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.

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.

Turbo-mixing in microplates.

How to effectively mix small volumes of liquids within microplate wells is a still underestimated and often neglected challenge. The method the authors introduce here relies on violent turbulent motion within a liquid caused by spotting an organic solvent drop onto its surface. The amount needed, less than 1 to 3 microL, is generally small enough not to alter bioactive molecules.

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.