Cold-induced phase separation for the simple and reliable extraction of sex hormones for subsequent LC-MS/MS analysis

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at ?30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

Comparative Network Biology Discovers Protein Complexes That Underline Cellular Differentiation in Anabaena sp.

The filamentous cyanobacterium Anabaena sp. PCC 7120 can differentiate into heterocysts to fix atmospheric nitrogen. During cell differentiation, cellular morphology and gene expression undergo a series of significant changes. To uncover the mechanisms responsible for these alterations, we built protein–protein interaction (PPI) networks for these two cell types by cofractionation coupled with mass spectrometry. We predicted 280 and 215 protein complexes, with 6322 and 2791 high-confidence PPIs in vegetative cells and heterocysts, respectively. Most of the proteins in both types of cells presented similar elution profiles, whereas the elution peaks of 438 proteins showed significant changes. We observed that some well-known complexes recruited new members in heterocysts, such as ribosomes, diflavin flavoprotein, and cytochrome c oxidase. Photosynthetic complexes, including photosystem I, photosystem II, and phycobilisome, remained in both vegetative cells and heterocysts for electron transfer and energy generation. Besides that, PPI data also reveal new functions of proteins. For example, the hypothetical protein Alr4359 was found to interact with FraH and Alr4119 in heterocysts and was located on heterocyst poles, thereby influencing the diazotrophic growth of filaments. The overexpression of Alr4359 suspended heterocyst formation and altered the pigment composition and filament length. This work demonstrates the differences in protein assemblies and provides insight into physiological regulation during cell differentiation.     

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

Phytogenic compounds from avocado (Persea americana L.) extracts; antioxidant activity,amylase inhibitory activity,therapeutic potential of type 2 diabetes

Diabetes is a worldwide public health disease. Currently, the most effective way to treat diabetes is to mitigate postprandial hyperglycemia by inhibiting carbohydrate hydrolysis enzymes in the digestive system. Plant extracts are rich in bioactive compounds, which can be used in diabetes treatment. This study aims to evaluate the polyphenols content in ethanolic extracts of avocado fruit and leaves (Persea americana Mill.). Additionally, their antioxidant activity using DPPH, while the inhibition ability of α-amylase was examined by reacting different amounts of the extracts with α-amylase compared to acarbose as standard inhibitor. The active compounds were detected in the extracts by LC/MS. The obtained results showed that the leaf extract recorded a significant content of total phenolic compounds compared to the fruit extract (178.95 and 145.7 mg GAE /g dry weight, respectively). The total flavonoid values ??ranged from 32.5 to 70.08 mg QE/g dry weight of fruit and leaves extracts, respectively. Twenty-six phytogenic compounds were detected in leaf and fruit extract by LC/MS. These compounds belong to fatty acids, sterols, triterpenes, phenolic acids, and flavonoids. The antioxidant activity of the extracts is due to the exist of phytogenic compounds, i.e., polyphenols and flavonoids. The antioxidant activity increased in a concentration dependant manner. Avocado fruit extract (1000 µg/mL) scavenged 95% of DPP? while leaf extract rummaged 91.03% of free radicals compared with Vit C and BHT. Additionally, higher α-amylase inhibitory activity was observed in fruit extract than the leaf extract, where the fruit and leaf extract (1000 μg/ml) inhibited the enzyme by 92.13% and 88.95%, respectively. The obtained results showed that the ethanolic extracts of avocado could have a significant impact on human health due to their high content of polyphenols.     

UHPLC-MS/MS-GNPS based phytochemical investigation of Equisetum arvense L. And evaluation of cytotoxicity against human melanoma and ovarian cancer cells

Equisetum arvense L. is widely used as a traditional medicine for the management of inflammation and cancer. In the present study, phyto-chemical analysis of E. arvense was carried out and its cytotoxic potential against human melanoma (MDA-MB-435) and ovarian cancer cells (OVCAR3) was evaluated. Phyto-chemical profile of E. arvense methanolic extract and its fractions was established employing UHPLC-MS/MS and Global Natural Product Social molecular networking. Cytotoxic activity was evaluated using absorbance assay (CellTiter-Blue® Cell Viability Assay). Overall, 22 compounds were identified in the crude extract and polarity-based fractions of E. arvense. Flavonoids, flavonoid-O-glycosides and phenolic acids were found to be the major classes of phyto-chemicals. In addition, the crude extract of E. arvense and its fractions were found active against the tested cell lines. The highest anti-cancer activity against OVCAR3 cells was exhibited by the n-hexane fraction. These results indicated that E. arvense is rich in flavonoids and might be used for the development of anti-cancer drugs against melanoma and ovarian cancers.     

Creatine and creatinine quantification in olympic athletes: dried blood spot analysis pilot study

Capillary dried blood spot (DBS) samples facilitate field-based collection without venipuncture. This pilot study aims to evaluate the viability of creatine (Cr) and creatinine (Crt) quantification using fresh capillary serum (Cr_S/Crt_S) and DBS samples (Cr_DBS/Crt_DBS), using Flow Injection Analysis Mass Spectrometry (FIA – MS). Nine Olympic Athletes provided a capillary blood sample to assess Cr_S/Crt_S and Cr_DBS/Crt_DBS quantified by FIA – MS. No difference between Crt_S (mean ± SD: 813.6 ± 102.4 μmol/L) and Crt_DBS (812.4 ± 108.1 μmol/L) was observed with acceptable variance [SEM 88.7; CV 10.7%; ICC 0.57 (CI 95% 0.06 – 0.84)] and agreement [very strong (Spearman: r = 0.77; p < 0.01) or strong (Pearson: r = 0.56; p = 0.04); Bland Altman: lower (-193) and upper (+196) limits of agreement]. Cr_S (mean ± SD: 691.8 ± 165.2 μmol/L) was significantly different to Cr_DBS (2911 ± 571.4 μmol/L) with unacceptable variance [SEM 171.6; CV 27%; ICC 0.002 (CI 95% -0.02 – 0.07)] and ‘weak’ agreement [Spearman: r = 0.21, p = 0.47 and Pearson: r = 0.06, p = 0.84; Bland Altman lower (-3367) and upper (-1072) limits of agreement]. Crt quantification is viable using both Crt_S and Crt_DBS (but not for Cr and Cr_S/Cr_DBS), with the DBS tissue handling technique offering several methodological and practice facing advantages. Future work should expand upon the sample size, explore sport/discipline relevant analytes across a full competitive season, including key training, recovery and performance blocks of their periodized performance plan.     

Determination of cefazedone in human plasma by high performance liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study on Chinese volunteers

A rapid, sensitive and simple high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for determination of cefazedone in human plasma using metronidazole as internal standard (IS). The chromatographic separation was achieved on an Ultimate XB-CN column (2.1 mm × 150 mm, 5 μm) with an isocratic mobile phase of acetonitrile and 20 mM ammonium acetate in 0.1% formic acid in water (15:85, v/v). Detection was performed using electrospray ionization in positive ion multiple reaction-monitoring mode (SRM), monitoring the transitions m/z 548.2 → 344.1 for cefazedone and m/z 172.2 → 128.1 for IS. Calibration curves were linear over a wide range of 0.20–401.12 μg/mL for cefazedone in plasma. The lower limit of quantification (LLOQ) was 0.20 μg/mL. The intra- and inter-day precisions were less than 7.2%. The average recovery of cefazedone was 90.8–91.0%. The validated method was successfully applied to the pharmacokinetic study of cefazedone in Chinese healthy volunteers following intravenous (IV) administration of 500, 1000 and 2000 mg cefazedone injection.     

Selective ϖ-1 oxidation of fatty acids by CYP147G1 from Mycobacterium marinum

Background

Cyp147G1 is one of 47 cytochrome P450 encoding genes in Mycobacterium marinum M, a pathogenic bacterium with a high degree of sequence similarity to Mycobacterium tuberculosis and Mycobacterium ulcerans. Cyp147G1 is one of only two of these cyp genes which are closely associated with a complete electron transfer system.

Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris

Lipid droplets (LD) are the main depot of non-polar lipids in all eukaryotic cells. In the present study we describe isolation and characterization of LD from the industrial yeast Pichia pastoris. We designed and adapted an isolation procedure which allowed us to obtain this subcellular fraction at high purity as judged by quality control using appropriate marker proteins. Components of P. pastoris LD were characterized by conventional biochemical methods of lipid and protein analysis, but also by a lipidome and proteome approach. Our results show several distinct features of LD from P. pastoris especially in comparison to Saccharomyces cerevisiae. P. pastoris LD are characterized by their high preponderance of triacylglycerols over steryl esters in the core of the organelle, the high degree of fatty acid (poly)unsaturation and the high amount of ergosterol precursors. The high phosphatidylinositol to phosphatidylserine of ~ 7.5 ratio on the surface membrane of LD is noteworthy. Proteome analysis revealed equipment of the organelle with a small but typical set of proteins which includes enzymes of sterol biosynthesis, fatty acid activation, phosphatidic acid synthesis and non-polar lipid hydrolysis. These results are the basis for a better understanding of P. pastoris lipid metabolism and lipid storage and may be helpful for manipulating cell biological and/or biotechnological processes in this yeast.