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. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap's unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process

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

NRC publication: Ye

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. We constructed a P. aeruginosa strain that produces higher concentrations of phenazines under anaerobic conditions by over-expressing the PqsE effector in a PQS negative ΔpqsC mutant. The engineered strain exhibited an improved electrical performance in microbial fuel cells (MFCs) and potentiostat-controlled electrochemical cells with an approximate five-fold increase of maximum current density relative to the parent strain. Electrochemical analysis showed that the current increase correlates with an over-synthesis of phenazines. These results therefore demonstrate that targeting microbial cell-to-cell communication by genetic engineering is a suitable technique to improve power output of bioelectrochemical systems

Electrical output of MFCs inoculated with various microbial strains.

<p>(A) Graph of averaged current density generated as a function of time by various microbial strains kept in incubator at 36°C. (B) Graph of MFC power curves with various microbial strains at 36°C. (C) Graph of averaged current density generated as a function of time by various microbial strains kept in incubator at 30°C.</p

Pyocyanin production by <i>P. aeruginosa</i> PAO1, ΔpqsC and ΔpqsC+pLG10.

<p>Pyocyanin production by <i>P. aeruginosa</i> PAO1, ΔpqsC and ΔpqsC+pLG10.</p

Observation of pyocyanin production in <i>P. aeruginosa</i> strains.

<p><i>P. aeruginosa</i> strains were grown in LB medium for 18 h, and aliquots of respective cultures were recorded to document the production of the blue-green pigment, pyocyanin.</p

Electrochemical analyses of various microbial strains.

<p>(A) Chronoamperometry of <i>P. aeruginosa</i> biofilms poised at potential of 400 mV vs. Ag/AgCl. (B) Differential pulse voltammetry of <i>P. aeruginosa</i> biofilms after 20 hr of growth poised at potential of 400 mV vs. Ag/AgCl. (C) Representative CVs of the four <i>P. aeruginosa</i> strains used in this study. The CV of the PAO1+sPqsE strain at 20 hr was truncated due to an off-scale condition of the potentiostat.</p

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. In this study, we have characterized the lipid profile of mammalian cell lines that have a defect in GPI anchor biosynthesis. Depending on which step of GPI anchor biosynthesis the cells were defective, we observed sphingolipid changes predominantly for very long chain monoglycosylated ceramides (HexCer). We found that the structure of the GPI anchor plays an important role in the control of HexCer levels. GPI anchor-deficient cells that generate short truncated GPI anchor intermediates showed a decrease in very long chain HexCer levels. Cells that synthesize GPI anchors but have a defect in GPI anchor remodeling in the ER have a general increase in HexCer levels. GPI-transamidase-deficient cells that produce no GPI-anchored proteins but generate complete free GPI anchors had unchanged levels of HexCer. In contrast, sphingomyelin levels were mostly unaffected. We therefore propose a model in which the transport of very long chain ceramide from the ER to Golgi is regulated by the transport of GPI anchor molecules