TY - GEN A1 - Tchipilov, Teodor A1 - Raysyan, Anna A1 - Weller, Michael G. T1 - Methods for the quantification of particle-bound protein – Application to reagents for lateral-flow immunoassays (LFIA) N2 - Protein immobilization for the functionalization of particles is used in various applications, including biosensors, lateral-flow immunoassays (LFIA), bead-based assays, and others. Common methods for the quantification of bound protein are measuring protein in the supernatant before and after coating and calculating the difference. This popular approach has the potential for a significant overestimation of the amount of immobilized protein since layers not directly bound to the surface (soft protein corona) are usually lost during washing and handling. Only the layer directly bound to the surface (hard corona) can be used in subsequent assays. A simplified amino acid analysis method based on acidic hydrolysis and RP-HPLC-FLD of tyrosine and phenylalanine (aromatic amino acid analysis, AAAA) is proposed to directly quantify protein bound to the surface of gold nano- and latex microparticles. The results are compared with indirect methods such as colorimetric protein assays, such as Bradford, bicinchoninic acid (BCA), as well as AAAA of the supernatant. For both particle types, these indirect quantification techniques show a protein overestimation of up to 1700% compared to the direct AAAA measurements. In addition, protein coating on latex particles was performed both passively through adsorption and covalently through EDC/sulfo-NHS chemistry. Our results showed no difference between the immobilization methodologies. This finding suggests that usual protein determination methods are no unambiguous proof of a covalent conjugation on particles or beads. KW - Soft protein corona KW - Hard protein corona KW - Gold particles KW - Nanoparticles KW - Mikroparticles KW - Antibody KW - Bioconjugation KW - Protein quantification KW - Supernatant KW - Sodium chloride method KW - Covalent conjugation KW - Latex particles KW - Lateral flow immunoassays PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-545365 DO - https://doi.org/10.20944/preprints202203.0332.v1 SP - 1 EP - 8 PB - MDPI CY - Basel AN - OPUS4-54536 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Tchipilov, Teodor A1 - Meyer, Klas A1 - Weller, Michael G. T1 - Quantitative 1H nuclear magnetic resonance (qNMR) of aromatic amino acids for protein quantification N2 - qNMR is a valuable technique for metrological studies due to the uniformity of its signal response for all chemical species of an isotope of interest, which enables compound-independent calibration. However, protein quantification remained challenging as large molecules produce wide, low-intensity signals that reduce the already low sensitivity. Combining qNMR with the hydrolysis of protein samples into amino acids circumvents many of these issues and facilitates the use of NMR spectroscopy for absolute protein and peptide quantification.In this work, different conditions have been tested for quantifying aromatic amino acids and proteins. First, we examined the pH-based signal shifts in the aromatic region. The preferable pH depends on the selection of the amino acids for quantification and which internal standard substance should be used to avoid peak overlap. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, have been applied as internal standards. The quantification of amino acids from an amino acid standard, as well as from a certified reference material (bovine serum albumin), was performed. Using the first two suggested internal standards, recovery was ~ 97 % for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98 ± 2 and 88 ± 4 %, respectively, at a protein concentration of 16 g/L or 250 µM. KW - Amino acid analysis KW - AAA KW - Protein hydrolysis KW - Metrology KW - Traceability KW - Reference materials KW - Internal standards KW - Calibration PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-564520 DO - https://doi.org/10.20944/preprints202211.0569.v1 SP - 1 EP - 13 PB - MDPI CY - Basel AN - OPUS4-56452 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Lemke, Nora A1 - El-Khatib, Ahmed H. A1 - Tchipilov, Teodor A1 - Jakubowski, N. A1 - Weller, Michael G. A1 - Vogl, Jochen T1 - Procedure providing SI‑traceable results for the calibration of protein standards by sulfur determination and its application on tau N2 - Quantitative proteomics is a growing research area and one of the most important tools in the life sciences. Well-characterized and quantified protein standards are needed to achieve accurate and reliable results. However, only a limited number of sufficiently characterized protein standards are currently available. To fill this gap, a method for traceable protein quantification using sulfur isotope dilution inductively coupled plasma mass spectrometry (ICP-MS) was developed in this study. Gel filtration and membrane filtration were tested for the separation of non-protein-bound sulfur in the protein solution. Membrane filtration demonstrated a better performance due to the lower workload and the very low sulfur blanks of 11 ng, making it well suited for high-purity proteins such as NIST SRM 927, a bovine serum albumin (BSA). The method development was accomplished with NIST SRM 927e and a commercial avidin. The quantified mass fraction of NIST SRM 927e agreed very well with the certified value and showed similar uncertainties (3.6%) as established methods while requiring less sample preparation and no species-specific standards. Finally, the developed procedure was applied to the tau protein, which is a biomarker for a group of neurodegenerative diseases denoted “tauopathies” including, e.g., Alzheimer’s disease and frontotemporal dementia. For the absolute quantification of tau in the brain of transgenic mice overexpressing human tau, a well-defined calibration standard was needed. Therefore, a pure tau solution was quantified, yielding a protein mass fraction of (0.328 ± 0.036) g/kg, which was confirmed by amino acid analysis. KW - Iinductively coupled plasma mass spectrometry KW - Isotope dilution KW - Quantitative protein analysis KW - Sulfur KW - SI traceability PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-545200 DO - https://doi.org/10.1007/s00216-022-03974-z VL - 414 SP - 4441 EP - 4455 PB - Springer Verlag AN - OPUS4-54520 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Reinmuth-Selzle, K. A1 - Tchipilov, Teodor A1 - Backes, A. T. A1 - Tscheuschner, Georg A1 - Tang, K. A1 - Ziegler, K. A1 - Lucas, K. A1 - Pöschl, U. A1 - Fröhlich-Nowoisky, J. A1 - Weller, Michael G. T1 - Determination of the protein content of complex samples by aromatic amino acid analysis, liquid chromatography-UV absorbance, and colorimetry N2 - Fast and accurate determination of the protein content of a sample is an important and non-trivial task of many biochemical, biomedical, food chemical, pharmaceutical, and environmental research activities. Different methods of total protein determination are used for a wide range of proteins with highly variable properties in complex matrices. These methods usually work reasonably well for proteins under controlled conditions, but the results for non-standard and complex samples are often questionable. Here, we compare new and well-established methods, including traditional amino acid analysis (AAA), aromatic amino acid analysis (AAAA) based on the amino acids phenylalanine and tyrosine, reversed-phase liquid chromatography of intact proteins with UV absorbance measurements at 220 and 280 nm (LC-220, LC-280), and colorimetric assays like Coomassie Blue G-250 dye-binding assay (Bradford) and bicinchoninic acid (BCA) assay. We investigated different samples, including proteins with challenging properties, chemical modifications, mixtures, and complex matrices like air particulate matter and pollen extracts. All methods yielded accurate and precise results for the protein and matrix used for calibration. AAA, AAAA with fluorescence detection, and the LC-220 method yielded robust results even under more challenging conditions (variable analytes and matrices). These methods turned out to be well-suited for reliable determination of the protein content in a wide range of samples, such as air particulate matter and pollen. KW - Air particulate matter KW - Aromatic amino acid analysis KW - Atmospheric aerosol KW - Chemical protein modification KW - Derivatization KW - Nitration KW - Nitrotyrosine KW - LC-UV absorbance KW - Pollen extract KW - Protein quantification KW - Protein test KW - Kjeldahl KW - Tyrosine KW - Phenylalanine KW - Hydrolysis KW - Bradford KW - BCA test KW - 280 nm KW - Air filter samples KW - Fluorescence KW - HPLC KW - Chromatography KW - Protein content KW - 150th anniversary of BAM KW - Topical collection: Analytical Methods and Applications in the Materials and Life Sciences PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-545924 UR - https://pubmed.ncbi.nlm.nih.gov/35320366/ DO - https://doi.org/10.1007/s00216-022-03910-1 SP - 1 EP - 14 PB - Springer Nature Limited CY - New York, Heidelberg AN - OPUS4-54592 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Tchipilov, Teodor A1 - Meyer, Klas A1 - Weller, Michael G. T1 - Quantitative 1H nuclear magnetic resonance (qNMR) of aromatic amino acids for protein quantification N2 - Hydrolysis of protein samples into amino acids facilitates the use of NMR spectroscopy for protein and peptide quantification. Different conditions have been tested for quantifying aromatic amino acids and proteins. The pH-dependent signal shifts in the aromatic region of amino acid samples were examined. A pH of 12 was found to minimize signal overlap of the four aromatic amino acids. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, were applied as internal standards. The quantification of amino acids from an amino acid standard was performed. Using the first two suggested internal standards, recovery was ~97% for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98% ± 2% and 88% ± 4%, respectively, at a protein concentration of 16 g/L or 250 µM. KW - Amino acid analysis KW - Aromatic amino acid analysis KW - AAA KW - AAAA KW - Protein hydrolysis KW - Hydrochloric acid KW - Metrology KW - Traceability KW - NIST KW - Reference materials KW - Internal standard KW - Calibration KW - Compound-independent calibration KW - Histidine KW - Tyrosine KW - Tryptophan KW - Phenylalanine KW - Terephthalic acid KW - Benzene-1,3,5-tricarboxylic acid KW - Bovine serum albumin (BSA) KW - Quantitative protein analysis KW - Phenylketonuria PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-570943 DO - https://doi.org/10.3390/mps6010011 VL - 6 IS - 1 SP - 1 EP - 13 PB - MDPI CY - Basel, Schweiz AN - OPUS4-57094 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Chapartegui-Arias, Ander A1 - Raysyan, A. A1 - Belenguer, A. M. A1 - Jaeger, Carsten A1 - Tchipilov, Teodor A1 - Prinz, Carsten A1 - Abad Andrade, Carlos Enrique A1 - Beyer, S. A1 - Schneider, Rudolf A1 - Emmerling, Franziska T1 - Tailored mobility in a zeolite imidazolate framework (ZIF) antibody conjugate N2 - Zeolitic imidazolate framework (ZIF) hybrid fluorescent nanoparticles and ZIF antibody conjugates have been synthesized, characterized, and employed in lateral-flow immunoassay (LFIA). The bright fluorescence of the conjugates and the possibility to tailor their mobility gives a huge potential for diagnostic assays. An enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase (HRP) as label, proved the integrity, stability, and dispersibility of the antibody conjugates, LC-MS/MS provided evidence that a covalent link was established between these metal-organic frameworks and lysine residues in IgG antibodies. KW - ZIF KW - ELISA PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-532096 DO - https://doi.org/10.1002/chem.202100803 SN - 0947-6539 SN - 1521-3765 VL - 27 IS - 36 SP - 9414 EP - 9421 PB - Wiley-VCH CY - Weinheim AN - OPUS4-53209 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -