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A rapid analytical optical biosensor-based immunoassay was developed and validated for the detection of okadaic acid (OA) and its structurally related toxins from shellfish matrix. The assay utilizes a monoclonal antibody which binds to the OA group of toxins in order of their toxicities, resulting in a pseudofunctional assay. Single-laboratory validation of the assay for quantitative detection of OA determined that it has an action limit of 120 [micro]g/kg, a limit of detection of 31 [micro]g/kg, and a working range of 31-174 [micro]g/kg. The midpoint on the standard matrix calibration curve is 80 [micro]g/kg, half the current regulatory limit. Inter-and intra-assay studies of negative mussel samples spiked with various OA concentrations produced average coefficient of variation (CV) and standard deviation (SD) values of 7.9 and 10.1, respectively. The assay was also validated to confirm the ability to accurately codetect and quantify dinophysistoxin-1 (DTX-1), DTX-2, and DTX-3 from shellfish matrix. Alkaline hydrolysis was not required for the detection of DTX-3 from matrix. Excellent correlations with the data generated by the biosensor method and liquid chromatography/tandem mass spectrometry (LC/ MS/MS) were obtained using a certified reference material ([R.sup.2] = 0.99), laboratory reference material, and naturally contaminated mussel samples ([R.sup.2] = 0.97). This new procedure could be used as a rapid screening procedure replacing animal-based tests for DSP toxins. 10.1021/ac902084a

Extensive prefractionation is now considered to be a necessary prerequisite for the comprehensive analysis of complex proteomes where the dynamic range of protein abundances can vary from ~[10.sup.6] for cells to ~[10.sup.10] for tissues such as blood. Here, we describe a high-resolution 2D protein separation system that uses a continuous free-flow electrophoresis (FFE) device to fractionate complex protein mixtures by solution-phase isoelectric focusing (IEF) into 96 well-defined pools, each separated by ~0.02-0.10 pH unit depending on the gradient created, followed by rapid (~46 min per analysis) reversed-phase high-performance liquid chromatography (RP-HPLC) of each FFE pool. Fractionated proteins are readily visualized in a virtual 2D format using software that plots protein loci, pI in the first dimension and relative hydrophobicity (i.e., RP-HPLC retention time) in the second dimension. By coupling a diode-array detector in line with a multiwavelength fluorescence detector, separated proteins can be monitored in the RP-HPLC eluent by both UV absorbance and intrinsic fluorescence simultaneously from a single experiment. Triplicate analyses of standard proteins using a pH 3-10 gradient conducted over a 3-day period revealed a high system reproducibility with a SD of 0.57 (0.05 pH unit) within the FFE pools and 0.003 (0.18 s) for protein retention times in the second-dimension RP-HPLC step. In addition, we demonstrate that the FFE-IEF/RP-HPLC separation strategy can also be applied to complex mixtures of low molecular weight compounds such as peptides. With the facile ability to measure the pH of the isoelectric focused pools, peptide pI values can be estimated and used to qualify peptide identifications made using either MS/MS sequencing approaches or pI discriminated peptide mass fingerprinting. The calculated peak capacity of this 2D liquid-based FFE-IEF/RP-HPLC system is 6720.

The possible adverse effects on health of diet-derived advanced glycation endproducts (AGEs) and advanced lipoxidation endproducts (ALEs) is of current interest. This study had the objective of determining the effects of the addition of AGE/ALE inhibitors and different types of sugar and cooking oil on Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) formation in model foods (sponge cakes). The cake baked using glucose produced the highest level of CML (2.07±0.24 mmol/mol lysine), whereas the cake baked using fructose produced the highest concentration of CEL (25.1±0.15 mmol/mol lysine). There were no significant differences between CML concentrations formed in the cakes prepared using different types of cooking oil, but significant differences (P<0.001) were observed between the cakes prepared using different proportions of cooking oil. The cakes containing oil generated greater concentrations of CML than sucrose. α-Tocopherol and rutin did not inhibit CML and CEL formation. In contrast, ferulic acid and thiamin, thiamin monophosphate, and thiamin pyrophosphate reduced CML and CEL formation.

The present study was designed to compare surface plasmon resonance (SPR) biosensor, enzyme-linked immunosorbent assay (ELISA), and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods for the analysis of Nε-(carboxymethyl)lysine (CML) in glucose-bovine serum albumin (BSA) model systems and to investigate the possible inhibitory effect of selected compounds (α-tocopherol, ferulic acid, rutin, thiamin, thiamin monophosphate, and thiamin pyrophosphate) on CML formation. The reported levels of CML detected were dependent upon the method of analysis employed. The highest reported concentrations were obtained with the SPR biosensor, whereas the lowest were found by ELISA. However, a high correlation was observed between these two immunochemical procedures. CML concentrations were dependent upon the type and concentration of the candidate CML inhibitor. All inhibitory compounds investigated, with the exception of α-tocopherol, decreased the level of CML formation in the glucose-BSA system.

A comprehensive analysis of the phosphoinositide interactome has been performed using analogues of PI(3,5)P2 and PI(4,5)P2 phosphatidyl phospholipids which were immobilized onto Affi-10 beads or incorporated into liposomes for use as affinity absorbents with cytosolic extracts from colonic carcinoma cell lines. Affinity/LC/MS/MS experiments allowed identification of 388 proteins/protein complexes that appeared to interact specifically with the phosphoinositide targets: a number of novel potential phosphoinositide interacting proteins have been identified.