Relative quantitation of transfer RNAs using liquid chromatography mass spectrometry and signature digestion products

Transfer ribonucleic acids (tRNAs) are challenging to identify and quantify from unseparated mixtures. Our lab previously developed the signature digestion approach for identifying tRNAs without specific separation. Here we describe the combination of relative quantification via enzyme-mediated isotope labeling with this signature digestion approach for the relative quantification of tRNAs. These quantitative signature digestion products were characterized using liquid chromatography mass spectrometry (LC-MS), and we find that up to 5-fold changes in tRNA abundance can be quantified from sub-microgram amounts of total tRNA. Quantitative tRNA signature digestion products must (i) incorporate an isotopic label during enzymatic digestion; (ii) have no m/z interferences from other signature digestion products in the sample and (iii) yield a linear response during LC-MS analysis. Under these experimental conditions, the RNase T1, A and U2 signature digestion products that potentially could be used for the relative quantification of Escherichia coli tRNAs were identified, and the linearity and sequence identify of RNase T1 signature digestion products were experimentally confirmed. These RNase T1 quantitative signature digestion products were then used in proof-of-principle experiments to quantify changes arising due to different culturing media to 17 tRNA families. This method enables new experiments where information regarding tRNA identity and changes in abundance are desired

Glycosylation Patterns of Proteins Studied by Liquid Chromatography-Mass Spectrometry and Bioinformatic Tools

Due to their extensive structural heterogeneity, the elucidation of glycosylation patterns in glycoproteins such as the subunits of chorionic gonadotropin (CG), CG-alpha and CG-beta remains one of the most challenging problems in the proteomic analysis of posttranslational modifications. In consequence, glycosylation is usually studied after decomposition of the intact proteins to the proteolytic peptide level. However, by this approach all information about the combination of the different glycopeptides in the intact protein is lost. In this study we have, therefore, attempted to combine the results of glycan identification after tryptic digestion with molecular mass measurements on the intact glycoproteins. Despite the extremely high number of possible combinations of the glycans identified in the tryptic peptides by high-performance liquid chromatography-mass spectrometry (> 1000 for CG-alpha and > 10.000 for CG-beta), the mass spectra of intact CG-alpha and CG-beta revealed only a limited number of glycoforms present in CG preparations from pools of pregnancy urines. Peak annotations for CG-alpha were performed with the help of an algorithm that generates a database containing all possible modifications of the proteins (inclusive possible artificial modifications such as oxidation or truncation) and subsequent searches for combinations fitting the mass difference between the polypeptide backbone and the measured molecular masses. Fourteen different glycoforms of CG-alpha, including methionine-oxidized and N-terminally truncated forms, were readily identified. For CG-beta, however, the relatively high mass accuracy of ± 2 Da was still insufficient to unambiguously assign the possible combinations of posttranslational modifications. Finally, the mass spectrometric fingerprints of the intact molecules were shown to be very useful for the characterization of glycosylation patterns in different CG preparations

Circadian Intraocular Pressure Profiles in Chronic Open Angle Glaucomas

Purpose: To evaluate circadian intraocular pressure (IOP) profiles in eyes with different types of chronic open-angle glaucoma (COAG) and normal eyes. Methods: This study included 3,561 circadian IOP profiles obtained from 1,408 eyes of 720 Caucasian individuals including glaucoma patients under topical treatment (1,072 eyes) and normal subjects (336 eyes). IOP profiles were obtained by Goldmann applanation tonometry and included measurements at 7 am, noon, 5 pm, 9 pm, and midnight. Results: Fluctuations of circadian IOP in the secondary open-angle glaucoma (SOAG) group (6.96±3.69 mmHg) was significantly (P<0.001) higher than that of the normal pressure glaucoma group (4.89±1.99 mmHg) and normal eyes (4.69±1.95 mmHg); but the difference between the two latter groups was not significant (P=0.47). Expressed as percentages, IOP fluctuations did not vary significantly among any of the study groups. Inter-ocular IOP difference for any measurement was significantly (P<0.001) smaller than the profile fluctuations. In all study groups except the SOAG group, IOP was highest at 7 am, followed by noon, 5 pm, and finally 9 pm or midnight. In the SOAG group, mean IOP measurements did not vary significantly during day and night. Conclusions: In contrast to normal eyes and eyes with primary open-angle glaucoma under topical antiglaucoma treatment, eyes with SOAG under topical treatment do not show the usual circadian IOP profile in which the highest IOP values occur in the morning, and the lowest in the evening or at midnight. These findings may have implications for timing of tonometry. Fluctuation of circadian IOP was highest in SOAG compared to other types of open angle glaucomas

Impact of different hydrophobic ion pairs of octreotide on its oral bioavailability in pigs

The objective of this study was to investigate the impact of different hydrophobic ion pairs (HIP) on the oral bioavailability of the model drug octreotide in pigs. Octreotide was ion paired with the anionic surfactants deoxycholate, decanoate and docusate differing in lipophilicity. These hydrophobic ion pairs were incorporated in self-emulsifying drug delivery systems (SEDDS) based on BrijO10, octyldodecanol, propylene glycol and ethanol in a concentration of 5 mg/ml. SEDDS were characterized regarding size distribution, zeta potential, stability towards lipase, log DSEDDS/release medium and mucus diffusion behavior. The oral bioavailability of octreotide was evaluated in pigs via LC-MS/MS analyses. Most efficient ion pairing was achieved at a molar ratio of 1:3 (peptide: surfactant). SEDDS containing the octreotide-deoxycholate, -decanoate and -docusate ion pair exhibited a mean droplet size of 152 nm, 112 nm and 191 nm and a zeta potential of − 3.7, − 4.6 and − 5.7 mV, respectively. They were completely stable towards degradation by lipase and showed a log DSEDDS/release medium of 1.7, 1.8 and 2.7, respectively. The diffusion coefficient of these SEDDS was in the range of 0.03, 0.11 and 0.17 × 10− 9 cm2/s, respectively. In vivo studies with these HIPs showed no improvement in the oral bioavailability in case of octreotide-decanoate. In contrast, octreotide-deoxycholate and octreotide-docusate SEDDS resulted in a 17.9-fold and 4.2-fold higher bioavailability vs. control. According to these results, hydrophobic ion pairing could be identified as a key parameter for SEDDS to achieve high oral bioavailability

Annotating Nontargeted LC-HRMS/MS Data with Two Complementary Tandem Mass Spectral Libraries

Tandem mass spectral databases are indispensable for fast and reliable compound identification in nontargeted analysis with liquid chromatography–high resolution tandem mass spectrometry (LC-HRMS/MS), which is applied to a wide range of scientific fields. While many articles now review and compare spectral libraries, in this manuscript we investigate two high-quality and specialized collections from our respective institutes, recorded on different instruments (quadrupole time-of-flight or QqTOF vs. Orbitrap). The optimal range of collision energies for spectral comparison was evaluated using 233 overlapping compounds between the two libraries, revealing that spectra in the range of CE 20–50 eV on the QqTOF and 30–60 nominal collision energy units on the Orbitrap provided optimal matching results for these libraries. Applications to complex samples from the respective institutes revealed that the libraries, combined with a simple data mining approach to retrieve all spectra with precursor and fragment information, could confirm many validated target identifications and yield several new Level 2a (spectral match) identifications. While the results presented are not surprising in many ways, this article adds new results to the debate on the comparability of Orbitrap and QqTOF data and the application of spectral libraries to yield rapid and high-confidence tentative identifications in complex human and environmental samples

The effects of midazolam on intraocular pressure in children during examination under sedation

Background: To obtain reliable and accurate measurements of the intraocular pressure (IOP) in children often requires sedation or anaesthesia. Therefore, we investigated the effects of oral midazolam on IOP in children. Methods: In a prospective study, IOP was measured in 72 eyes of 36 cooperative children without glaucoma requiring general anaesthesia (mean age 3.5±1.3 years, body weight ≤20 kg) by using a Perkins hand-held tonometer. Measurements of IOP were performed before, and 15 and 30 min after sedation with orally administered midazolam (1 mg/kg) given as preoperative medication, and 5 and 15 min after induction of general anaesthesia. The individual IOP courses were analysed. Results: In all of the cooperative children, IOP measurement was possible after sedation with midazolam. Mean IOP was 11.2±0.3 mmHg before sedation, 10.9±0.2 mmHg at 15 min, and 10.7±0.3 mmHg 30 min after administration of midazolam. This small decrease was not statistically significant, whilst the IOP decline at 5 and 15 min after induction of general anaesthesia was statistically significant (p<0.0001). Conclusion: Sedation with midazolam can be assumed to be an applicable, well-tolerated, safe method for IOP measurements in children

Isradipine therapy in Cacna1dIle772Met/+ mice ameliorates primary aldosteronism and neurologic abnormalities

Somatic gain-of-function mutations in the L-type calcium channel CaV1.3 (CACNA1D gene) cause adrenal aldosterone-producing adenomas and micronodules. De novo germline mutations are found in a syndrome of primary aldosteronism, seizures, and neurologic abnormalities (PASNA) as well as in autism spectrum disorder. Using CRISPR/Cas9, we here generated mice with a Cacna1d gain-of-function mutation found in both adenomas and PASNA syndrome (Cacna1dIle772Met/+). These mice show reduced body weight and increased mortality from weaning to approximately 100 days of age. Male mice do not breed, likely due to neuromotor impairment, and the offspring of female mice die perinatally, likely due to lack of maternal care. Mice generated by in vitro fertilization showed elevated intracellular calcium in the aldosterone-producing zona glomerulosa, an elevated aldosterone/renin ratio, and persistently elevated serum aldosterone on a high-salt diet as signs of primary aldosteronism. Anesthesia with ketamine and xylazine induced tonic-clonic seizures. Neurologic abnormalities included hyperlocomotion, impaired performance in the rotarod test, impaired nest building, and slight changes in social behavior. Intracellular calcium in the zona glomerulosa, aldosterone levels, and rotarod performance responded to treatment with the calcium channel blocker isradipine, with implications for the therapy of patients with aldosterone-producing lesions and with PASNA syndrome