Biomedical subjects

Margarete Schulze

Publications and source records attributed to Margarete Schulze.

3 recordsLinked to original sources

PubMed2006

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. In this way the proteins are conserved in their native states and are distributed in 2400 liquid fractions with high recovery rates and sufficient reproducibility. The resulting fractions are subjected to protein quantitation and identification. Spectrophotometrical and immunological methods and enzyme activity measurements are used for quantitation. To identify proteins, the fractions are subjected to MALDI-MS, and their tryptic digests to both MALDI- and LC-ESI-MS/MS. All preparation steps except the first are applied in parallel to sets of multiples of 96 samples. The procedure may be refined by adding more separation steps and may be adapted to various protein amounts and to various proteomes. Moreover, the method offers the opportunity to investigate functional protein complexes. The method was applied to separate the normal human serum proteome. Within 255 fractions exhibiting the highest protein concentrations, 742 proteins were identified by LC-ESI-MS/MS peptide sequence tags.

Blood Proteins↗

PubMed2006

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. The sum of normalized peak heights, S(n), or the normalized mean peak height, M(n), reflects the concentration of the respective protein. For fractions containing various proteins, S(n) and M(n) can be used to compare concentrations of a protein between different fractions. For fractions with one predominating protein, they can be used to estimate concentration ratios between fractions, or to quantify the fractional protein concentration after calibration with pure protein solutions. Initial native fractionation retains the possibility to apply all conventional analytic procedures. Moreover, it renders the method relatively robust to MS mass accuracy. The method was validated with albumin, transferrin, alpha1-antitrypsin, and immunoglobulin G within highly complex chromatographic fractions of pathological and normal sera, which contained the respective intact native protein in dominating as well as minor concentrations. The correlation found between S(n) and the protein concentration as determined with ELISA showed that the method can be applied to select markers for distinguishing between normal and pathological serum samples.

Blood Proteins↗

PubMed2004

An improved method for checking HTS/uHTS liquid-handling systems.

An efficient method is presented to determine precision and accuracy of multichannel liquid-handling systems under conditions near to application. The method consists of gravimetrical determination of accuracy and optical determination of precision based on the dilution of absorbing and fluorescent dye solutions in microplates. Mean delivery volume per well can be determined with precision better than a 0.04% coefficient of variation (CV). Optical signal precision, CV(S), is improved by multiwavelength measurements. Precision of absorbance measurement yields a better resolution than precision of fluorescence measurement (0.3% and 1.5%, respectively), indicating that absorbance measurements should be preferred. From CV(S), an upper bound of the precision of the volumes delivered is derived. Method performance is demonstrated with the dispenser CyBi-Drop and the pipettor CyBi-Well using different ejection principles; with commonly used fluids; with 96-, 384-, and 1536-well microplates; and with photometric and fluorometric indicators. Precision of the volumes delivered, as obtained with optimized methods, all plate formats, and both devices, is better than 2% CV with 2 microL set volume and about 1% CV with higher set volumes.

Calibration↗