1.5 Proteinanalytik
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- 1.7 Organische Spuren- und Lebensmittelanalytik (5)
Paper des Monats
- ja (1)
Reusable enzyme carriers are valuable for proteomic workflows, yet many supports are expensive or lack robustness. This study describes the covalent immobilization of recombinant trypsin on micrometer-sized corundum particles and assesses their performance in protein digestion and antibody analysis. The corundum surface was cleaned with potassium hydroxide, silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde. Recombinant trypsin was then attached, and the resulting imines were reduced with sodium cyanoborohydride. Aromatic amino acid analysis (AAAA) estimated an enzyme loading of approximately 1 µg/mg. Non-specific adsorption of human plasma proteins was suppressed by blocking residual aldehydes with a Tris-glycine-lysine buffer. Compared with free trypsin, immobilization shifted the temperature optimum from 50 to 60 °C and greatly improved stability in 1 M guanidinium hydrochloride. Activity remained above 80 % across several reuse cycles, and storage at 4 °C preserved functionality for weeks. When applied to digesting the NISTmAb, immobilized trypsin provided peptide yields and sequence coverage comparable to soluble enzyme and outperformed it at elevated temperatures. MALDI-TOF MS analysis of Herceptin digests yielded fingerprint spectra that correctly identified the antibody and achieved >60 % sequence coverage. The combination of low cost, robustness and analytical performance makes corundum-immobilized trypsin an attractive option for research and routine proteomic workflows.
Multichannel biomarker detection using photonic integrated circuits on ultra compact CMOS chips
(2025)
This work presents the development of a real-time, multichannel detector for biomolecules based on photonic integrated circuits with super-compact CMOS chips. The presentation focuses on using micro-ring-resonators for bioassays, enabling highly integrated and scalable biosensing-solutions. The talk also covers major challenges like including microfluidic integration, data analysis, custom surface functionalization, and chip packaging.
A novel method for screening one-bead-one-peptide libraries is presented and discussed. The approach comprises the following steps: 1. The peptide library is synthesized on peptide beads with a 4-(hydroxymethyl)benzoic acid (HMBA) linker. 2. The beads are placed on glass slides equipped with electrically conductive double-sided adhesive tape using a precision sieve. 3. The chip is incubated with fluorescence-labeled target molecules (e.g., antibodies or receptors) and scanned with a fluorescence scanner to identify the positive beads. 4. The peptides are cleaved from the polymer beads using ammonia gas. 5. The MALDI matrix is applied using a spray gun. 6. The positive beads are sequenced using MALDI-ToF-MS.
In dieser Arbeit wurden zwei Trypsinvarianten, der Wildtyp (wtT) und eine thermostabile Variante (tsT), rekombinant hergestellt. Die Herstellung von Proteinen in Bakterien kann zu Fehlfaltungen und Einschlusskörperchen führen, weshalb eine geeignete Faltungsstrategie notwendig ist. In dieser Arbeit konnte eine schrittweise Vorgehensweise erfolgreich angewendet werden.
Nach der Ni-NTA-Aufreinigung wurde zusätzlich eine substratspezifische Benzamidin-Aufreinigung eingesetzt, um die Aktivität zu verbessern. Außerdem wurden die rekombinanten Trypsine auf Korund immobilisiert. Korund ist ein kostengünstiges Material, das nach Silanisierung und Glutaraldehyd-Kopplung für die Bindung von Proteinen geeignet gemacht wurde. Dabei konnte gezeigt werden, dass mehr wtT als tsT gebunden wurde, was sich auch in den Kopplungsdichte widerspiegelte.
Die immobilisierten Enzyme wurden in Bezug auf Aktivität, Wiederverwendbarkeit, Lagerfähigkeit und Temperaturstabilität untersucht. Beide Varianten zeigten vergleichbare Leistungen, wobei tsT bei höheren Temperaturen eine etwas bessere Stabilität aufwies. Für den Antikörperverdau wurden sowohl thermische als auch chemische Denaturierungen bei verschiedenen Temperaturen ( 37 °C und 60 °C) getestet. Die Analyse mit MALDI-TOF-MS zeigte, dass gute Werte für die Sequenzabdeckung erzielt werden konnten. Besonders die chemische Denaturierung führte zu besseren Ergebnissen mit weniger Selbstverdau.
Beim Vergleich mit kommerziell immobilisiertem Crude-Trypsin zeigten die rekombinanten Varianten eine höhere Spezifität und bessere Sequenzabdeckung bei gleichzeitig geringerem Selbstverdau. Die Wiederverwendbarkeit wurde durch fünf Verdauzyklen bei 37 °C überprüft. Dabei blieb die Aktivität weitgehend stabil, auch wenn die Peakintensität in den MALDI-Spektren mit jedem Zyklus abnahm. Eine eindeutige Zuordnung der Fingerprints des Antikörpers aus einer Bibliothek von über 90 Antikörpern war trotz dessen gegeben.
Objective: Resolve how peroxynitrite and O3/NO2 reshape tyrosine chemistry in Phl p 5 and modulate TLR4 activation. Design: Recombinant Phl p 5 underwent defined ONOO−:Y titrations and O3/NO2 exposures. ND, HOY-D, and dityrosine cross-links were quantified; modified residues were assigned; TLR4 responses were benchmarked to native.
Key results: Y285 exhibited highest susceptibility across pathways; Y236 remained unmodified. ND peaked at ONOO−:Y = 3:1 and at O3/NO2 = 10/200 ppb. The strongest hydroxylation arose with 200 ppb O3, predominating at Y112 and Y285. Cross-linking patterns diverged: ONOO− increased domain-1 connectivity while suppressing head/tail links; NO2 shifted cross-linking toward head/tail positions.
Biological effect: ONOO− did not raise TLR4 activity, whereas 200 ppb O3 produced a ~6% increase. Conclusion: Phl p 5 modification is residue- and chemistry-specific. Ozone-driven hydroxylation correlates with higher TLR4 signaling, while peroxynitrite-driven nitration/cross-linking leaves TLR4 unchanged. These relationships pinpoint reactive hotspots and suggest exposure-dependent mechanisms in pollutant-enhanced allergenicity.
Aus sicherer Entfernung
(2025)
Da chemische Gefahrstoffe im Boden eine ernsthafte Bedrohung für Leben und Gesundheit darstellen, das Grundwasser kontaminieren und langfristige Umweltschäden verursachen können, ist ihre frühzeitige Erkennung von großer Bedeutung. Um solche Gefahrstoffe aus sicherer Entfernung detektieren zu können, wurde bei der Bundesanstalt für Materialforschung und -prüfung (BAM) im Rahmen eines Forschungsvorhabens seit Anfang 2022 an einem neuen Ansatz zur Erkennung und Überwachung chemischer Gefahrstoffe gearbeitet.
We report the full-length nucleotide and amino acid sequences of the light (κ) and heavy chain of the IgG2c anti-CCMV monoclonal antibody (hybridoma clone BAM-CCMV-29-81), generated against Cowpea Chlorotic Mottle Virus (CCMV). Sequencing of hybridoma clone was performed using a cost-effective Sanger-based workflow that includes DNA-level subclass determination and peptide mass fingerprint confirmation. The κ-chain sequence was assigned to IGKV4-6101 / IGKJ101, while the heavy chain was assigned to IGHV2-601 / IGHD1-101 / IGHJ3*01 with IgG2c constant regions from the NOD strain. Both chains were validated against RNA Illumina sequencing, confirming 100% identity. The sequences are available in GenBank under accession numbers PX123807 (κ-chain) and PX123808 (heavy chain).
Cost-Effective Method for Full-Length Sequencing of Monoclonal Antibodies from Hybridoma Cells
(2025)
Background: Monoclonal antibodies play an important role in therapeutic and analytical applications. For recombinant expression, the coding sequences of the variable regions of the heavy and light chains are required. In addition, cloning antibody sequences, including constant regions, reduces the impact of hybridoma cell loss and ensures preservation of the naturally occurring full antibody sequence. Method: We combined amplification of IgG antibody variable regions from hybridoma mRNA with an advanced method for fulllength cloning of monoclonal antibodies in a simple two-step workflow. Following Sanger sequencing and evaluation of consensus sequences, the best matching variable, diversity,
and joining (V-(D-)J) gene segments were identified according to identity scores from IgBLAST reference sequences. Simultaneously, the mouse IgG subclass was determined at the DNA level based on isotype-specific sequence patterns in the CH1 domain. Knowing the DNA sequence of V-(D-)J recombination responsible for the complementary determining region 3 (CDR 3), variable region-specific primers were designed and used to amplify the corresponding antibody constant regions. Results: To verify the approach, we applied it to the hybridoma clone BAM-CCMV-29-81 and obtained identical full-length antibody
sequences as with RNA Illumina sequencing. Further validation at the protein level using an established MALDI-TOF MS-fingerprinting protocol showed that five out of six genetically encoded CDR domains of the monoclonal antibody BAM-CCMV-29-81 could be efficiently correlated. Conclusion: This simple, streamlined method enables the cost-effective determination of the full-length sequence of monoclonal antibodies from hybridoma cell lines, with the added benefit of obtaining the DNA sequence of the antibody ready for recombinant expression.
This work presents the real-time, multichannel detection of the biomolecule Neutravidine using photonic integrated circuits on ultra-compact monolithic CMOS chips. The development focuses on implementing bioassays directly on CMOS platforms, enabling highly integrated and scalable biosensing. The presentation will cover key aspects including microfluidic integration, data analysis, surface functionalization, and chip packaging.
Photonic micro-ring resonators (MRR) are widely studied for their high sensitivity across applications like environmental monitoring, healthcare, and chemical analysis. Their evanescent field sensing requires partially unembedded waveguides compatible with CMOS processing. Our approach uses local backside etching with an additional buried oxide (BOX) etch to release waveguides while preserving the back-end of line (BEOL) structure, enabling spatial separation of the sensing area and electronics. The BOX etch critically affects sensor performance, as waveguide surface roughness can alter MRR properties and coupling. We analyzed MRR design variations, comparing wet and dry etching techniques for their effects on optical performance across rib and strip waveguides in quasi-TE and quasi-TM modes. Wafer-level measurements show that backside-released MRR achieve high extinction ratios with slightly reduced quality factors, advancing high-sensitivity photonic sensors.