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H. Okamoto, H. Shimada, K. Matsuno, K. Tanahashi, J. Utsumi, T. Iwata; Enrichment and Isolation of Low Molecular Weight Protein in Vitreous Humor Using a Newly Developed Protein Separator. Invest. Ophthalmol. Vis. Sci. 2008;49(13):777. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mass spectrometer (MS) is a powerful tool to discover biomarker protein in clinical proteomics. However, abundant high molecular proteins (>50kDa) such as albumin and IgG often disturb precise detection of low content, low molecular weight (LMW) proteins (<30kDa). The removal of high content proteins prior to mass measurement is one of the big issues remaining in clinical proteomics analysis. We develop an ultra high performance hollow fiber based protein separator to remove much abundant high molecular weight (HMW) proteins and enrich LMW proteins.
Human vitreous humor (VH) was obtained from patients with macular hole (MH, 16cases), diabetic macular edema (DME, 16cases) and proliferate diabetic retinopathy (PDR, 16cases). Sample solution contained 305 ug of protein was injected into the protein separator to remove HMW proteins. Separated LMW samples were then fractionated by reverse phase column and digested with trypsin. Digested samples were separated by 2D-HPLC followed by ion trap MS/MS. DTA files obtained from MS were fed into two protein identification softwares (Bioworks and Phenyx).
By two independent softwares, 86, 76 and 79 proteins were identified from each LMW samples with MH, DME and PDR. 41 of these proteins were common among three diseases and several proteins were specific to each disease. Some of disease specific proteins have not been reported as VH protein.
This new protein separator will benefit identification of LMW biomarkers from low protein concentration samples such as VH. The combination of protein separator and MS has the potential to become a powerful tool to identify biomarker proteins at even low concentration.
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