Quantitative analysis of human cerebrospinal fluid proteins using a combination of cysteine tagging and amine-reactive isobaric labeling.
Highly complex and dynamic protein mixtures are hardly comprehensively resolved by direct shotgun proteomic analysis. As many proteins of biological interest are of low abundance, numerous analytical methodologies have been developed to reduce sample complexity and go deeper into proteomes. The present work describes an analytical strategy to perform cysteinyl-peptide subset enrichment and relative quantification through successive cysteine and amine-isobaric tagging. A cysteine-reactive covalent capture tag (C³T) allowed derivatization of cysteines and specific isolation on a covalent capture (CC) resin. The 6-plex amine-reactive tandem mass tags (TMT) served for relative quantification of the targeted peptides. The strategy was first evaluated on a model protein mixture with increasing concentrations to assess the specificity of the enrichment and the quantitative performances of the workflow. It was then applied to human cerebrospinal fluid (CSF) from post-mortem and ante-mortem samples. These studies confirmed the specificity of the C³T and the CC technique to cysteine-containing peptides. The model protein mixture analysis showed high precision and accuracy of the quantification with coefficients of variation and mean absolute errors of less than 10% on average. The CSF experiments demonstrated the potential of the strategy to study complex biological samples and identify differential brain-related proteins. In addition, the quantification data were highly correlated with a classical TMT experiment (i.e., without C³T cysteine-tagging and enrichment steps). Altogether, these results legitimate the use of this quantitative C³T strategy to enrich and relatively quantify cysteine-containing peptides in complex mixtures.
Biomedical Proteomics Group, Department of Structural Biology and Bioinformatics, University of Geneva, Geneva, Switzerland.
Priscille
P
Giron
Loïc
L
Dayon
Natacha
N
Turck
Christine
C
Hoogland
Jean-Charles
JC
Sanchez
D000588:Amines; D000818:Animals; D001923:Brain Chemistry; D002555:Cerebrospinal Fluid / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007553:Isotope Labeling / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D054464:Peroxiredoxins / Q000032:analysis / Q000378:metabolism; D011180:Postmortem Changes; D018709:Statistics, Nonparametric
D000588:Amines; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; C515974:PRDX5 protein, human; D054464:Peroxiredoxins; D003545:Cysteine