Analyzing RNA posttranscriptional modifications to decipher the epitranscriptomic code.

The discovery of RNA silencing has revealed that non-protein-coding sequences (ncRNAs) can cover essential roles in regulatory networks and their malfunction may result in severe consequences on human health. These findings have prompted a general reassessment of the significance of RNA as a key player in cellular processes. This reassessment, however, will not be complete without a greater understanding of the distribution and function of the over 170 variants of the canonical ribonucleotides, which contribute to the breathtaking structural diversity of natural RNA. This review surveys the analytical approaches employed for the identification, characterization, and detection of RNA posttranscriptional modifications (rPTMs). The merits of analyzing individual units after exhaustive hydrolysis of the initial biopolymer are outlined together with those of identifying their position in the sequence of parent strands. Approaches based on next generation sequencing and mass spectrometry technologies are covered in depth to provide a comprehensive view of their respective merits. Deciphering the epitranscriptomic code will require not only mapping the location of rPTMs in the various classes of RNAs, but also assessing the variations of expression levels under different experimental conditions. The fact that no individual platform is currently capable of meeting all such demands implies that it will be essential to capitalize on complementary approaches to obtain the desired information. For this reason, the review strived to cover the broadest possible range of techniques to provide readers with the fundamental elements necessary to make informed choices and design the most effective possible strategy to accomplish the task at hand.

Unbiased Proteomic and Phosphoproteomic Analysis Identifies Response Signatures and Novel Susceptibilities After Combined MEK and mTOR Inhibition in BRAFV600E Mutant Glioma.

The mitogen-activated protein kinase pathway is one of the most frequently altered pathways in cancer. It is involved in the control of cell proliferation, invasion, and metabolism, and can cause resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer, and glioma, are driven by a constitutively activating missense mutation (V600E) in the v-Raf murine sarcoma viral oncogene homolog B (BRAF) component of the pathway. Mitogen-activated protein kinase kinase (MEK) inhibition is initially effective in targeting these cancers, but reflexive activation of mammalian target of rapamycin (mTOR) signaling contributes to frequent therapy resistance. We have previously demonstrated that combination treatment with the MEK inhibitor trametinib and the dual mammalian target of rapamycin complex 1/2 inhibitor TAK228 improves survival and decreases vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of this combination therapy and understand the ensuing tumor response, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228. We identified 13,313 proteins and 30,928 localized phosphosites, of which 12,526 proteins and 17,444 phosphosites were quantified across all samples (data available via ProteomeXchange; identifier PXD022329). We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the mitogen-activated protein kinase and mTOR pathways. Combination therapy also increased apoptotic signaling, suppressed angiogenesis signaling, and broadly suppressed the activity of the cyclin-dependent kinases. In response to combination therapy, both epidermal growth factor receptor and class 1 histone deacetylase proteins were activated. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies new targets for the development of rational combination therapies for BRAF-driven tumors.

Early postnatal metabolic profile in neonates with critical CHDs.

BACKGROUND: Cyanotic CHD is a life-threatening condition that presents with low oxygen saturation in the newborn period. Hypoxemia might cause alterations in the metabolic pathways. In the present study, we aimed to evaluate the early postnatal amino acid and carnitine/acylcarnitine profiles of newborn infants with cyanotic CHD. METHODS: A single centre case-control study was conducted. Twenty-seven patients with cyanotic CHD and 54 healthy newborn controls were enrolled. As part of the neonatal screening programme, results of amino acid and carnitine/acylcarnitine were recorded and compared between groups. RESULTS: Twenty-seven neonates with cyanotic CHD and 54 healthy newborns as controls were enrolled in the study. Cyanotic CHD neonates had higher levels of alanine, phenylalanine, leucine/isoleucine, citrulline, ornithine, C5, C5-OH; but lower levels of C3, C10, C12, C14, C14:1, C16, C16.1, C18, C5-DC, C6-DC, C16-OH, C16:1-OH when compared with the healthy controls. CONCLUSION: This study showed that there are differences between patients with cyanotic CHD and healthy controls in terms of postnatal amino acid and carnitine/acylcarnitine profiles.

C-SEQer: An Open-Source de Novo Glycan Identification Tool in C+.

Glycans play an important role in many biochemical processes, including protein function and cell signaling. Mass spectrometry (MS) provides the potential for high-throughput, high-sensitivity analysis of glycans but relies heavily on computational interpretation of experimental results. Open-source, stand-alone algorithms for de novo glycan MS analysis are few. One such algorithm, Sweet-SEQer, is available in Python. Glycan analysis of mass spectra can easily involve high volumes of data where Python's performance in time and memory is a noticeable bottleneck. This manuscript describes C-SEQer, a new implementation of the Sweet-SEQer algorithm in C++, which produces the same output as the original algorithm in approximately 15-fold less time with substantially less memory usage. The implementation is freely available with an MIT license.

Optimal Dissociation Methods Differ for N- and O-Glycopeptides.

Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area of N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods in terms of identifications, indicating that ETD-based methods are not required for routine N-glycoproteomics even if they can generate higher quality spectra. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.

Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2.

The mitochondrial and chloroplast mRNAs of the majority of land plants are modified through cytidine to uridine (C-to-U) RNA editing. Previously, forward and reverse genetic screens demonstrated a requirement for pentatricopeptide repeat (PPR) proteins for RNA editing. Moreover, chloroplast editing factors OZ1, RIP2, RIP9 and ORRM1 were identified in co-immunoprecipitation (co-IP) experiments, albeit the minimal complex sufficient for editing activity was never deduced. The current study focuses on isolated, intact complexes that are capable of editing distinct sites. Peak editing activity for four sites was discovered in size-exclusion chromatography (SEC) fractions ≥ 670 kDa, while fractions estimated to be approximately 413 kDa exhibited the greatest ability to convert a substrate containing the editing site rps14 C80. RNA content peaked in the ≥ 670 kDa fraction. Treatment of active chloroplast extracts with RNase A abolished the relationship of editing activity with high-MW fractions, suggesting a structural RNA component in native complexes. By immunoblotting, RIP9, OTP86, OZ1 and ORRM1 were shown to be present in active gel filtration fractions, though OZ1 and ORRM1 were mainly found in low-MW inactive fractions. Active editing factor complexes were affinity-purified using anti-RIP9 antibodies, and orthologs to putative Arabidopsis thaliana RNA editing factor PPR proteins, RIP2, RIP9, RIP1, OZ1, ORRM1 and ISE2 were identified via mass spectrometry. Western blots from co-IP studies revealed the mutual association of OTP86 and OZ1 with native RIP9 complexes. Thus, RIP9 complexes were discovered to be highly associated with C-to-U RNA editing activity and other editing factors indicative of their critical role in vascular plant editosomes.

De novo sequencing of proteins by mass spectrometry.

INTRODUCTION: Proteins are crucial for every cellular activity and unraveling their sequence and structure is a crucial step to fully understand their biology. Early methods of protein sequencing were mainly based on the use of enzymatic or chemical degradation of peptide chains. With the completion of the human genome project and with the expansion of the information available for each protein, various databases containing this sequence information were formed. AREAS COVERED: De novo protein sequencing, shotgun proteomics and other mass-spectrometric techniques, along with the various software are currently available for proteogenomic analysis. Emphasis is placed on the methods for de novo sequencing, together with potential and shortcomings using databases for interpretation of protein sequence data. EXPERT OPINION: As mass-spectrometry sequencing performance is improving with better software and hardware optimizations, combined with user-friendly interfaces, de-novo protein sequencing becomes imperative in shotgun proteomic studies. Issues regarding unknown or mutated peptide sequences, as well as, unexpected post-translational modifications (PTMs) and their identification through false discovery rate searches using the target/decoy strategy need to be addressed. Ideally, it should become integrated in standard proteomic workflows as an add-on to conventional database search engines, which then would be able to provide improved identification.

The occurrence of inositolphosphoceramides in spirulina microalgae.

Spirulina microalga (Arthrospira platensis) is an interesting phototrophic organism because of its high content of nutrients including proteins, lipids, essential amino acids, antioxidants, vitamins, polysaccharides, and minerals. Hydrophilic interaction liquid chromatography (HILIC) coupled to linear ion trap (LIT) and Orbitrap Fourier transform mass spectrometry (FTMS) via ESI was employed for the separation and characterization of lipid species in A. platensis. Inositolphosphoceramides (IPC) are minor but important constituents of spirulina; their investigation was accomplished by HILIC-ESI-MS including collision-induced dissociation (MS2 , MS3 ) of deprotonated molecules in the LIT analyzer and a schematic fragmentation pattern is described. All four commercial spirulina samples revealed the occurrence of the same IPC species at m/z 796.6 (d18:0/16:0;1), 810.6 (d18:0/17:0;1), 824.6 (d18:0/18:0;1), and 826.6 (d18:0/17:0;2) but in diverse relative abundance. This study sets the stage for future investigations on IPC in other algae and microalgae.

Analysis of Phospholipids, Lysophospholipids, and Their Linked Fatty Acyl Chains in Yellow Lupin Seeds (Lupinus luteus L.) by Liquid Chromatography and Tandem Mass Spectrometry.

Hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled to either Fourier-transform (FT) orbital-trap or linear ion-trap tandem mass spectrometry (LIT-MS/MS) was used to characterize the phospholipidome of yellow lupin (Lupinus luteus) seeds. Phosphatidylcholines (PC) were the most abundant species (41 ± 6%), which were followed by lyso-forms LPC (30 ± 11%), phosphatidylethanolamines (PE, 13 ± 4%), phosphatidylglycerols (PG, 5.1 ± 1.7%), phosphatidic acids (PA, 4.9 ± 1.8%), phosphatidylinositols (PI, 4.7 ± 1.1%), and LPE (1.2 ± 0.5%). The occurrence of both isomeric forms of several LPC and LPE was inferred by a well-defined fragmentation pattern observed in negative ion mode. An unprecedented characterization of more than 200 polar lipids including 52 PC, 42 PE, 42 PA, 35 PG, 16 LPC, 13 LPE, and 10 PI, is reported. The most abundant fatty acids (FA) as esterified acyl chains in PL were 18:1 (oleic), 18:2 (linoleic), 16:0 (palmitic), and 18:3 (linolenic) with relatively high contents of long fatty acyl chains such as 22:0 (behenic), 24:0 (lignoceric), 20:1 (gondoic), and 22:1 (erucic). Their occurrence was confirmed by reversed-phase (RP) LC-ESI-FTMS analysis of a chemically hydrolyzed sample extract in acid conditions at 100 °C for 45 min.

Exploration of Rapid Evaporative-Ionization Mass Spectrometry as a Shotgun Approach for the Comprehensive Characterization of Kigelia Africana (Lam) Benth. Fruit.

Rapid evaporative-ionization mass spectrometry (REIMS) coupled with an electroknife as a sampling device was recently employed in many application fields to obtain a rapid characterization of different samples without any need for extraction or cleanup procedures. In the present research, REIMS was used to obtain a metabolic profiling of the Kigelia africana fruit, thus extending the applicability of such a technique to the investigation of phytochemical constituents. In particular, the advantages of REIMS linked to a typical electrosurgical handpiece were applied for a comprehensive screening of this botanical species, by exploiting the mass accuracy and tandem MS capabilities of a quadrupole-time of flight analyzer. Then, 78 biomolecules were positively identified, including phenols, fatty acids and phospholipids. In the last decade, Kigelia africana (Lam.) Benth. fruit has attracted special interest for its drug-like properties, e.g., its use for infertility treatments and as anti-tumor agent, as well as against fungal and bacterial infections, diabetes, and inflammatory processes. Many of these properties are currently correlated to the presence of phenolic compounds, also detected in the present study, while the native lipid composition is here reported for the first time and could open new directions in the evaluation of therapeutic activity.

Characterization of microbial mixtures by mass spectrometry.

MS applications in microbiology have increased significantly in the past 10 years, due in part to the proliferation of regulator-approved commercial MALDI MS platforms for rapid identification of clinical infections. In parallel, with the expansion of MS technologies in the "omics" fields, novel MS-based research efforts to characterize organismal as well as environmental microbiomes have emerged. Successful characterization of microorganisms found in complex mixtures of other organisms remains a major challenge for researchers and clinicians alike. Here, we review recent MS advances toward addressing that challenge. These include sample preparation methods and protocols, and established, for example, MALDI, as well as newer, for example, atmospheric pressure ionization (API) techniques. MALDI mass spectra of intact cells contain predominantly information on the highly expressed house-keeping proteins used as biomarkers. The API methods are applicable for small biomolecule analysis, for example, phospholipids and lipopeptides, and facilitate species differentiation. MS hardware and techniques, for example, tandem MS, including diverse ion source/mass analyzer combinations are discussed. Relevant examples for microbial mixture characterization utilizing these combinations are provided. Chemometrics and bioinformatics methods and algorithms, including those applied to large scale MS data acquisition in microbial metaproteomics and MS imaging of biofilms, are highlighted. Select MS applications for polymicrobial culture analysis in environmental and clinical microbiology are reviewed as well.

Microfluidic capillary zone electrophoresis mass spectrometry analysis of alkaloids in Lobelia cardinalis transgenic and mutant plant cell cultures.

Application of a microfluidic CE* device for CZE-MS allows for fast, rapid, and in-depth analysis of large sample sets. This microfluidic CZE-MS device, the 908 Devices ZipChip, involves minimal sample preparation and is ideal for small cation analytes, such as alkaloids. Here, we evaluated the microfluidic device for the analysis of alkaloids from Lobelia cardinalis hairy root cultures. Extracts from wild-type, transgenic, and selected mutant plant cultures were analyzed and data batch processed using the mass spectral processing software MZmine2 and the statistical software Prism 8. In total 139 features were detected as baseline resolved peaks via the MZmine2 software optimized for the electrophoretic separations. Statistically significant differences in the relative abundance of the primary alkaloid lobinaline (C27 H34 N2 ), along with several putative "lobinaline-like" molecules were observed utilizing this approach. Additionally, a method for performing both targeted and untargeted MS/MS experiments using the microfluidic device was developed and evaluated. Coupling data-processing software with CZE-MS data acquisition has enabled comprehensive metabolomic profiles from plant cell cultures to be constructed within a single working day.

Prevalence and predictors of vitamin D deficiency in a nationally representative sample of adults participating in the 2011-2013 Australian Health Survey.

Vitamin D deficiency is recognised as a public health problem globally, and a high prevalence of deficiency has previously been reported in Australia. This study details the prevalence of vitamin D deficiency in a nationally representative sample of Australian adults aged ≥25 years, using an internationally standardised method to measure serum 25-hydroxyvitamin D (25(OH)D) concentrations and identifies demographic and lifestyle factors associated with vitamin D deficiency. We used data from the 2011-2013 Australian Health Survey (n 5034 with complete information on potential predictors and serum 25(OH)D concentrations). Serum 25(OH)D concentrations were measured by a liquid chromatography-tandem MS that is certified to the reference measurement procedures developed by the National Institute of Standards and Technology, Ghent University and the US Centers for Disease Control and Prevention. Vitamin D deficiency and insufficiency were defined as serum 25(OH)D concentrations <50 nmol/l and 50 to <75 nmol/l, respectively. Overall, 20 % of participants (19 % men; 21 % women) were classified as vitamin D deficient, with a further 43 % classified as insufficient (45 % men; 42 % women). Independent predictors of vitamin D deficiency included being born in a country other than Australia or the main English-speaking countries, residing in southern (higher latitude) states of Australia, being assessed during winter or spring, being obese, smoking (women only), having low physical activity levels and not taking vitamin D or Ca supplements. Given our increasingly indoor lifestyles, there is a need to develop and promote strategies to maintain adequate vitamin D status through safe sun exposure and dietary approaches.

Production and characterization of glycolipid biosurfactant from Achromobacter sp. (PS1) isolate using one-factor-at-a-time (OFAT) approach with feasible utilization of ammonia-soaked lignocellulosic pretreated residues.

With the ever growing increase in the demands of biosurfactants, the present study was focused in developing a set of parameters influencing biosurfactant production using one-factor-at-a-time (OFAT) approach in chemically defined medium from an indigenous isolate of Achromobacter sp. (PS1). Subsequently, the feasibility of biosurfactant production was examined using influential OFAT parameters in same medium, replacing only carbon source with lignocellulosic hydrolyzed sugars. These sugars were obtained from ammonia (15% v/v) soaking pretreatment of lignocellulosic residues (7.5% solid loading at 70 °C for 72 h) with subsequent saccharification using lignocellulolytic enzymes. OFAT influential parameters observed were dextrose (3-4% w/v); C/N ratio 8.3 using sodium nitrate and beef extract; 2 × 10-5 grams equivalents Fe2+; 1500 mM PO43- in minimal salt medium (MSM) at pH 7.0, 120 rpm, 30 °C resulting in 4.13 ± 0.12 g/L rhamnolipid in 192 h with 30.42 mN/m surface tension and 136 mg/L critical micelle concentration (CMC). Biosurfactant was characterized using tandem-MS and NMR as rhamnolipid with six-congeners, Rha-C10-C10 and Rha-Rha-C10-C10 being the most abundant. Rhamnolipid showed broad range stability at temperatures (30-121 °C), pH (6-12), and salinity (0.5-5% w/v) of NaCl. In Rice-straw (RS) hydrolysate, maximum glucan (73.10%) and xylan (91.13%) were obtained and the RS-hydrolysate medium with a total of 4.55% (w/v) sugars under optimum OFAT parameters (other than dextrose) showed at par production of 3.55 ± 0.06 g/L of rhamnolipid in 192 h with YBS/S (biosurfactant yield per gram of sugar consumed) of 0.08 g/g and YBS/CDW (biosurfactant yield per gram of cell biomass) of 0.68 g/g.

Searching for Potential Lipid Biomarkers of Parkinson's Disease in Parkin-Mutant Human Skin Fibroblasts by HILIC-ESI-MS/MS: Preliminary Findings.

Early diagnosis of neural changes causing cerebral impairment is critical for proposing preventive therapies for Parkinson's disease (PD). Biomarkers currently available cannot be informative of PD onset since they are characterized by analysing post-mortem tissues from patients with severe degeneration of the substantia nigra. Skin fibroblasts (SF) are now recognized as a useful model of primary human cells, capable of reflecting the chronological and biological aging of the subjects. Here a lipidomic study of easily accessible primary SF is presented, based on hydrophilic interaction liquid chromatography coupled to electrospray ionization and mass spectrometry (HILIC/ESI-MS). Phospholipids (PL) from dermal fibroblasts of five PD patients with different parkin mutations and healthy control SF were characterized by single and tandem MS measurements using a hybrid quadrupole-Orbitrap and a linear ion trap mass analysers. The proposed approach enabled the identification of more than 360 PL. Univariate statistical analyses highlight abnormality of PL metabolism in the PD group, suggesting down- or up-regulation of certain species according to the extent of disease progression. These findings, although preliminary, suggest that the phospholipidome of human SF represents a source of potential biomarkers for the early diagnosis of PD. The dysregulation of ethanolamine plasmalogens in the circulatory system, especially those containing polyunsaturated fatty acids (PUFA), might be likely associated with neurodegeneration.

Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance between Healthy Adults.

Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter-individual variability reported, confident, and reliable insights have been hindered. Using label-free quantitative (LFQ)-proteomics analysis, a reproducible, quantifiable investigation of the 1U mL-1 thrombin-induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ-proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet-derived exosomes. Vesicles in the exosomal-size range were confirmed in healthy-human PR and reduced numbers of similar-sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha-granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low-population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet-related disease.

Functional assessment of creatine transporter in control and X-linked SLC6A8-deficient fibroblasts.

Creatine transporter is currently the focus of renewed interest with emerging roles in brain neurotransmission and physiology, and the bioenergetics of cancer metastases. We here report on amendments of a standard creatine uptake assay which might help clinical chemistry laboratories to extend their current range of measurements of creatine and metabolites in body fluids to functional enzyme explorations. In this respect, short incubation times and the use of a stable-isotope-labeled substrate (D3-creatine) preceded by a creatine wash-out step from cultured fibroblast cells by removal of fetal bovine serum (rich in creatine) from the incubation medium are recommended. Together, these measures decreased, by a first order of magnitude, creatine concentrations in the incubation medium at the start of creatine-uptake studies and allowed to functionally discriminate between 4 hemizygous male and 4 heterozygous female patients with X-linked SLC6A8 deficiency, and between this cohort of eight patients and controls. The functional assay corroborated genetic diagnosis of SLC6A8 deficiency. Gene anomalies in our small cohort included splicing site (c.912G > A [p.Ile260_Gln304del], c.778-2A > G and c.1495 + 2 T > G), substitution (c.407C > T) [p.Ala136Val] and deletion (c.635_636delAG [p.Glu212Valfs*84] and c.1324delC [p.Gln442Lysfs*21]) variants with reduced creatine transporter function validating their pathogenicity, including that of a previously unreported c.1324delC variant. The present assay adaptations provide an easy, reliable and discriminative manner for exploring creatine transporter activity and disease variations. It might apply to drug testing or other evaluations in the genetic and metabolic horizons covered by the emerging functions of creatine and its transporter, in a way, however, requiring and completed by additional studies on female patients and blood-brain barrier permeability properties of selected compounds. As a whole, the proposed assay of creatine transporter positively adds to currently existing measurements of this transporter activity, and determining on a large scale the extent of its exact suitability to detect female patients should condition in the future its transfer in clinical practice.

Mechanism of irreversible inhibition of Mycobacterium tuberculosis shikimate kinase by ilimaquinone.

Ilimaquinone (IQ), a marine sponge metabolite, has been considered as a potential therapeutic agent for various diseases due to its broad range of biological activities. We show that IQ irreversibly inactivates Mycobacterium tuberculosis shikimate kinase (MtSK) through covalent modification of the protein. Inactivation occurred with an apparent second-order rate constant of about 60 M-1 s-1. Following reaction with IQ, LC-MS analyses of intact MtSK revealed covalent modification of MtSK by IQ, with the concomitant loss of a methoxy group, suggesting a Michael-addition mechanism. Evaluation of tryptic fragments of IQ-derivatized MtSK by MS/MS demonstrated that Ser and Thr residues were most frequently modified with lesser involvement of Lys and Tyr. In or near the MtSK active site, three residues of the P-loop (K15, S16, and T17) as well as S77, T111, and S44 showed evidence of IQ-dependent derivatization. Accordingly, inclusion of ATP in IQ reactions with MtSK partially protected the enzyme from inactivation and limited IQ-based derivatization of K15 and S16. Additionally, molecular docking models for MtSK-IQ were generated for IQ-derivatized S77 and T111. In the latter, ATP was observed to sterically clash with the IQ moiety. Out of three other enzymes evaluated, lactate dehydrogenase was derivatized and inactivated by IQ, but pyruvate kinase and catalase-peroxidase (KatG) were unaffected. Together, these data suggest that IQ is promiscuous (though not entirely indiscriminant) in its reactivity. As such, the potential of IQ as a lead in the development of antitubercular agents directed against MtSK or other targets is questionable.

Charge-induced geometrical reorganization of DNA oligonucleotides studied by tandem mass spectrometry and ion mobility.

Mass spectrometry is applied as a tool for the elucidation of molecular structures. This premises that gas-phase structures reflect the original geometry of the analytes, while it requires a thorough understanding and investigation of the forces controlling and affecting the gas-phase structures. However, only little is known about conformational changes of oligonucleotides in the gas phase. In this study, a series of multiply charged DNA oligonucleotides (n = 15-40) has been subjected to a comprehensive tandem mass spectrometric study to unravel transitions between different ionic gas-phase structures. The nucleobase sequence and the chain length were varied to gain insights into their influence on the geometrical oligonucleotide organization. Altogether, 23 oligonucleotides were analyzed using collision-induced fragmentation. All sequences showed comparable correlation regarding the characteristic collision energy. This value that is also a measure for stability, strongly correlates with the net charge density of the precursor ions. With decreasing charge of the oligonucleotides, an increase in the fragmentation energy was observed. At a distinct charge density, a deviation from linearity was observed for all studied species, indicating a structural reorganization. To corroborate the proposed geometrical change, collisional cross-sections of the oligonucleotides at different charge states were determined using ion mobility-mass spectrometry. The results clearly indicate that an increase in charge density and thus Coulomb repulsion results in the transition from a folded, compact form to elongated structures of the precursor ions. Our data show this structural transition to depend mainly on the charge density, whereas sequence and size do not have an influence.

Effect of aging on muscle mitochondrial substrate utilization in humans.

Previous studies have implicated age-associated reductions in mitochondrial oxidative phosphorylation activity in skeletal muscle as a predisposing factor for intramyocellular lipid (IMCL) accumulation and muscle insulin resistance (IR) in the elderly. To further investigate potential alterations in muscle mitochondrial function associated with aging, we assessed basal and insulin-stimulated rates of muscle pyruvate dehydrogenase (VPDH) flux relative to citrate synthase flux (VCS) in healthy lean, elderly subjects and healthy young body mass index- and activity-matched subjects. VPDH/VCS flux was assessed from the (13)C incorporation from of infused [1-13C] glucose into glutamate [4-13C] relative to alanine [3-13C] assessed by LC-tandem MS in muscle biopsies. Insulin-stimulated rates of muscle glucose uptake were reduced by 25% (P<0.01) in the elderly subjects and were associated with ∼70% (P<0.04) increase in IMCL, assessed by 1H magnetic resonance spectroscopy. Basal VPDH/VCS fluxes were similar between the groups (young: 0.20±0.03; elderly: 0.14±0.03) and increased approximately threefold in the young subjects following insulin stimulation. However, this increase was severely blunted in the elderly subjects (young: 0.55±0.04; elderly: 0.18±0.02, P=0.0002) and was associated with an ∼40% (P=0.004) reduction in insulin activation of Akt. These results provide new insights into acquired mitochondrial abnormalities associated with aging and demonstrate that age-associated reductions in muscle mitochondrial function and increased IMCL are associated with a marked inability of mitochondria to switch from lipid to glucose oxidation during insulin stimulation.