultrahigh-resolution orthogonal electrospray (ESI)-TOF analysis and found to be 13 610.6066 Da, in excellent agreement with the established sequence. To our knowledge, this is the first time that the entire primary structure of a protein with a molecular weight {$>$}13 kDa has been established by mass spectrometric top-down sequencing.},
added-at = {2024-05-17T13:01:35.000+0200},
address = {United States},
author = {Resemann, Anja and Wunderlich, Dirk and Rothbauer, Ulrich and Warscheid, Bettina and Leonhardt, Heinrich and Fuchser, Jens and Kuhlmann, Katja and Suckau, Detlev},
biburl = {https://www.bibsonomy.org/bibtex/27b077035b1e7c12c4e65ce7ab7e46e40/warscheidlab},
doi = {10.1021/ac1000515},
interhash = {9908142386328244368cce24717b4b48},
intrahash = {7b077035b1e7c12c4e65ce7ab7e46e40},
issn = {1520-6882 0003-2700},
journal = {Analytical chemistry},
keywords = {Analysis Antibodies/*chemistry,Spectrometry Chains/*chemistry,Sequence Desorption-Ionization/*methods,to_read Heavy Immunoglobulin Laser Mass Matrix-Assisted Protein,Single-Chain},
langid = {english},
month = apr,
number = 8,
pages = {3283--3292},
pmid = {20329751},
timestamp = {2024-05-17T13:01:35.000+0200},
title = {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.},
volume = 82,
year = 2010
}