Quantitative proteomics using 16O/18O labeling and linear ion trap mass spectrometry.
Quantitative proteomics using stable isotopic 16O/18O labeling has emerged as a very powerful tool, since it has a number of advantages over other methods, including the simplicity of chemistry, the constant mass tag at the C termini and its general applicability. However, due to the small mass difference between labeled and unlabeled peptide species, this approach has usually been restricted to high-resolution mass spectrometers. In this study we explored whether the high-resolution scanning mode, together with the extremely high scanning speed of the linear IT allows the 16O/18O-labeling method to be used for accurate, large-scale quantitative analysis of proteomes. A protocol, including digestion, desalting, labeling, MS and quantitative analysis was developed and tested using protein standards and whole proteome extracts. Using this method we were able to identify and quantify 140 proteins from only 10 mug of a proteome extract from mesenchymal stem cells. Relative expression changes larger than twofold can be identified with this method at the 95% confidence level. Our results demonstrate that accurate quantitative analysis using 16O/18O labeling can be performed in the practice using linear IT MS, without compromising large-scale peptide identification efficiency.
Protein Chemistry and Proteomics Laboratory, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
Daniel
D
López-Ferrer
Antonio
A
Ramos-Fernández
Salvador
S
Martínez-Bartolomé
Predestinación
P
García-Ruiz
Jesús
J
Vázquez
D002478:Cells, Cultured; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D010100:Oxygen / Q000378:metabolism; D010103:Oxygen Isotopes / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D010103:Oxygen Isotopes; D010446:Peptide Fragments; D020543:Proteome; D010100:Oxygen