PROCAL: A Set of 40 Peptide Standards for Retention Time Indexing,Column Performance Monitoring,and Collision Energy Calibration

Beyond specific applications, such as the relative or absolute quantification of peptides in targeted proteomic experiments, synthetic spike‐in peptides are not yet systematically used as internal standards in bottom‐up proteomics. A number of retention time standards have been reported that enable chromatographic aligning of multiple LC–MS/MS experiments. However, only few peptides are typically included in such sets limiting the analytical parameters that can be monitored. Here, we describe PROCAL (ProteomeTools Calibration Standard), a set of 40 synthetic peptides that span the entire hydrophobicity range of tryptic digests, enabling not only accurate determination of retention time indices but also monitoring of chromatographic separation performance over time. The fragmentation characteristics of the peptides can also be used to calibrate and compare collision energies between mass spectrometers. The sequences of all selected peptides do not occur in any natural protein, thus eliminating the need for stable isotope labeling. We anticipate that this set of peptides will be useful for multiple purposes in individual laboratories but also aiding the transfer of data acquisition and analysis methods between laboratories, notably the use of spectral libraries.     

Extraction,Distribution and Diversity of Phenolic Compounds in Most Widespread Boraginaceae Species from Macedonia

A systematic study of extraction efficiency of polyphenolic compounds from the most widespread Boraginaceae species was carried out. The optimal extraction was achieved with 50 % (V/V) methanol for phenolic acids and flavonoids, 0.2 % (V/V) HCl in 50 % (V/V) methanol for anthocyanins and pure water for flavan-3-ols. The distribution and diversity of polyphenolic compounds in plant material obtained from wild-growing Anchusa officinalis, Cynoglossum creticum Mill., Echium vulgare, Echium italicum, and Onosma heterophylla Griseb. species from Macedonia was also assessed. These widespread Boraginaceae species contain phenolic acid derivatives, flavonoids, flavan-3-ols and anthocyanins and in total 31 of them were identified, from which 22 were first identified in the representative species, and 6,8-di-C-glucosides of apigenin and luteolin were identified for the first time in Boraginaceae. The profiles of polyphenolic compounds for each sample were obtained and their phytochemical profile established. The potential for further bioactivity studies of Anchusa officinalis and Cynoglossum creticum containing up to 24577.05 μg/g and 14304.15 μg/g of total polyphenols were assumed to be highest, followed by Echium vulgare (from 6382.61 to 14114.33 μg/g), Onosma heterophylla (9463.97 μg/g) and Echium (4108.14 μg/g).     

Methanol Extracts of 28 Hieracium Species from the Balkan Peninsula – Comparative LC–MS Analysis,Chemosystematic Evaluation of their Flavonoid and Phenolic Acid Profiles and Antioxidant Potentials

Introduction

Hieracium s. str. represents one of the largest and most complex genera of flowering plants. As molecular genetics seems unlikely to disentangle intricate relationships within this reticulate species complex, analysis of flavonoids and phenolic acids, known as good chemosystematic markers, promise to be more reliable. Data about pharmacological activity of Hieracium species are scarce.

Objective

Evaluation of the chemosystematic significance of flavonoids and phenolic acids of methanol extracts of aerial flowering parts of 28 Hieracium species from the Balkans. Additionally, investigation of antioxidant potentials of the extracts.

Methods

Comparative qualitative and quantitative analysis of flavonoids and phenolic acids was performed by LC–MS. Multivariate statistical data analysis included non‐metric multidimensional scaling (nMDS), unweighted pair‐group arithmetic averages (UPGMA) and principal component analysis (PCA). Antioxidant activity was evaluated using three colorimetric tests.

Results

Dominant phenolics in almost all species were luteolin type flavonoids, followed by phenolic acids. Although the investigated Hieracium species share many compounds, the current classification of the genus was supported by nMDS and UPGMA analyses with a good resolution to the group level. Hieracium naegelianum was clearly separated from the other investigated species. Spatial and ecological distances of the samples were likely to influence unexpected differentiation of some groups within H. sect. Pannosa. The vast majority of dominant compounds significantly contributed to differences between taxa. The antioxidant potential of the extracts was satisfactory and in accordance with their phenolics composition.

Conclusions

Comparative LC–MS analysis demonstrated that flavonoids and phenolic acids are good indicators of chemosystematic relationships within Hieracium, particularly between non‐hybrid species and groups from the same location. Copyright © 2017 John Wiley & Sons, Ltd.     

Quantitative profiling of endocannabinoids and related compounds in rat brain using liquid chromatography-tandem electrospray ionization mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous identification and quantification of eight endocannabinoid (EC) or related "entourage" compounds in rat brain tissue. Analytes were extracted and purified from rat brain tissue using an ethyl acetate/hexane solvent extraction, followed by a solid phase extraction (SPE) protocol. Chromatographic separation was achieved using a gradient elution, with a mobile phase of acetonitrile, formic acid, and ammonium acetate, at pH 3.6. A Thermo Hypersil C8 HyPurity Advance column (100x2.1 mm i.d., 3 microm) was used with a flow rate of 0.3 ml/min). Anandamide (AEA), 2-arachidonyl glycerol (2-AG), 2-arachidonylglyceryl ether (noladin ether), O-arachidonyl ethanolamide (virodhamine), 2-linoleoyl glycerol (2-LG), arachidonyl glycine, oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA) were quantified by positive ion tandem electrospray ionization mass spectrometry. Internal standards were deuterated AEA, deuterated 2-AG, and heptadecanoyl ethanolamide (HEA). Linearity was proven over the range of 25 fmol to 250 pmol, with a limit of detection of 25 fmol on column for all analytes except 2-AG, noladin ether, and 2-LG (250 fmol). This corresponded to a limit of quantification in biological tissue of 10 pmol/g for all analytes except 2-AG (100 pmol/g). Intra- and interday precision in biological tissue was routinely approximately 20% or lower, and accuracy was between 65% and 155%. This method was used to quantitatively profile regional differences in nine discrete rat brain regions for AEA, 2-AG, 2-LG, OEA, PEA, noladin ether, virodhamine, and arachidonyl glycine.     

Enantioselective analyses of chloroquine and hydroxychloroquine in rat liver microsomes through chiral liquid chromatography–tandem mass spectrometry

An efficient, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) chiral analysis method was established for determination of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes. Effects of polysaccharide chiral stationary phases and basic additives on chiral separations of two analytes were discussed in detail. Amylose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase showed the best separation performance for them with acetonitrile-diethylamine-ethanol-diethylamine mixture (90:0.1:10:0.1, v/v/v/v) among four chiral stationary phases. Then, multiple reaction monitoring mode was selected as the data acquisition for determination of two pairs of enantiomers. The proposed LC–MS/MS chiral analysis method was validated in terms of linearity, accuracy, precision, and specificity. Good linearity with correlation coefficient over 0.998 was obtained in the concentration range of 0.05–5 μM. Limits of quantification for chloroquine and hydroxychloroquine enantiomers were 5.0 and 1.0 nM, respectively. The recoveries ranged from 81.14% to 111.09%. The intra-day and inter-day relative standard deviation were less than 6.5%. Moreover, concentrations of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes were determined through the proposed LC–MS/MS analysis method. After incubated with rat liver microsomes for 10 min, the enantiomeric factor of hydroxychloroquine decreased from 0.50 to 0.45 (p < 0.001). In brief, our developed determination method for chloroquine and hydroxychloroquine enantiomers through LC–MS/MS spectrometry showed the characteristics of high-efficiency, fast speed, and very low detection limit, and would be greatly beneficial for screening and quantitation of them in biological matrices.     

LC–MS/MS and chiroptical spectroscopic analyses of multidimensional metabolic systems of chiral thalidomide and its derivatives

Enantiomeric thalidomide undergoes various kinds of biotransformations including chiral inversion, hydrolysis, and enzymatic oxidation, which results in several metabolites, thereby adding to the complexity in the understanding of the nature of thalidomide. To decipher this complexity, we analyzed the multidimensional metabolic reaction networks of thalidomide and related molecules in vitro . Characteristic patterns in the amount of various metabolites of thalidomide and related molecules generated during a combination of chiral inversion, hydrolysis, and hydroxylation were observed using liquid chromatography–tandem mass spectrometry and chiroptical spectroscopy. We found that monosubstituted thalidomide derivatives exhibited different time‐dependent metabolic patterns compared with thalidomide. We also revealed that monohydrolyzed and monohydroxylated metabolites of thalidomide were likely to generate mainly by a C‐5 oxidation of thalidomide and subsequent ring opening of the hydroxylated metabolite. Since chirality was conserved in most of these metabolites during metabolism, they had the same chirality as that of nonmetabolized thalidomide. Our findings will contribute toward understanding the significant pharmacological effects of the multiple metabolites of thalidomide and its derivatives.     

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next‐Generation Sequencing Technologies for Cell Line Development

Early analytical clone screening is important during Chinese hamster ovary (CHO) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. Sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. Next‐generation sequencing (NGS) technologies emerged as alternatives for CHO clone analytics. We report an efficient NGS workflow applying the targeted locus amplification (TLA) strategy for genomic screening of antibody expressing CHO clones. In contrast to previously reported RNA sequencing approaches, TLA allows for targeted sequencing of genomic integrated transgenic DNA without prior locus information, robust detection of single‐nucleotide variants (SNVs) and transgenic rearrangements. During clone selection, TLA/NGS revealed CHO clones with high‐level SNVs within the antibody gene and we report in another case the utility of TLA/NGS to identify rearrangements at transgenic DNA level. We also determined detection limits for SNVs calling and the potential to identify clone contaminations by TLA/NGS. TLA/NGS also allows to identify genetically identical clones. In summary, we demonstrate that TLA/NGS is a robust screening method useful for routine clone analytics during cell line development with the potential to process up to 24 CHO clones in less than 7 workdays.