Tobias Preckel

Efficient Fractionation and Improved Protein Identification by Peptide OFFGEL Electrophoresis

The sample fractionation steps conducted prior to mass detection are critically important for the comprehensive analysis of complex protein mixtures. This paper illustrates the effectiveness of OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator for the fractionation of peptides. An Escherichia coli tryptic digest was separated in 24 fractions, and peptides were identified by reversed-phase liquid chromatography on a microfluidic device with mass spectrometric detection. About 90% of the identified individual peptides were found in only one or two fractions. The distribution of the calculated isoelectric points for the peptides identified in each fraction was especially narrow in the acidic pH range. Standard deviations approached the size of the pH segment covered by the respective fraction. The experimental peptide isoelectric point measured by OFFGEL electrophoresis was used as an additional filter for validation of peptide identifications.

Cytometric analysis of protein expression and apoptosis in human primary cells with a novel microfluidic chip-based system.

Work with primary cells is inherently limited by source availability and life span in culture. Flow cytometry offers extensive analytical opportunities but generally requires high cell numbers for an experiment.

Single-cell analysis of yeast, mammalian cells, and fungal spores with a microfluidic pressure-driven chip-based system.

Cytomics aims at understanding the function of cellular systems by analysis of single cells. Recently, there has been a growing interest in single cell measurements being performed in microfluidic systems. These systems promise to integrate staining, measurement, and analysis in a single system. One important aspect is the limitation of allowable cell sizes due to microfluidic channel dimensions. Here we want to demonstrate the broad applicability of microfluidic chip technology for the analysis of many different cell types.

Detection of Cellular Parameters Using a Microfluidic Chip-Based System:

Lab-on-a-chip technology achieves a reduction of sample and reagent volume and automates complex laboratory processes. Here, we present the implementation of cell assays on a microfluidic platform using disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow inside networks of microfluidic channels. Cells are hydrodynamically focused and pass the fluorescence detector in single file. Initial applications are the determination of protein expression and apoptosis parameters. The microfluidic system allows unattended measurement of six samples per chip. Results obtained with the microfluidic chips showed good correlation with data obtained using a standard flow cytometer.

Metrological sharp shooting for plasma proteins and peptides: The need for reference materials for accurate measurements in clinical proteomics and in vitro diagnostics to generate reliable results

Reliable study results are necessary for the assessment of discoveries, including those from proteomics. Reliable study results are also crucial to increase the likelihood of making a successful choice of biomarker candidates for verification and subsequent validation studies, a current bottleneck for the transition to in vitro diagnostic (IVD). In this respect, a major need for improvement in proteomics appears to be accuracy of measurements, including both trueness and precision of measurement. Standardization and total quality management systems (TQMS) help to provide accurate measurements and reliable results. Reference materials are an essential part of standardization and TQMS in IVD and are crucial to provide metrological correct measurements and for the overall quality assurance process. In this article we give an overview on how reference materials are defined, prepared and what role they play in standardization and TQMS to support the generation of reliable results. We discuss how proteomics can support the establishment of reference materials and biomarker tests for IVD applications, how current reference materials used in IVD may be beneficially applied in proteomics, and we provide considerations on the establishment of reference materials specific for proteomics. For clarity, we solely focus on reference materials related to serum and plasma.

High-Throughput Protein and DNA Analysis Based on Microfluidic On-Chip Electrophoresis

Microfluidic technology applied to on-chip electrophoresis provides high-throughput DNA or protein analysis in an automated, unattended mode, which is currently not possible with any other technology. The 5100 Automated Lab-on-a-Chip Platform automates all the required experimental steps, including sample loading from multiple sample plates, electrophoresis, staining/destaining, and detection. The analysis of the digital data is completely automated as well and the results together with all other information, such as sample names, are directly fed into a database. The article describes in detail the design of the microfluidic system, including instrumentation, chips, DNA and protein assays, as well as the structure and the main features of the software.

Analysis of Multiple Apoptosis Parameters Using a Microfluidic Chip-Based System

We have developed miniaturized assays for detection of phosphatidylserine exposure, caspase activity and DNA laddering using lab-on-a-chip technology. A readout system capable of two-color fluorescence detection was used to run disposable glass chips with various analytical functionalities. The chips contain an interconnected network of microchannels that permits either electrophoretic separations of biomolecules or simple flow cytometric analysis of cells.