RESUMO
The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers requires therefore either the specific depletion of high abundance proteins with immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe the depletion of seven abundant rat plasma proteins with an immunoaffinity column with coupled antibodies directed against albumin, IgG, transferrin, IgM, haptoglobin, fibrinogen and alpha1-anti-trypsin. The IgY-R7-LC2 (Beckman Coulter) column showed high specificity for the targeted proteins and was able to efficiently remove most of the albumin, IgG and transferrin from rat plasma samples as judged by Western blot analysis. Depleted rat plasma protein samples were analyzed by SELDI-TOF MS, 2D SDS-PAGE and 2D-LC and compared to non-depleted plasma samples as well as to the abundant protein fraction that was eluted from the immunoaffinity column. Analysis of the depleted plasma protein fraction revealed improved signal to noise ratios, regardless of which proteomic method was applied. However, only a small number of new proteins were observed in the depleted protein fraction. Immunoaffinity depletion of abundant plasma proteins results in the significant dilution of the original sample which complicates subsequent analysis. Most proteomic approaches require specialized sample preparation procedures during which significant losses of less abundant proteins and potential biomarkers can occur. Even though abundant protein depletion reduces the dynamic range of the plasma proteome by about 2-3 orders of magnitude, the difference between medium-abundant and low abundant plasma proteins is still in the range of 7-8 orders of magnitude and beyond the dynamic range of current proteomic technologies. Thus, exploring the plasma proteome in greater detail remains a daunting task.
Assuntos
Proteínas Sanguíneas/análise , Proteômica/métodos , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Western Blotting , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/métodos , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida , Fibrinogênio/análise , Fibrinogênio/química , Fibrinogênio/isolamento & purificação , Haptoglobinas/análise , Haptoglobinas/química , Haptoglobinas/isolamento & purificação , Imunoglobulina G/análise , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Imunoglobulina M/análise , Imunoglobulina M/química , Imunoglobulina M/isolamento & purificação , Ratos , Reprodutibilidade dos Testes , Albumina Sérica/análise , Albumina Sérica/química , Albumina Sérica/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transferrina/análise , Transferrina/química , Transferrina/isolamento & purificação , alfa 1-Antitripsina/análise , alfa 1-Antitripsina/química , alfa 1-Antitripsina/isolamento & purificaçãoRESUMO
The purpose of this study was to investigate the mechanisms by which carotenoids [xanthophylls vs. beta-carotene(beta-C)] are taken up by retinal pigment epithelial (RPE) cells. The human RPE cell line, ARPE-19, was used. When ARPE-19 cells were fully differentiated (7-9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent 2-fold higher than beta-C (P < 0.05). At 9 weeks, cellular uptakes were 1.6, 2.5, and 3.2%, respectively, for beta-C, LUT, and ZEA. Similar extents were observed when carotenoids were delivered in either Tween 40 or "chylomicrons" produced by Caco-2 cells. Differentiated ARPE-19 cells did not exhibit any detectable beta-C 15,15'-oxygenase activity or convert exogenous beta-C into vitamin A. When using specific antibodies against the lipid transporters cluster determinant 36 (CD36) and scavenger receptor class B type I (SR-BI), cellular uptake of beta-C and ZEA were significantly decreased (40-60%) with anti-SR-BI but not with anti-CD36. Small interfering RNA transfection for SR-BI led to marked knockdown of SR-BI protein expression (approximately 90%), which resulted in decreased beta-C and ZEA uptakes by 51% and 87%, respectively. Thus, the present data show that RPE cells preferentially take up xanthophylls versus the carotene by a process that appears to be entirely SR-BI-dependent for ZEA and partly so for beta-C. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina.