Christian Wenz

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.

Interlaboratory study to evaluate the robustness of capillary electrophoresis-mass spectrometry for peptide mapping

A collaborative study on the robustness and portability of a capillary electrophoresis-mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis-mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath-flow capillary electrophoresis-mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin-digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3-12 and 9-29% RSD, respectively. These results demonstrate that capillary electrophoresis-mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis-mass spectrometry applications in biopharmaceutical analysis and related fields.

A fluorescent derivatization method of proteins for the detection of low‐level impurities by microchip capillary gel electrophoresis

A novel pre‐chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water‐soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on‐chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver‐stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre‐column derivatization include a shorter analysis time and an increased robustness and ease of use.

Immunoprecipitation combined with microchip capillary gel electrophoresis: Detection and quantification of β-galactosidase from crude E. coli cell lysate.

A sensitive and selective analytical method for the determination and quantification of endogenous β‐galactosidase in crude E. coli cell lysates by immunoprecipitation combined with automated microchip capillary gel electrophoresis (IP‐MCGE) with laser‐induced fluorescence (LIF) detection was developed. Total cell lysates were derivatized minimally with a fluorescence dye, incubated with anti‐β‐galactosidase antibodies, and the antigen/antibody complex was precipitated with protein G‐coated magnetic beads. After capturing the complex, it was eluted from the beads under denaturing conditions and loaded directly onto a multisample microchip for analysis. The effects of antibody selection and the importance of preclearing steps were studied in detail. For quantification, an external calibration through spiking pure β‐galactosidase into E. coli lysate was performed. Recovery rates of immunoprecipitation after spiking experiments and the amount of unknown endogenous β‐galactosidase in E. coli lysates were determined. As proof of principle, E. coli cultures grown on nutrition media with several glucose/lactose ratios were analyzed. Differences in the expression level of β‐galactosidase could be detected and measured with the developed method. Detected amounts of β‐galactosidase in different culture media correlated with the β‐galactosidase activities in these cultures.

Microchip CGE linked to immunoprecipitation as an alternative to Western blotting

A novel approach for protein identification is presented, which combines the specificity of an immunoprecipitation approach with the sensitivity of protein detection in microchip CGE. This method involves derivatization of the sample proteins with a fluorescent dye, target protein isolation with specific antibodies and Protein A coated magnetic beads, and automated sizing and quantification of the eluted samples on microchips. The performance of the new technique was demonstrated with glutathion‐S‐transferase‐ and polyHistidine‐tagged target proteins in an Escherichia coli background. A specific detection of target proteins was possible down to 0.001% or 1 ng target protein in a background of 100 μg E. coli protein. With this approach, proteins ranging from 10 to 220 kDa could be identified with a panel of different target‐specific antibodies. The reproducibility of the method was very similar to standard microchip CGE methods. In a direct comparison to Western blotting, a similar sensitivity and specificity of both techniques was observed. However, the new approach compares favorably to Western blotting in terms of time‐to‐result, usability and labor intensity, antibody consumption and access to quantitative data.

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.

Challenges of glycoprotein analysis by microchip capillary gel electrophoresis

Glycosylations severely influence a proteins biological and physicochemical properties. Five exemplary proteins with varying glycan moieties were chosen to establish molecular weight (MW) determination (sizing), quantitation, and sensitivity of detection for microchip capillary gel electrophoresis (MCGE). Although sizing showed increasing deviations from literature values (SDS‐PAGE or MALDI‐MS) with a concomitant higher degree of analyte glycosylation, the reproducibility of MW determination and accuracy of quantitation with high sensitivity and reliability were demonstrated. Additionally, speed of analysis together with the low level of analyte consumption render MCGE attractive as an alternative to conventional SDS‐PAGE.

Sensitive detection of C-reactive protein in serum by immunoprecipitation-microchip capillary gel electrophoresis.

Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP-MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R(2) = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP-MCGE bears potential for point-of-care diagnosis.

Microchip capillary gel electrophoresis combined with lectin affinity enrichment employing magnetic beads for glycoprotein analysis

AbstractDue to the constant search for reliable methods to investigate glycoproteins in complex biological samples, an alternative approach combining affinity enrichment with rapid and sensitive analysis on-a-chip is presented. Glycoproteins were specifically captured by lectin-coated magnetic beads, eluted by competitive sugars, and investigated with microchip capillary gel electrophoresis (MCGE), i.e., CGE-on-a-chip. We compared our results to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data, which turned out to be in very good agreement. While SDS-PAGE offers the possibility of subsequent mass spectrometric analysis of captured and separated analytes, MCGE scores with time savings, higher throughput, and lower sample consumption as well as quality control (QC) and process analytical technology (PAT) applicability. Due to these advantages, a lectin-based glycoprotein capture protocol can easily be optimized. In our case, two different types of magnetic beads were tested and compared regarding lectin binding. The selectivity of our strategy was demonstrated with a set of model glycoproteins, as well as with human serum and serum depleted from high-abundance proteins. The specificity of the capturing method was investigated revealing to a certain degree an unspecific binding between each sample and the beads themselves, which has to be considered for any specific enrichment and data interpretation. In addition, two glycoproteins from Trichoderma atroviride, a fungus with mycoparasitic activity and only barely studied glycoproteome, were enriched by means of a lectin and so identified for the first time. Graphical abstractGlycoproteins from biological samples were detected by microchip capillary gel electrophoresis after lectin affinity enrichment using magnetic beads and elution with respective competitive monosaccharides

Immunoprecipitation and the High Sensitivity Protein 250 Assay: Combining specific and sensitive detection of proteins with the Agilent 2100 Bioanalyzer

A new method for the targeted analysis of proteins is presented, which combines the specificity of immunopre cipitation with the sensitivity of protein detection on microchips using the High Sensitivity Protein 250 assay for the Agilent 2100 Bioanalyzer. As as an alternative to Western blotting, this method is valuable for researchers engaged, for example, in protein expression and purification in pharma, biotech, or academic labs. Advantages of this new method in comparison to Western blotting are: • More reliable results: higher specificity and sensitivity • Better accuracy and precision: less manual steps and direct availability of quantitative data • Increased productivity: 3 hours versus 1 day analysis time • Lower spending for antibodies: 10× less primary and no secondary antibody are needed • Lower reagent consumption: environmentally friendly process