High sensitivity LC-MS profiling of antibody-drug conjugates with difluoroacetic acid ion pairing

ABSTRACT

Reversed-phase liquid chromatography (RPLC) separations of proteins using optical detection generally use trifluoroacetic acid (TFA) because it is a strong, hydrophobic acid and a very effective ion-pairing agent for minimizing chromatographic secondary interactions. Conversely and in order to avoid ion suppression, analyses entailing mass spectrometry (MS) detection is often performed with a weaker ion-pairing modifier, like formic acid (FA), but resolution quality may be reduced. To gain both the chromatographic advantages of TFA and the enhanced MS sensitivity of FA, we explored the use of an alternative acid, difluoroacetic acid (DFA). This acid modifier is less acidic and less hydrophobic than TFA and is believed to advantageously affect the surface tension of electrospray droplets. Thus, it is possible to increase MS sensitivity threefold by replacing TFA with DFA. Moreover, we have observed DFA ion pairing to concomitantly produce higher chromatographic resolution than FA and even TFA. For this reason, we prepared and used MS-quality DFA in place of FA and TFA in separations involving IdeS digested, reduced NIST mAb and a proprietary antibody-drug conjugate (ADC), aiming to increase sensitivity, resolution and protein recovery. The resulting method using DFA was qualified and applied to two other ADCs and gave heightened sensitivity, resolution and protein recovery versus analyses using TFA. This new method, based on a purified, trace metal free DFA, can potentially become a state-of-the-art liquid chromatography-MS technique for the deep characterization of ADCs.     

An Evidence for the Repair of Double-Strand Scissions in DNA of Micrococcus radiodurans after Alpha Bombardment

The conversion of prochaetoglobosins as plausible precursors into mycotoxin chaetoglobosin A (1) in a cell-free system of Chaetomium subaffine was unsuccessful. However, reductase activity of the 20-keto-analogues (1), and prochaetoglobosins II (5) and III (6) were found in a microsomal fraction of this fungi. Two new metabolites of chaetoglobosins, named chaetoglobosin F_ex (2) and 20-dihydro-chaetoglobosin A (3), were also isolated from the same micro-organisms. Their structures were elucidated by spectroscopic data and chemical transformation.     

Phenylalanine ammonia-lyase and cell wall peroxidase are cooperatively involved in the extensive formation of ferulate network in cell walls of developing rice shoots

The relationship between the formation of cell wall-bound ferulic acid (FA) and diferulic acid (DFA) and the change in activities of phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) was studied in rice shoots. The length and the fresh mass of shoots increased during the growth period from day 4 to 6, while coleoptiles ceased elongation growth on day 5. The amounts of FA and DFA isomers as well as cell wall polysaccharides continued to increase during the whole period. The activities of PAL and CW-PRX greatly increased in the same manner during the period. There were close correlations between the PAL activity and ferulate content or between the CW-PRX activity and DFA content. The expression levels of investigated genes for PAL and putative CW-PRX showed good accordance with the activities of these enzymes. These results suggest that increases in PAL and CW-PRX activities are cooperatively involved in the formation of ferulate network in cell walls of rice shoots and that investigated genes may be, at least in part, associated with the enzyme activities. The substantial increase in such network probably causes the maturation of cell walls and thus the cessation of elongation growth of coleoptiles.     

Purification and characterization of a heat stable inulin fructotransferase (DFA I-producing) from Arthrobacter pascens a62-1

An inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter pascens a62-1 was purified and the properties of the enzyme were investigated. The enzyme was purified from culture supernatant of the microorganism 58.5 fold with a yield of 8.32% using Super Q Toyopearl chromatography and butyl Toyopearl chromatography. It showed maximum activity at pH 5.5 and 45 °C and was stable up to 75 °C. This heat stability was highest in the inulin fructotransferases (DFA I-producing) reported until now. The molecular mass of the enzyme was estimated to be 37,000 by SDS-PAGE and 60,000 by gel filtration, and was considered to be a dimer. The N-terminal amino acid sequence (20 amino acid residues) was determined as Ala-Asn-Thr-Val-Tyr-Asp-Val-Thr-Thr-Trp-Ser-Gly-Ala-Thr-Ile-Ser-Pro-Tyr-Val-Asp.     

NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium isolated in cloud water

Bacillus sp. 3B6, bacterium isolated from cloud water, was incubated on sucrose for exopolysaccharide production. Dialysis of the obtained mixture (MWCO 500) afforded dialyzate (DIM) and retentate (RIM). Both were separated by size exclusion chromatography. RIM afforded eight fractions: levan exopolysaccharide (EPS), fructooligosaccharides (FOSs) of levan and inulin types with different degrees of polymerization (dp 2–7) and monosaccharides fructose:glucose = 9:1. Levan was composed of two components with molecular mass ∼3500 and ∼100 kDa in the ratio 2.3:1. Disaccharide fraction contained difructose anhydride DFA IV. 1-Kestose, 6-kestose, and neokestose were identified as trisaccharides in the ratio 2:1:3. Fractions with dp 4–7 were mixtures of FOSs of levan (2,6-βFruf) and inulin (1,2-βFruf) type. DIM separation afforded two dominant fractions: monosaccharides with fructose: glucose ratio 1:3; disaccharide fraction contained sucrose only. DIM trisaccharide fraction contained 1-kestose, 6-kestose, and neokestose in the ratio1.5:1:2, penta and hexasaccharide fractions contained FOSs of levan type (2,6-βFruf) containing α-glucose. In the pentasaccharide fraction also the presence of a homopentasaccharide composed of 2,6-linked βFruf units only was identified. Nystose, inulin (1,2-βFruf) type, was identified as DIM tetrasaccharide. Identification of levan 2,6-βFruf and inulin 1,2-βFruf type oligosaccharides in the incubation medium suggests both levansucrase and inulosucrase enzymes activity in Bacillus sp. 3B6.     

Discrimination of the sibling species Apodemus flavicollis and A. sylvaticus (Rodentia,Muridae)

Karyotyping and several molecular methods have allowed successful identification of two morphologically similar wide-ranging Western Palearctic species, the yellow-necked field mouse Apodemus flavicollis (Melchior, 1934) and the long-tailed wood mouse A. sylvaticus (Linnaeus, 1758), but reliable species diagnosis on the basis of morphometric characters is particularly problematic. Although they are easily morphologically distinguishable in Central and Northern Europe, this is not the case in southern parts of their distribution areas. Despite that, we have successfully discriminated A. flavicollis and A. sylvaticus from Serbia (Southern Europe) using geometric and traditional morphometric methods on a data set for ventral crania of specimens previously genotyped by the Inter Simple Sequence Repeat-PCR (ISSR-PCR). Discrimination power of applied approaches was more or less similar. The majority of our results were consistent with those obtained for specimens collected across the Czech Republic (Central Europe). Morphological differences observed herein, as well as those already reported between A. flavicollis and A. sylvaticus from the central and northern parts of their distribution areas, could be the outcome of their biology, i.e. ecological discrepancies, different assumed evolutionary scenarios considering biogeography, phylogeny, history and ontogeny.     

Use of native woodlands and traditional olive groves by foraging bats on a Mediterranean island: consequences for conservation

We recorded bat activity on Zakynthos island (Greece) to test the hypotheses that (1) olive ( Olea europea ) groves and native woodlands provide comparable foraging habitat for insectivorous bats, (2) lower foraging activity occurs in olive groves treated with insecticide chemicals. We acoustically sampled bat activity (passes per minute) in four wooded habitats (organic and non-organic olive groves, oak woodland ( Quercus ilex and Quercus coccifera ) and pine ( Pinus halepensis ) woodland from June to August 2005. Habitat type did not affect overall bat activity. A single application of insecticide chemicals annually did not affect activity over traditional olive groves. Habitat use on the island differed in several ways from that reported in studies at mainland sites. Most strikingly, pine woodland supported higher bat activity than expected relative to other habitat types, and we recorded unexpectedly high levels of M. capaccinii activity in woodland habitats. We suggest that traditional olive groves buffer some bat species from the effects of deforestation. Conservation plans for Mediterranean bats should consider the biodiversity value of these groves along with the need to conserve small woodland patches. Finally, understanding island-specific patterns of habitat use is essential to bat conservation on small off-shore islands.     

Molecular cloning of levan fructotransferase gene from Arthrobacter ureafaciens K2032 and its expression in Escherichia coli for the production of difructose dianhydride IV

AIMS: To clone and overexpress a novel levan fructotransferase gene lftA from Arthrobacter ureafaciens K2032. METHODS AND RESULTS: The lftA gene, encoding a levan fructotransferase (LFTase) of 521 amino acids (aa) residues, was cloned from the genomic DNA of A. ureafaciens K2032, and overexpressed in Escherichia coli. The recombinant LFTase overexpressed in E. coli was then used to produce a difructose dianhydride (DFA IV) from levan. DFA IV crystals with 97% purity could be obtained from the reaction mixture in 83.7% yield by using a natural crystallization method. CONCLUSIONS: The lftA gene cloned from A. ureafaciens K2032 encode a novel levan fructotransferase which produces difructose dianhydride (DFA IV) from levan. SIGNIFICANCE AND IMPACT OF THE STUDY: Levan fructotransferase is a useful enzyme with great promise in the production of DFA IV and various fructosides.     

Amphotericin B Resistance Assay for the Detection of Cholesterol Biosynthesis Inhibitors

Three different types of cholesterol biosynthesis inhibitors, lovastatin, ketoconazole, and 25-hydroxycholesterol, showed their respective resistance against amphotericin B (AmB) cytotoxicity in Chinese hamster ovary (CHO)-K1 cells. The negative correlation between the acquisition of AmB resistance and the decrease of cellular cholesterol content by the inhibitor was confirmed.     

Cell wall oxalate oxidase modifies the ferulate metabolism in cell walls of wheat shoots

Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide.