Purification and characterization of Fe-containing superoxide dismutase from Methanobrevibacter arboriphilus strain AZ

Superoxide dismutase (SOD) was purified from cells of the strict anaerobic methanogenic archaeon Methanobrevibacter arboriphilus strain AZ. The four-step purification procedure resulted in enzyme with specific activity of 3970 units/mg and yield of 22%. It was shown that the SOD is a Fe-containing homotetramer composed of subunits of 21.2 kD each. Sodium azide (13.5 mM), unlike KCN, inhibits the activity of the SOD. Hydrogen peroxide (0.5 mM) inactivates the enzyme, which is consistent with the properties of the known Fe-containing SODs from methanogenic Archae

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. Because mRNA and protein abundances rarely correlate well, mRNA profiles need to be complemented with the information on protein expression. The work presented here analyzed the proteomes of adult zebrafish gonads by a multidimensional protein identification technology, generating the to-date most populated lists of proteins expressed in mature zebrafish gonads. The acquired proteomics data partially confirmed existing transcriptomics information for several genes, including several novel transcripts. However, disagreements between mRNA and protein abundances were often observed, further stressing the necessity to assess the expression on different levels before drawing conclusions on a certain gene's expression and function. Several gene groups expressed in a sexually dimorphic way in zebrafish gonads were identified. Their potential importance for gonad development and function is discussed. The data gained in the current study provide a basis for further work on elucidating processes occurring during zebrafish development with use of high-throughput proteomic

Methods for Collecting Milk from Mice

Mouse models offer unique opportunities to study mammary gland biology and lactation. Phenotypes within the mammary glands, especially those caused by genetic modification, often arise during lactation, and their study requires the collection of adequate volumes of milk. We describe two approaches for collecting milk from lactating mice. Both methods are inexpensive, are easy to use in the laboratory or classroom, are non-invasive, and yield adequate volumes of milk for subsequent analyses

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