B. Desharnais 1* , M.-J. Lajoie 2 , J. Laquerre 2 , S. Savard 2 , P. Mireault 2 , C. D. Skinner 1
1Concordia University, 2Laboratoire de sciences judiciaires et de médecine légale
The presence of endogenous substances in biological matrices used for calibration standards and quality controls (QCs) can compromise validation steps and quantitative analyses. Several approaches to overcome this problem have been suggested, but they create serious problems with regards to the accuracy of the analytes or production capacity. We present here a solution that efficiently addresses this problem. The endogenous analyte concentration is estimated via a standard-addition type process. This estimated concentration is then added to the known spiked concentration for every sample treated, yielding the analyte concentration actually present in the samples. These corrected concentrations are then used in data analysis software (MultiQuant, Mass Hunter) as the actual concentration. This yields an accurate quantification of the analyte, free from interference of the endogenous contribution. This correction has been applied in a production setting on two BHB quantification methods (GC-MS and LC-MS/MS), allowing the rectification of BHB biases of up to 27 μg/mL. The additional error introduced by this correction procedure is minimal, although the exact amount will be highly method-dependent. The endogenous concentration correction process has been automated with an R script. The final procedure is therefore highly efficient, only adding 4 mouse clicks to the data analysis operations.
B. Garneau 1* , B. Desharnais 2 , A. Beauchamp-Doré 3 , C. Lavallée 3 , P. Mireault 3 , A. Lajeunesse 1
1Université du Québec à Trois-Rivières, 2Concordia University, 3Laboratoire de sciences judiciaires et de médecine légale
In recent years, North America has experienced a considerable growth in mortality related to misuse of prescribed opioids. This “opioids crisis” gained even greater momentum with the apparition of novel synthetic opioids (NSO). These new psychoactive substances (NPS) are derived from prescribed opioids or failed pharmaceuticals, some examples include carfentanil, furanylfentanyl and U-47700. NSO present a high degree of structural similarity and often low biological concentrations, resulting in a clear analytical challenge for forensic toxicology laboratories. The present work describes the development of an LC-MS/MS screening method for 57 NSO using a Design of Experiments (DOE) approach to optimize instrumental conditions (flow rate, column temperature, mobile phase composition, etc.). Biological matrices (cardiac and femoral blood, urine) are extracted by protein precipitation and analysed on an Agilent 1200 HPLC coupled to a Sciex 5500 QTrap operated in positive electrospray ionization mode with multiple reaction monitoring (MRM). Fatal opioids intoxication cases analyzed with this method will be presented, including one where an impressive combination of 15 NSO was detected. Ultimately, this method will allow an assessment of the increasing NSO prevalence in cases handled by the Laboratoire de sciences judiciaires et de médecine légale (LSJML).
S. Pfammatter * , E. Bonneil , P. Thibault
IRIC - Universite de Montreal
The identification of endogenous peptides presented by major histocompatibility complex class I (MHC I) by LC-MS/MS present significant analytical challenges due to their low abundance and the lower charge state distribution compared to tryptic peptides. To enhance the detection of MHC I peptides, we evaluated the use of TMT labeling on a set of 440 synthetic MHC I peptides. TMT labelling enhanced the formation of multiply-charge peptide ions and resulted in a ~20% gain in identification compared to underivatized peptides. Moreover, TMT peptide labeling provided a higher identification score (Ø77 for TMT-label vs. Ø50 for underivatized peptides). Next, we evaluated the analytical benefits of high field asymmetric waveform ion mobility (FAIMS). With 50 million B-LCL cells we identified 4564 MHC I peptides with FAIMS compared to 3755 MHC I peptides without. We extended the application of FAIMS and isobaric peptide labeling to the target analysis of MHC I peptides from only 2 million B-LCL cells. MHC I peptides were derivatized with TMT-126 while increasing amounts of MHCs synthetic peptides were labeled with TMT-128 to TMT-131 to generate a calibration curve. TMT signal amplification obtained using synthetic peptides enabled the more precise quantification with FAIMS compared to regular MS3 method.
T. Geib * , L. Sleno
Hepatotoxicity of over-the-counter medications, such as acetaminophen (APAP), is resulting in major drug-induced adverse effects. APAP is known to form reactive metabolites, and covalently bind to cysteine residues of hepatic proteins. We have investigated the protein binding of APAP to different glutathione S-transferase (GST) isoforms by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
In vitro activation of APAP to N-acetyl p-benzoquinone imine (NAPQI) was performed with rat liver microsomes or human CYP3A4 Supersomes, while adding recombinant GSTs. Further sample preparation employed tryptic or peptic digestion, coupled to LC-MS/MS analysis. Results between solid-phase extraction and offline LC fractionation have been compared. We have developed two LC-MS/MS based strategies and compared them for target identification.
The first screening technique used a traditional bottom-up proteomics workflow using data-dependent acquisition (DDA) with database searching, with custom modifications for APAP. Then, a targeted multiple reaction monitoring (MRM) method for all cysteine-containing peptides of the individual GST isoforms was optimized. Several cysteine sites were detected as modified in the tested GSTs. Comparison of the performance of DDA and MRM analyses showed that MRM represents a complementary technique to untargeted DDA, with increased site identification and higher throughput.
A. Bain * , T. C. Preston
Absorbing aerosol particles are of interest in climate science since they absorb and reemit radiation from the sun leading to atmospheric warming. Single particle studies are of great importance as they remove the effects of ensemble averaging and allow for precise determination of particle size and composition. The successes seen in the optical trapping and characterization of single weakly absorbing aerosol particles have proven to be difficult to extend to strongly absorbing particles. Here we present an optical set-up capable of trapping single absorbing particles. We collect white light scattering spectra of trapped dye-doped polystyrene beads. We show that the real and imaginary parts of the refractive index of a particle can be determined through the use of a Lorentzian oscillator model, the Kramers-Kronig relations, and broadband light scattering measurements. The size of a single particle can be simultaneously be found by maximizing the correlation between a collected spectrum and a library of simulated spectra.
A series of propylenedioxythiophenes (ProDOT) were synthesized and polymerized by both chemical and electrochemical oxidation. The resulting conjugated polymers sustained electronic conduction along their backbone. This is a suitable property for using the conjugated polymers in electronic devices such as biosensors, electronic skin, OLED and photovoltaics. Owing to the solubility of ProDOT monomers in commonly used solvents, they can be copolymerized with conventionally used EDOT. Here, ProDOT derivatives were used to functionalized small elastomers prior to their crosslinking. This was to develop polymers having both electronic conduction from the ProDOT and elastomeric properties from the matrix. These properties were characterized with a stretch bench and a four-point probe station. Surface analyses by AFM and profilometry showed a interesting morphologies that will be presented.
A. Dominguez-Huerta * , I. Perepichka , C.-J. Li
Amino acids are bio-renewable, C-chiral, nitrogen sources with important applications in chemistry and biology. Our laboratory has developed novel methodologies through which these building blocks can be arylated or cyclohexylated by using 2-cyclohexen-1-one or phenol, respectively. By using a phenolic compound as the N-alkylating reagent we can circumvent the need of alkyl halides and protecting groups, while generating water as the sole by-product. In addition, phenol represents a sustainable alternative to current alkylating reagents due to its wide availability as the main constituent of lignin. Upon optimization we found that N -arylation is achieved using 2-cyclohexen-1-one as the coupling partner with up to 74% yield; while phenol is successfully used to N-cyclohexylate 17 out of the 20 natural a-amino acids as well as small peptides in excellent yields in water at room temperature.
B. Keenan 1* , P. Douglas 1 , A. Breckenridge , K. Johnston 3
1McGill University, 2University of Wisconsin-Superior, 3Independent Scholar
Coprostanol, the main 5β-stanol found in human faeces, is a potential useful proxy for human populations that can be analysed alongside other palaeoenvironmental proxies in lake sediments. I will present initial results of faecal stanol analyses from sediment cores from Laguna Itzan in Guatemala, as a proxy for the population of Itzan, an ancient Maya population centre.
We find variable amounts of faecal stanols throughout the Itzan sediment core, potentially implying changes in human population through time. We see a peak in the coprostanol/cholestanol ratio at about 639 BCE. with lower ratios in later Maya archaeological periods. This is in contrast to archaeological estimates indicating peak local populations during the Late Classic period (~600 to 800 CE). We see evidence for settlement in the area by at least 1350 BCE, which is earlier than has been inferred from archaeological records. These discrepancies will require further exploration, and may indicate that the coprostanol record is influenced by other factors.
These data indicate that faecal stanols could be a powerful tool for tracing human population dynamics in tropical lake watersheds that can be used to complement archaeological datasets, and to link human populations with environmental change.
N. Truong * , B. J. Atienza , F. Williams
University of Alberta
The cleavage of C−O bonds is an important reaction in organic synthesis, especially in the degradation and transformation of natural compounds. A number of tactics for this transformation have been reported involving the use of acids, bases or transition metals. Among these approaches, boron tribromide is widely used in stoichiometric amounts for this conversion because of its effectiveness under mild conditions and its tolerance towards many functional groups. However, synthetic applications of boron tribromide has been limited to symmetrical dialkyl or aryloxy ethers, perhaps because of unpredictable regioselectivity with other substrates. In contrast, BCl3 does rapidly forms stable coordination complexes with unactivated ethers under the same conditions. Taking the best properties of BCl3 and BBr3 together, we discovered that disproportionation of BBr3 and BCl3 followed by reaction with unsymmetrical alkyl ethers can predictably yield dialkoxy boron chlorides, which quickly convert to the corresponding alcohols with high efficiency and regioselectivity. This presentation will outline the detailed study of this reaction including a broad substrate scope.
I. Nad 1* , P. Mattar 2
1University of Ottawa, 2Univeristy of Ottawa
BACKGROUND: The nuclear lamina regulates genome organization. Lamin A and Lamin B receptor are key proteins required for heterochromatin tethering. Adult mouse rod photoreceptors express neither of these proteins, resulting in a unique nuclear architecture which makes them a convenient model for studying molecular determinants of genome organization.
OBJECTIVE: We propose to use mouse rod photoreceptors as an assay system for tethering sufficiency. We hypothesize that the C-terminus of Lamin A binds heterochromatin. Our goals are to identify the critical domain responsible for heterochromatin tethering, and to elucidate the genomic and transcriptomic consequences behind it.
METHODS: To determine how tethering proteins affect genome organization, we ectopically express them in mouse rods. We inject the plasmid DNA into the eye and transfect it by in vivo electroporation. ATAC-seq and RNA-seq will help to determine the genomic and transcriptomic changes of the observed phenotype changes of Lamin A.
RESULTS: Lamin A causes reorganization of the rod nucleus and is sufficient for heterochromatin tethering. GFP-tagged Lamin A doesn’t affect the genome.
CONCLUSIONS: The Lamin A C-terminus might be the key for heterochromatin tethering. N-terminal occlusion also appears to be function-blocking, suggesting that there are multiple domains containing affinities for different types of heterochromatin.
D. Abou Samhadaneh 1* , A. Smart 1 , L. Sanchez 1 , J. A. Capobianco 2 , U. Stochaj 1
1McGill University, 2Concordia University
Lanthanide-doped upconverting nanoparticles (UCNPs) are promising tools for cancer therapeutics because of their unique physico-chemical properties. They are commonly used in imaging and photodynamic therapy. However, their effect on living cells remains largely undefined. The exposure of cells to environmental insults, such as nanoparticles, can activate various stress responses and, at the same time, impair protein homeostasis. Stress responses often alter cell fate, which ultimately can cause cell death. These features may compromise UCNP-dependent applications in living cells. Yet, particle properties could also be explored for targeted cell killing.
Using non-transformed and cancer cells, we assessed the effects of UCNPs on cellular stress responses and nuclear trafficking. We focused on nucleoli, the compartments for rRNA synthesis, and evaluated nucleolar proteins that are required for ribosome biogenesis. UCNPs altered the abundance of several essential nucleolar resident proteins, thereby changing the overall nucleolar organization. Moreover, UCNPs diminished rRNA synthesis in cancer cells, suggesting that nucleolar function was compromised. UCNPs also affected key components that are required for transport in and out of the nucleus. Taken together, our results demonstrate that UCNPS impair rRNA synthesis and cellular trafficking. This knowledge is important for the proper development of UCNPs for cancer therapy.
O. Moujaber * , D. Abou Samhadaneh , U. Stochaj
Organ functions decline during aging, and the most profound changes occur in the kidney. Kidneys are continuously exposed to oxidative stress. They are particularly vulnerable to physiological and environmental damage.
The proper response to stress is crucial for cell and organ survival. To survive different forms of insult, eukaryotic cells form cytoplasmic stress granules (SGs). This process is conserved among divergent species. Aging impairs the stress response, but little is known about the underlying mechanisms.
It is our goal to define how aging compromises the kidney’s ability to cope with stress. To this end, we developed two models of renal proximal tubule cell aging. They are based on the chemical or pharmacological induction of senescence. We demonstrated that both model systems display hallmarks of aging.
Using these models, we assessed SG formation and stress-induced signaling. We showed that aging impairs SG assembly. Moreover, our studies uncovered and characterized the underlying mechanisms at the molecular level.
Taken together, our research provides a better understanding of the senescence-dependent changes in kidney physiology. We identified new biomarkers that can score the stress response in kidney cells. Long-term, this information will help to develop new diagnostic and therapeutic tools to evaluate cellular aging.
K. Szereszewski * , H. Hadj-Moussa , A. Watts , K. Storey
Our research program is aimed at discovering the cellular and molecular mechanisms that allow both vertebrates and invertebrate extremophiles to cope with severe environmental stresses. Our specific interests include gene regulation responses to metabolic rate depression. Novel areas of research include microRNA regulation in new and interesting animals, novel neuronal mitochondrial peptide regulation, as well as the circadian rhythms of hibernators.
L. Lahens * , A. Dion-Fortier , H. Cabana , P. Segura
Université de Sherbrooke
Trace organic contaminants are mostly studied in rivers and wastewater, but data is lacking on their presence and concentrations in lakes. These contaminants can be persistent organic pollutants, such as pesticides, as well as contaminants of emerging concern, such as pharmaceuticals, personal care products and industrial additives.
Here, we present a multi-residue method developed to quantify 50 contaminants. This method will be applied to evaluate the contamination of around 300 lakes, representative of the distribution of lakes in Canada regarding ecozones, size and human activities impact, sampled over the country within the Lake Pulse Network.
The molecules of interest will be extracted from lake water by solid phase extraction (SPE) before being analysed using ultra performance liquid chromatography coupled to triple quadrupole mass spectrometry (UPLC-QqQMS) in positive or negative electrospray ionisation.
The method’s figures of merit, such as the SPE recoveries (49 – 105%), method detection limits (13 – 294 ng/L), precision (2 – 33%) and accuracy (1 – 25% for most compounds) will be presented. First results on the contaminants concentrations in around a hundred lakes show that at least one compound was identified in 70% of the lakes. The link between contamination and land use will be discussed.
F. Charih 1* , Y. B. Ruiz- Blanco 2 , K. K. Biggar 1 , J. R. Green 1
1Carleton University, 2University of Duisburg-Essen
Lysine methylation is a post-translational modification of proteins that mediates a myriad of cellular processes including DNA damage repair, cell signaling, and metabolism. Recent research suggests that this vastly understudied modification may be more prevalent than previously thought. Unfortunately, the identification and characterization of lysine methylation sites is technically challenging and resource-intensive. For this reason, the elucidation of the methyllysine proteome has remained elusive. Computational approaches have the potential to facilitate rational experiment design and to guide experiments aiming to identify such sites. Machine learning approaches have shown promise for a variety of applications in biochemistry such as protein-protein interaction prediction or protein function prediction, but have had limited success in predicting lysine methylation sites. Here, we present MethylSight, an alignment-free method for predicting lysine methylation sites in proteins. MethylSight combines the ProtDCal feature extractor with a support vector machine classifier to predict sites with a recall and precision that surpass the current state of the art. The MethylSight web server is freely available, and provides researchers with a streamlined tool to investigate putative lysine methylation sites. Altogether, MethylSight is an invaluable resource for researchers interested in this important post-translational modification.