Proteomic sample preparation by microdialysis: easy, speedy, and nonselective.

Microdialysis tools have been developed for parallelized medium exchange designated for sample volumes from 10 to 100 μl, compatible with the microplate format, and guaranteeing maximum recoveries without selectivity. These tools are applicable to both protein and peptide analysis. Moreover, they may be used for binding studies as well as for reconcentration and as unique sample containers for complex operating sequences allowing contemporaneous processing and high throughput.

Long-term serum proteomes are quite similar under high- and low-flux hemodialysis treatment.

Purpose: Aim of the study was to identify long-term differences of middle and high-molecular-weight serum constituents under high- and low-flux hemodialysis treatments. Thus, the entire predialytic serum proteomes had to be analyzed using identical hemodialysis membrane material but with different cut-off values.

Methods And Results: A cross-over study and a global native chromatographic proteomic approach were used to analyze serum compositions of 16 patients suffering from end-stage renal disease.

Spinal antinociceptive effects of cyclooxygenase inhibition during inflammation: Involvement of prostaglandins and endocannabinoids.

Both cyclooxygenase-1 and -2 are expressed in the spinal cord, and the spinal COX product prostaglandin E(2) (PGE(2)) contributes to the generation of central sensitization upon peripheral inflammation. Vice versa spinal COX inhibition is considered an important mechanism of antihyperalgesic pain treatment. Recently, however, COX-2 was shown to be also involved in the metabolism of endocannabinoids.

Searching biomarker candidates in serum using multidimensional native chromatography. I. Enhanced separation method.

The microplate-based method developed by our group for non-denaturing multidimensional proteome separation was improved on using improved column arrays and a newly developed robot. Currently size exclusion, anion exchange and lectin affinity chromatography are combined orthogonally. Different samples run simultaneously to enhance reliability of intercomparison.

Searching biomarker candidates in serum using multidimensional native chromatography. II Method evaluation with Alport syndrome and severe inflammation.

Biomarker search using multidimensional native liquid fractionation of serum in microplates was evaluated. From different donors, homologous sample fractions with UV absorbance depending on state of illness were selected, and their constituents were identified and quantitated by MS. Analysis of sera of patients with Alport syndrome and severe inflammation proved the reliability of the method by confirming characteristic alterations.

Extended FMRFamides in dipteran insects: conservative expression in the neuroendocrine system is accompanied by rapid sequence evolution.

Extended FMRFamides are found throughout the central nervous system (CNS) of insects and exhibit diverse physiological effects on different target organs, such as muscles, intestine, and the nervous system. The genes encoding for extended FMRFamides are known from a number of flies, including Drosophila species, and the pest insects Lucilia cuprina, Calliphora vomitoria, and Musca domestica. No data, however, exist about the expression of the numerous paralogs of the latter three species, and studies on Drosophila melanogaster resulted in controversial findings.

Multidimensional chromatography: validation and efficient fishing for biomarkers and fractions containing them using the VisualCockpit software package.

2-D native LC yields thousands of fractions especially when applied to sera of different origin. Checking reproducibility of repeated separation of the same serum or searching for biomarker candidates and fractions containing them requires finding, selection, and comparison of interesting data subsets out of huge data volumes. An innovative software package is applied that markedly enhances simplicity, velocity, and reliability of (i) check of reproducibility of the separation method and (ii) analysis of proteomes pertaining to different disease states.

Robust protein quantitation in chromatographic fractions using MALDI-MS of tryptic peptides.

A method is introduced to evaluate protein concentrations using the height sum of all MALDI-MS peaks that unambiguously match theoretic tryptic peptide masses of the protein sought after. The method uses native chromatographic protein fractionation prior to digestion but does not require any depletion, labeling, derivatization, or preparation of a compound similar to the analyte. All peak heights of tryptic peptides are normalized with the peak height of a unique standard peptide added to the MALDI-MS samples.

Multidimensional proteomics of human serum using parallel chromatography of native constituents and microplate technology.

A versatile, multidimensional, and non-denaturing proteome separation procedure using microplate technology is presented, yielding a digitized image of proteome composition. In the first dimension, the sample under study is separated into 96 fractions by size exclusion chromatography (SEC). In the second dimension, the fractions of the first dimension are transferred by the liquid-handling device CyBi-Well (CyBio AG, Jena, Germany) to 96 parallel anion exchange chromatography columns.

UV measurements in microplates suitable for high-throughput protein determination.

An UV spectrophotometric method for protein determination using microplates is described. Using the SPECTRAmax PLUS reader, the UVStar 96- and 384-well microplates and a 96 or 384 parallel channel liquid handling technique, large-scale determinations can be performed with intraassay precision better than 3% CV (coefficient of variation) in the range from 1 to 8000 microg of protein/ml, measuring at 205, 215, and 280 nm and using different volume-dependent light-path lengths. Since the absorbance coefficient at 205 nm is found to be 30 ml/(mgxcm) for eight different proteins with a CV of 5.