Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody-Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry.

The therapeutic efficacy and pharmacokinetics of antibody-drug conjugates (ADCs) in general, and antibody-oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis.

Microwave-Assisted Protein Staining, Destaining, and In-Gel/In-Solution Digestion of Proteins.

Rapid evolution of state-of-the-art proteomic analyses has encompassed development of high-throughput analytical instrumentation and bioinformatic tools. However, recently there has been a particular emphasis on increasing the throughput of sample preparation, which has become one of the rate-limiting steps in protein characterization workflows. Researchers have been investigating alternative methods to conventional convection oven incubations to try and reduce sample preparation time for protein characterization.

Engineering PQS biosynthesis pathway for enhancement of bioelectricity production in pseudomonas aeruginosa microbial fuel cells.

The biosynthesis of the redox shuttle, phenazines, in Pseudomonas aeruginosa, an ubiquitous microorganism in wastewater microflora, is regulated by the 2-heptyl-3,4-dihydroxyquinoline (PQS) quorum-sensing system. However, PQS inhibits anaerobic growth of P. aeruginosa.

Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels.

Glycosylphosphatidylinositol (GPI) anchor biosynthesis takes place in the endoplasmic reticulum (ER). After protein attachment, the GPI anchor is transported to the Golgi where it undergoes fatty acid remodeling. The ER exit of GPI-anchored proteins is controlled by glycan remodeling and p24 complexes act as cargo receptors for GPI anchor sorting into COPII vesicles.

Microwave-assisted protein staining, destaining, and in-gel/in-solution digestion of proteins.

Rapid evolution of state-of-the-art proteomic analyses has encompassed development of high-throughput analytical instrumentation and bioinformatic tools. However, recently, there has been a particular emphasis on increasing the throughput of sample preparation, which has become one of the rate-limiting steps in protein characterization workflows. Researchers have been investigating alternative methods to conventional convection oven incubations to try and reduce sample preparation time for protein characterization.

Global proteomics analysis of testis and ovary in adult zebrafish (Danio rerio).

The molecular mechanisms controlling sex determination and differentiation in zebrafish (Danio rerio) are largely unknown. A genome-wide analysis may provide comprehensive insights into the processes involved. The mRNA expression in zebrafish gonads has been fairly well studied, but much less data on the corresponding protein expression are available, although the proteins are considered to be more relevant markers of gene function.

Binding of silver nanoparticles to bacterial proteins depends on surface modifications and inhibits enzymatic activity.

Here we describe results from a proteomic study of protein-nanoparticle interactions to further the understanding of the ecotoxicological impact of silver nanoparticles (AgNPs) in the environment. We identified a number of proteins from Escherichia coli that bind specifically to bare or carbonate-coated AgNPs. Of these proteins, tryptophanase (TNase) was observed to have an especially high affinity for both surface modifications despite its low abundance in E.

On the acquisition of +1 charge states during high-throughput proteomics: Implications on reproducibility, number and confidence of protein identifications.

Modern high-throughput methods for the proteome analysis are gradually replacing more traditional 2D gel-based techniques. Almost immediately after the introduction of high-throughput proteomics techniques in 2001, reproducibility of the results became an issue. Extensive discussion in the literature led to the conclusion that certain "undersampling" exhibited during measurements could be due to the stochastic nature of the data-dependent sampling, routinely used with current mass spectrometry equipment.

Analysis of environmental stress response on the proteome level.

Thousands of man-made chemicals are annually released into the environment by agriculture, transport, industries, and other human activities. In general, chemical analysis of environmental samples used to assess the pollution status of a specific ecosystem is complicated by the complexity of the mixture, and in some cases by the very low toxicity thresholds of chemicals present. In that sense, a proteomics approach, capable of detecting subtle changes in the level and structure of individual proteins within the whole proteome in response to the altered surroundings, has obvious applications in the field of ecotoxicology.

Proteomics for the analysis of environmental stress responses in organisms.

Thousands of man-made chemicals are constantly released into the environment by agricultural and industrial production processes, traffic, and countless other human activities. Hence, very complex mixtures of anthropogenic chemicals and the transformation products of non-persistent compounds can be found in the aquatic environment. They reflect regional input but are also influenced by long-range transport.

Effect of cadmium on the interaction of 17beta-estradiol with the rainbow trout estrogen receptor.

The widely reported negative effects of xenoestrogens on the endocrine system of aquatic organisms gave raise to public concern and led to a number of screening and testing initiatives on the international level. Recent studies indicated that not only organic chemicals but also certain heavy metals, including cadmium, can mimic the effects of the endogenous estrogen receptor agonist 17beta-estradiol (E2) and lead to estrogen receptor activation. While the effects of cadmium on the endocrine system and its potential to harm living organisms are no longer in doubt, the exact mode of action is still essentially unknown.

Identification of the estrogen receptor Cd-binding sites by chemical modification.

The widely reported interactions of the estrogen receptor (ER) with endocrine disrupting chemicals (EDCs) present in the environment gave raise to public concern and led to a number of screening and testing initiatives on the international level. Recent studies indicated that certain heavy metals, including cadmium, can mimic the effects of the endogenous estrogen receptor agonist 17beta-estradiol, and lead to estrogen receptor activation. Previous studies of the chimeric proteins, which incorporate the ligand-binding domain of the human ER, identified Cys 381, Cys 447, Glu 523, His 524 and Asp 538 as possible sites of interactions with cadmium.

Recovery of intact proteins from silver stained gels.

Silver stained proteins of a wide molecular weight (MW) range (20-116 kDa) were successfully recovered by both electroblot and electroelution. The recovery was demonstrated for nanogram loads of proteins separated by SDS-PAGE and visualized by silver staining methods compatible and incompatible with mass spectrometry (MS). It was shown that the alcohol/acid and glutaraldehyde fixation steps present in a number of staining procedures did not prevent recovery of intact proteins from gels.

Microwave-assisted protein staining: mass spectrometry compatible methods for rapid protein visualisation.

The effects of microwave irradiation on the staining of electrophoresed and electroblotted proteins have been assessed using currently available detection methods. Although the absorption of microwave radiation was found to be uneven, band intensity following microwave-assisted protein staining (MAPS) was comparable and in some cases exceeded the intensity of the bands visualised by the original staining methods. It was found that microwave treatment drastically reduced the duration of the staining protocols for visualisation of the proteins separated by both one- and two-dimensional electrophoresis.