title: Determination of isotopic labeling of proteins by precursor ion scanning liquid chromatography/tandem mass spectrometry of derivatized amino acids applied to nuclear magnetic resonance studies.
abstract: BACKGROUND
  A method has been developed for the quantitation of isotopic labeling of proteins using liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the application of protein nuclear magnetic resonance (NMR) studies. NMR relies on specific isotopic nuclei, such as (13)C and (15)N, for detection and, therefore, isotopic labeling is an important sample preparation step prior to in-depth structural characterization of proteins. The goal of this study was to develop a robust quantitative assay for assessing isotopic labeling in proteins while retaining information on the extent of labeling for individual amino acids.
  
  
  METHODS
  Complete digestion of proteins by acid hydrolysis was followed by derivatization of free amino acids with 6-aminoquinolyl N-hydroxysuccinimidyl carbamate (AQC) forming derivatives having identical MS/MS fragmentation behavior. Precursor ion scanning on a hybrid quadrupole-linear ion trap platform was used for amino acid analysis and determining isotopic labeling of proteins.
  
  
  RESULTS
  Using a set of isotope-labeled amino acid standards mixed with their unlabeled counterparts, the method was validated for accurately measuring % isotopic contribution. We then applied the method for determining the (13)C isotopic content of algal proteins during a feeding study using (13)C(6)-glucose- or (13)C-bicarbonate-supplemented culture media as well as the level of labeling in mussel byssal threads obtained after feeding with labeled algae.
  
  
  CONCLUSIONS
  This method is ideally suited for assessing the extent of protein labeling prior to NMR studies, where the isotopic labeling is a determining factor in the quality of resulting protein spectra, and can be applied to a multitude of different biological samples.
authors.affiliation: Université du Québec à Montréal, Chemistry Department, Montréal, QC, Canada.
authors.forename: André
authors.initials: A
authors.lastname: LeBlanc
authors.forename: Alexandre A
authors.initials: AA
authors.lastname: Arnold
authors.forename: Bertrand
authors.initials: B
authors.lastname: Genard
authors.forename: Jean-Bruno
authors.initials: JB
authors.lastname: Nadalini
authors.forename: Marc-Olivier Séguin
authors.initials: MO
authors.lastname: Heine
authors.forename: Isabelle
authors.initials: I
authors.lastname: Marcotte
authors.forename: Réjean
authors.initials: R
authors.lastname: Tremblay
authors.forename: Lekha
authors.initials: L
authors.lastname: Sleno
mesh_terms: D020418:Algal Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes / Q000032:analysis / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D016014:Linear Models; D009682:Magnetic Resonance Spectroscopy; D058086:Microalgae / Q000378:metabolism; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D020418:Algal Proteins; D000596:Amino Acids; D002247:Carbon Isotopes