Quantitative Analysis of Human Cerebrospinal Fluid Proteins Using a Combination of Cysteine Tagging and Amine-Reactive Isobaric Labeling

Abstract

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.

Quantitative Mass Spectrometry Analysis Using PAcIFIC for the Identification of Plasma Diagnostic Biomarkers for Abdominal Aortic Aneurysm

“…Then, t-test was applied to duplicated values of intensity ratios for each protein in order to evaluate differences of protein abundance. Significant proteins were considered when p-value<0.05 and fold change >12%, since variation in technical replicates for isobaric labeling has been estimated to that value [41], [42].…”

Comparative Proteomics for the Evaluation of Protein Expression and Modifications in Neurodegenerative Diseases

“…Nowadays, the quantitative differential proteomics, which measure the relative and/or absolute amount of proteins or specific PTMs, is based on two methodological approaches that differ for the gel-based or liquid chromatography (LC)-based step of proteins/ peptides fractionation prior to the mass spectrometry (MS) analysis and protein identification (for a detailed methodological review, see Craft, Chen, & Nairn, 2013;Giron, Dayon, Turck, Hoogland, & Sanchez, 2011;van Gool & Hendrickson, 2012). The gel-based methodologies, mainly two-dimensional electrophoresis (2DE), are still powerful and largely used, and they rely on intact proteins separation, protein quantification by gel-image analysis, digestion of relevant spots with trypsin, and then tandem MS analysis for protein identification performed either by MALDI-TOF/ TOF (matrix-assisted laser desorption ionization-time of flight) or by ESI-MS/MS (electron spray ionization-MS), whereas the liquid chromatography (LC)-based approach is characterized by a protein digestion that precedes the peptides separation in liquid-phase chromatography and a direct ionization by ESI into tandem MS for protein identifications.…”

“…Both approaches have advantages and disadvantages; in particular, the 2DE gel-based analysis is limited in the number and type of proteins that can be resolved, but conversely is able to reveal the presence of protein fragments and isoforms undetectable if peptides are analyzed as for LC-MS/MS; on the contrary, the MS approaches coupled with LC are able to resolve, without type-specific limitation, large number of proteins (Huang et al, 2007;Yuan & Desiderio, 2005a;Zhang et al, 2008). Usually, the relative quantitation of protein expression is reported as a fold change and can be obtained, within the two approaches, either by label-based or by label-free methods: typically 2DE gel staining versus fluorescence-labeled protein 2DE for gel-based approach, and label-free quantitative MS versus isotope-coded affinity tags for LC-MS approach (Andreev et al, 2012;Craft et al, 2013;Giron et al, 2011;Huang et al, 2007;van Gool & Hendrickson, 2012;Yuan & Desiderio, 2005a;Zhang et al, 2008). Absolute quantitation can be determined by addition in the samples of internal standards; in particular, the addition of stable isotopic standard is suitable in the LC-MS approach with the so-called iTRAQ (isobaric tag for relative and absolute quantitation) (reviewed in Craft et al, 2013).…”

Recent advances in quantitative neuroproteomics

“…They identified 1,212 proteins in total, with 745 low-abundance proteins in CSF detected only after the peptide library treatment. Giron et al combined cysteine-containing peptide enrichment with TMT isobaric labeling of human CSF [218]. Stoop et al used label-free quantitation of health CSF samples identifying sets of CSF proteins with low or high variation that can be used for the wider community when selecting potential biomarkers [219].…”