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C. Lam, K.-K. Li, K.-M. Chun, Y.-Y. Tse, W.-S. Tam, S.-W. Shan, C.-H. To; Application of Two-Dimensional Nano-LC Tandem Mass Spectrometry for Screening the Differential Retinal Protein Expressions in the Lens-Induced Myopic Chicks. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1743.
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© ARVO (1962-2015); The Authors (2016-present)
To apply a recent Stable Isotope Labeling Experiment (SILE) - Isotope Coded Protein Label (ICPL) for quantitative study the retinal protein changes in lens-induced myopic Leghorn chicks using nanoLC ESI MS/MS approach.
The retinal tissues were harvested from early emmetropising chick eyes after -10D lens wears. After reduction and alkylation, equal amount of the samples from myopic eyes and fellow control eyes were differentially labeled with 12C-Nic-reagent (light isotope) and 13C-Nic-reagent (heavy isotope) respectively. The ICPL labeled retinal samples were combined and pooled with protein standard mixtures. They were enzymatically digested overnight using trypsin and endoproteinase GluC. A total of 60ug mixture of the proteolytic digested peptides were separated by a 2D nano-flow liquid chromatography system using a "SALT-Plug" approach. Automatic precursor ion selection and MS-fragmentation of ICPL tagged peptides was performed in an ion-trap tandem MS operated in collision-induced dissociation mode. Peptides labeling for isotope pairing and protein quantification were performed in WARP-LCTM software. Protein identifications were searched against the NCBInr databases in BiotoolTM software.
Using the integrated LC-MS control and WARP-LCTM data processing for SILE pairs evaluation, the present setup not only provided high statistical match for protein identification but also gave multiple isotopic peptide pairs for more confident protein quantification. Over hundreds of proteins could be normally detected and identified in each nano-LC injection using the "SALT-Plug" gradient fractionation. The averaged Heavy / Light isotopes ratio of the control paired tagged retinal lysate was found at or below 1.5 folds across SALT gradients. Applying the same threshold, a list of differentially expressed proteins which possibly involving the myopic eye growth could be screened. Most significant retinal protein changes after the lens treatment were those up-regulated proteins. They involve broad biological functions such as catalytic activity, glycolysis, protein binding, signal transduction and cytoskeletal changes.
ICPL is an useful isotope labeling technique to study the differentially expressed SILE pairs within complex protein mixtures. Our results demonstrated a workable non-gel platform to study the differential protein expressions in chick myopia by detection and fragmentation of pre-labeled SILE pairs by high-throughput NanoLC-MS.
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