Cell fractionation - an important tool for compartment proteomics

In order to maximize coverage in proteome studies, a successful approach is the fractionation of cellular compartments. For providing evidence for the most reliable and efficient separation technique, we compared four different procedures for subcellular fractionation of Jurkat cells. The analysis of fractions by LTQ-Orbitrap yielded between 559 and 1195 unambiguously identified unique proteins. The assumed correct localisation of the proteins was defined using Scaffold3 according to GO annotations, with the highestreliability (~80%) for the cytoplasmic fraction and the lowest (~20%) for the cytoskeletal fraction. This comparison revealed evidence for the efficiencies of separating subcellular fractions and will thereby facilitate the decision on which procedure might be the best match to a specific research question and contribute to the emerging field of compartment proteomics

Antibody Responses in Humans Infected with Newly Emerging Strains of West Nile Virus in Europe

Abstract Infection with West Nile Virus (WNV) affects an increasing number of countries worldwide. Although most human infections result in no or mild flu-like symptoms, the elderly and those with a weakened immune system are at higher risk for developing severe neurological disease. Since its introduction into North America in 1999, WNV has spread across the continental United States and caused annual outbreaks with a total of 36,000 documented clinical cases and 3c1,500 deaths. In recent years, outbreaks of neuroinvasive disease also have been reported in Europe. The WNV strains isolated during these outbreaks differ from those in North America, as sequencing has revealed that distinct phylogenetic lineages of WNV concurrently circulate in Europe, which has potential implications for the development of vaccines, therapeutics, and diagnostic tests. Here, we studied the human antibody response to European WNV strains responsible for outbreaks in Italy and Greece in 2010, caused by lineage 1 and 2 strains, respectively. The WNV structural proteins were expressed as a series of overlapping fragments fused to a carrier-protein, and binding of IgG in sera from infected persons was analyzed. The results demonstrate that, although the humoral immune response to WNV in humans is heterogeneous, several dominant peptides are recognized

Antibody responses in humans infected with newly emerging strains of West Nile Virus in Europe

Infection with West Nile Virus (WNV) affects an increasing number of countries worldwide. Although most human infections result in no or mild flu-like symptoms, the elderly and those with a weakened immune system are at higher risk for developing severe neurological disease. Since its introduction into North America in 1999, WNV has spread across the continental United States and caused annual outbreaks with a total of 36,000 documented clinical cases and ,1,500 deaths. In recent years, outbreaks of neuroinvasive disease also have been reported in Europe. The WNV strains isolated during these outbreaks differ from those in North America, as sequencing has revealed that distinct phylogenetic lineages of WNV concurrently circulate in Europe, which has potential implications for the development of vaccines, therapeutics, and diagnostic tests. Here, we studied the human antibody response to European WNV strains responsible for outbreaks in Italy and Greece in 2010, caused by lineage 1 and 2 strains, respectively. The WNV structural proteins were expressed as a series of overlapping fragments fused to a carrier-protein, and binding of IgG in sera from infected persons was analyzed. The results demonstrate that, although the humoral immune response to WNV in humans is heterogeneous, several dominant peptides are recognized

Structure of the E-ectodomain of WNV.

<p>The sequences corresponding to the peptides E_71–100, E_231–260 and E_371–400 are highlighted in color. DI, DII and DIII indicate the respective domains.</p

Optimizing antibody binding to selected peptides.

<p>ELISA plates were coated with increasing amounts of the indicated peptides fused to GST and incubated with human sera: WNV positive sera I2, I4; G11, G12, G15 and negative serum N5. Values are the mean of two independent experiments (performed in duplicate), error bars represent the standard deviation.</p

Analysis of binding properties of human sera with recombinant E ectodomain in an ELISA.

<p>Sera used in this study were incubated with the recombinant E-ectodomain protein (lineage 1). Mean values of two independent experiments (performed in duplicate) are shown, and error bars represent the standard deviation.</p

Competition of antibody binding between selected peptides and recombinant E-ectodomain.

<p>The indicated sera were pre-incubated with recombinant peptides E_71–100, E_231–260, E_371–400 (all fused to GST) or GST alone, before they were bound to recombinant E-ectodomain of WNV. Values are derived from at least two independent experiments (performed in duplicate). Error bars represent the standard deviation. Statistical analysis to evaluate the difference between the signals of the competitor peptide and the control competitor (GST alone) were performed by using a Mann-Whitney Rank Sum Test (asterisks, p<0,05).</p

Amino acid sequences of the fragments used in this study (lineage 1 WNV strain Ita09).

<p>Amino acid sequences of the fragments used in this study (lineage 1 WNV strain Ita09).</p