Exactive Mass Spectrometer Research Articles

Catalytic ethanolysis of Dahuangshan lignite and directional upgrading of two derived soluble mixtures

An in-situ and ex-situ cascade upgrade strategy was designed and used for upgrading Dahuangshan lignite-derived soluble mixtures (LDSMs) to obtain value-added chemicals and clean liquid fuels (CLFs), respectively. In the H2-free catalytic ethanolysis, Ni/mordenite can effectively activate ethanol and promote the transfer of various free radicals including H·, CH3CH2·, and CH3CH2O·, which synchronously facilitates the cleavage of bridged bonds in Dahuangshan lignite and the in-situ removal of heteroatoms (HAs) from LDSMs. As a result, the total yield of LDSMs increased from 40.6 to 66.5 wt%, wherein that of ethanol-soluble portion (ESP) increased from 21.4 to 32.4 wt%. Interestingly, alkanes and arenes with higher relative content (RC, 38.1%) were detected in ESPC (for catalytic ethanolysis), while arenols (24.7%) dominate ESPN (for ethanolysis). The analysis with a quadrupole exactive orbitrap mass spectrometer further confirmed the significant decrease in the relative abundance of HAs, especially Ox class species, in ESPC. In addition, ESPN and ESPC with different properties were further used for enriching derived chemicals and for obtaining CLFs by catalytic hydroconversion (CHC), respectively. After the CHC, the total RCs of alkanes and cyclanes in the soluble portion (SP) are as high as 71.3%, and the content of HA-containing organic compounds was further decreased. Therefore, such SP containing less HAs is an attractive CLF precursor. A ternary mixed solvent was also designed for rapidly separating the SP from ESPN. The total RC of alkanes and arenes in PE5 as well as arenols in M5 are as high as 79.4 and 70.9%, respectively. Molecular dynamics simulations were further used to reveal the rapid enrichment mechanisms with ternary mixed solvents.

Abstract 624: Clinical evaluation of the MasSpec Pen technology for rapid diagnosis and margin assessment in pancreatic cancer surgery

Abstract Precise removal of pancreatic ductal adenocarcinoma (PDAC) with microscopically negative margins, commonly assessed by frozen section analysis, is associated with longer disease-free survival. However, histologic complexities and tissue-processing artifacts can render frozen section analysis of PDAC margins a challenging and time-consuming task, with reported accuracies dependent on the skillset and subspecialty of the pathologist on call. We developed the MasSpec Pen, a handheld device coupled to a mass spectrometer, for rapid (~15 s) and nondestructive molecular analysis and diagnosis of tissues. The MasSpec Pen supplies a discrete water droplet onto a tissue's surface, allowing diagnostic metabolites and lipids to be extracted into the droplet and then transmitted into a mass spectrometer for analysis. Here, we evaluate the performance of the MasSpec Pen for intraoperative diagnosis of PDAC in human pancreatic and bile duct margins. Pancreatic and bile duct tissue samples (N=157) were obtained from the Cooperative Human Tissue Network and Baylor College of Medicine and stored at -80°C prior to analysis. A Q Exactive mass spectrometer (Thermo Scientific) coupled to the MasSpec Pen was used for analysis of thawed samples in the negative ion mode. Tissues were then cryo-sectioned, H&E stained, and blindly evaluated by a pathologist. Based on the distinct molecular profiles acquired, we generated two statistical classifiers using lasso penalized logistic regression for distinguishing PDAC from healthy pancreas and bile duct tissue based on a sparse set of molecular features indicative of disease state. For distinguishing normal pancreas from PDAC, an overall accuracy of 91.5%, sensitivity of 95.5%, and specificity of 89.7% was achieved for training, validation, and test sets. Classification results for discriminating normal bile duct from PDAC had an overall accuracy of 95%, sensitivity of 92%, and specificity of 100% in training and validation. We have begun clinical testing of the MasSpec Pen in human surgeries following its successful translation to an operating room at Texas Medical Center. To date, the MasSpec Pen has been used to analyze in vivo and fresh ex vivo tissue in 19 pancreatic surgeries. When predicting on 64 intraoperative analyses using classification models built on banked data, 93.8% agreement with final postoperative pathology reports was achieved. While further validation studies are needed, our results show that the MasSpec Pen can distinguish PDAC from normal pancreas and bile duct tissues with high accuracy and is compatible for in vivo use, suggesting this technology may be valuable for near real-time margin evaluation during pancreatic oncologic surgeries. Citation Format: Mary E. King, Jialing Zhang, John Q. Lin, Kyana Y. Garza, Rachel J. DeHoog, Clara L. Feider, Alena V. Bensussan, Marta Sans, Anna Krieger, Sunil Badal, Michael F. Keating, Sadhna Dhingra, Wendong Yu, George Van Buren, Christopher Pirko, Kirtan Brahmbhatt, William E. Fisher, James W. Suliburk, Livia S. Eberlin. Clinical evaluation of the MasSpec Pen technology for rapid diagnosis and margin assessment in pancreatic cancer surgery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 624.

Matrix ions as internal standard for high mass accuracy matrix-assisted laser desorption/ionization mass spectrometry imaging.

High mass accuracy is indispensable for reliable identification in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) imaging. Ubiquitous matrix ions can serve as reference masses for mass calibration if their sum formula is known. Here we report an overview of ions generated on tissue by 11 common MALDI matrices for use in internal or external mass calibration. Matrices covered in this study were applied onto coronal mouse brain sections using a pneumatic sprayer setup. MALDI imaging was performed on a Q Exactive HF orbital trapping mass spectrometer coupled to an AP-SMALDI 10 source. Measurements were conducted with high mass resolution (240 k full width at half maximum at m/z 200) and high mass accuracy with a root mean square mass error of better than 1.5ppm achieved via internal mass calibration using matrix ions. MALDI MS imaging was used to investigate ions generated on tissue by 11 common MALDI matrices. An example of using matrix ions for internal mass calibration in MALDI imaging of drug substances and lipids in murine lung sections is presented. Tables containing the cluster composition, sum formulae, and the measured and theoretical m/z ratios of the identified ions were compiled for each matrix. Using matrix ions as reference masses for internal and external mass calibration in MALDI MS imaging is an effective and elegant way to achieve sub-ppm mass accuracy as it makes use of ubiquitous signals present in every MALDI MS spectrum without the need for an additional calibration standard.

Untargeted Fecal Metabolomics to Investigate the Role of the Microbiome and Nutrients in Osteoarthritis

Abstract Objectives The objective of this study was to determine if perturbations in gut microbial composition and the gut metabolome could be linked to individuals with obesity and osteoarthritis (OA). Methods Fecal samples were collected from 92 participants with obesity recruited from the Johnston County Osteoarthritis Project. OA cases (n = 59) had radiographic hand plus knee OA, defined as involvement of at least 3 joints across both hands, and a Kellgren-Lawrence (KL) grade 2–4 in at least one knee. Controls (n = 33) were without hand OA and with KL grade 0–1 knees. Fecal metabolomes were analyzed by a UHPLC/Q Exactive HFx mass spectrometer. Microbiome composition was determined in fecal samples by 16S ribosomal RNA amplicon sequencing (rRNA-seq). Stepwise logistic regression models were built to determine predictors of OA status. Spearman correlations were performed to determine associations between metabolites and microbiota in OA or healthy individuals. Results Untargeted metabolomics analysis indicated that OA cases had significantly higher levels of di- and tri-peptides (P < 0.05), and significant perturbations (P < 0.1) in microbial metabolites. Pathway analysis revealed several significantly perturbed pathways (P < 0.05) associated with OA, including leukotriene metabolism, amino acid metabolism and fatty acid utilization. Logistic regression models selected metabolites associated with the microbiota and leaky gut syndrome as significant predictors of OA status, particularly when combined with the 16S rRNA sequencing data. Omega-3/6 polyunsaturated fatty acids (PUFAs) levels were significantly correlated with the phyla Bacteriodetes and Firmicutes. Conclusions Adults with obesity and OA have distinct fecal metabolomes characterized by perturbations in microbial metabolites, PUFAs, and protein digestion compared with healthy controls. These metabolic perturbations suggest a role of intestinal inflammation and leaky gut in OA. Funding Sources Supported by the Arthritis Foundation, the National Center for Advancing Translational Sciences (NCATS) (UL1TR002489), and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (P30AR072580).

Label-Free Quantitative Proteomic Analysis of Three Strains of Viscerotropic Leishmania Isolated from Patients with Different Epidemiological Types of Visceral Leishmaniasis in China.

There are three epidemiological types of visceral leishmaniasis in China, which are caused by Leishmania strains belonging to the L. donovani complex. The mechanisms underlying their differences in the population affected, disease latency, and animal host, etc., remain unclear. We investigated the protein abundance differences among Leishmania strains isolated from three types of visceral leishmaniasis endemic areas in China. Promastigotes of the three Leishmania strains were cultured to the log phase and harvested. The protein tryptic digests were analyzed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free quantitative analysis. The MS experiment was performed on a Q Exactive mass spectrometer. Raw spectra were quantitatively analyzed with the MaxQuant software (ver 1.3.0.5) and matched with the reference database. Differentially expressed proteins were analyzed using the bioinformatics method. The MS analysis wasrepeatedthreetimes for each sample. A total of 5012 proteins were identified across the KS-2, JIASHI-5 and SC6 strains in at least 2 of the three samples replicate. Of them, 1758 were identified to be differentially expressed at least between 2 strains, including 349 with known names. These differentially expressed proteins with known names are involved in biological functions such as energy and lipid metabolic process, nucleotide acid metabolic process, amino acid metabolic process, response to stress, cell membrane/cytoskeleton, cell cycle and proliferation, biological adhesion and proteolysis, localization and transport, regulation of the biological process, and signal transduction. The differentially expressed proteins and their related biological functions may shed light on the pathogenicity of Leishmania and targets for the development of vaccines and medicines.

Proteome and phospho-proteome profiling for deeper phenotype characterization of colorectal cancer heterogeneity.

e15536 Background: The rise of precision oncology therapeutics requires deep understanding of the molecular mechanisms implicated in cancer biology. Colorectal cancer (CRC) is one of the first solid tumors to be molecularly characterized by defined genes and pathways. Advances in tumor profiling have revealed a profound molecular heterogeneity in CRC leading to the definition of several consensus molecular subtypes (CMS). However, this molecular heterogeneity is still largely defined on the genomic and transcriptomics level. To complement the understanding of genetically defined molecular subgroups, we performed large-scale deep proteomic and phospho-proteomic profiling of CRC patient biopsies and adjacent healthy control tissue, which has enabled to explore the phenotype and obtain more functional insights in cancer biology. Methods: Sample processing from 5-10 mg of tissue per sample was performed using a liquid handling robot. Phospho-peptide enrichment was carried out with a Kingfisher Flex device and MagReSyn Ti-IMAC magnetic beads. Data-Independent Acquisition (DIA) LC-MS/MS was performed on multiple platforms consisting of a Thermo Scientific Q Exactive HF-X mass spectrometer coupled to a Waters M-Class LC. Chromatography was operating at 5 µL/min, and separation was achieved using 45 min (whole proteome) and 60 min (phospho-proteome) gradients. Results: Indivumed has built IndivuType, the world’s first multi-omics database for individualized cancer therapy, analyzing the highest quality cancer biospecimens to generate the most comprehensive dataset, including genomics, transcriptomics, proteomics, and clinical outcome information. Enabled by the DIA technology, a mass spectrometric method developed by Biognosys that obtains peptide fragmentation data in a highly parallelized way with high sensitivity, more than 7,000 proteins in the whole proteome and 20,000 phospho-peptides in the phospho-proteome workflow were profiled across more than 900 resected tissue samples of various CMS of CRC. The resulting proteome and phospho-proteome data were integrated into the IndivuType database and cross-analyzed with genomic and transcriptomic markers. Through this combined analysis, novel insights in clinically relevant signaling pathways in CRC subtypes were revealed. Conclusions: The deep phenotypic profiling of cancer samples, using next generation proteomics and phospho-proteomics, has enabled us to go beyond the genomic level in the characterization of tumor molecular heterogeneity. This multi-omics approach provides a solid foundation to advance the understanding of cancer biology, unravel key molecular events, and support the identification of novel therapeutic targets for precision medicine in CRC.

Rapid Identification of Chemical Constituents in Artemisia argyi Lévi. et Vant by UPLC-Q-Exactive-MS/MS

Artemisia argyi Lévi. et Vant is a traditional Chinese medicine with a long history, and its buds and seedlings can be used as vegetables. However, the investigations on the chemical constituents of A. argyi are not sufficient. In this paper, ultra-high performance liquid chromatography tandem hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive-MS/MS) was used to identify the chemical constituents of A. argyi. The Q Exactive mass spectrometer was used to collect MS and MS2 data. Finally, 125 compounds were preliminarily identified in A. argyi by comparing the retention time and accurate molecular weight with standard databases such as MZVault, MZCloud, and BGI Library (self-built standard Library by BGI Co., Ltd), including flavonoids, phenylpropanoids, terpenoids, and organic acids.

Metabolic Profiles of 5F-MDMB-PICA in Human Urine, Serum and Hair Samples Using LC-Q Exactive HF-MS.

In 2020, 5F-MDMB-PICA (5F-MDMB-2201) was one of the most common synthetic cannabinoids (SCs) identified in drugs seized by the Beijing Drug Control Agency, and it was categorized as Schedule II by the United Nations Office on Drugs and Crime in March 2020. It is difficult to detect 5F-MDMB-PICA in biological matrices due to its fast metabolic rate in vivo. In this work, 5F-MDMB-PICA metabolic profiles were investigated by liquid chromatography--quadrupole exactive high field orbitrap mass spectrometer (LC-Q Exactive HF MS), with accurate mass measurements in human urine, serum and pubic hair. To obtain intact metabolites, solid-phase extraction for urine and serum and direct ultrasonic extraction for pubic hair were applied to clean the samples without enzymatic hydrolysis. The differences in 5F-MDMB-PICA metabolism in the three different matrices were compared for the first time to determine the best detection biomarkers for monitoring 5F-MDMB-PICA misuse. Urine samples were determined to be the preferred biological material for identifying 5F-MDMB-PICA abuse. Forty-seven intact metabolites were detected in human urine, the ester hydrolyzed with glucuronidated metabolite in urine samples can be used as the primary biomarker to identify drug misuse. Fifteen metabolites were found in serum samples. Ester hydrolysis was considered to be the major metabolic pathway, and a large number of metabolites were involved with it. Zero metabolites apart from the parent drug were detected in pubic hair samples. Twenty-eight new metabolites and their metabolic pathways were characterized and tentatively identified by LC-QE-HF-MS, and a new potential biomarker (M5 ester hydrolysis + propionic acid) was also identified.

Metabolic Evaluation of Urine from Patients Diagnosed with High Grade (HG) Bladder Cancer by SPME-LC-MS Method.

Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. Thin-film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research. Moreover, this study’s results may support a better understanding of bladder cancer development and progression mechanisms.

Transmission-Mode MALDI Mass Spectrometry Imaging of Single Cells: Optimizing Sample Preparation Protocols.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) makes it possible to simultaneously visualize the spatial distribution of dozens to hundreds of different biomolecules (e.g., phospho- and glycolipids) in tissue sections and in cell cultures. The implementation of novel desorption and (post-)ionization techniques has recently pushed the pixel size of this imaging technique to the low micrometer scale and below and thus to a cellular and potentially sub-cellular level. However, to fully exploit this potential for cell biology and biomedicine, sample preparation becomes highly demanding. Here, we investigated the effect of several key parameters on the quality of the sample preparation and achievable spatial resolution, that include the washing, drying, chemical fixation, and matrix coating steps. The incubation of cells with formalin for about 5 min in combination with isotonic washing and mild drying produced a robust protocol that largely preserved not only cell morphologies, but also the molecular integrities of amine group-containing cell membrane phospholipids (phosphatidylethanolamines and -serines). A disadvantage of the chemical fixation is an increased permeabilization of cell membranes, resulting in leakage of cytosolic compounds. We demonstrate the pros and cons of the protocols with four model cell lines, cultured directly on indium tin oxide (ITO)-coated glass slides. Transmission (t-)mode MALDI-2-MSI enabled on a Q Exactive plus Orbitrap mass spectrometer was used to analyze the cultures at a pixel size of 2 μm. Phase contrast light microscopy and scanning electron microscopy were used as complementary methods. The protocols described could prove to be an important contribution to the advancement of single-cell MALDI imaging, especially for the characterization of cell-to-cell heterogeneities at a molecular level.

Simultaneous Determination of Acrylamide, 5-Hydroxymethylfurfural, and Heterocyclic Aromatic Amines in Thermally Processed Foods by Ultrahigh-Performance Liquid Chromatography Coupled with a Q Exactive HF-X Mass Spectrometer

In this study, a rapid and reliable method based on ultrahigh-performance liquid chromatography coupled with Q Exactive HF-X mass spectrometry (UHPLC-QE/MS) was established for the simultaneous quantification and validation of acrylamide, 5-hydroxymethylfurfural, and 14 heterocyclic aromatic amines in thermally processed foods. With the optimization of the pretreatment method, all 16 hazardous compounds with different polarities were simultaneously extracted and purified by one-step purification. By studying various acquisition modes in detail, full MS + PRM detection using an electrospray ionization source in the positive mode gives an excellent-shaped chromatographic peak and thereby achieves a better quantitative ability for analytes in the matrix. This method demonstrated good quantification recovery in the range of 68.85-146.42%. The limits of quantification were within the range from 0.1 to 50 ng/mL. With the method proposed, the simultaneous determination of 16 hazardous compounds in different thermally processed foods was successfully applied. The all-fragment-ion approaches at high resolution have the ability to reduce false-positive peak detections arising from peak alignment software in the detection of samples significantly. The proposed isotope dilution UHPLC-QE/MS method was validated and demonstrated to be sensitive, accurate, and precise for the simultaneous quantification of multiple contaminants in one injection.

Extraction, Radical Scavenging Activities, and Chemical Composition Identification of Flavonoids from Sunflower (Helianthus annuus L.) Receptacles.

This study was focused on extraction, radical scavenging activities, and chemical composition identification of total flavonoids in sunflower (Helianthus annuus L.) receptacles (TFSR). We investigated the optimal extract parameters of TFSR using response surface methodology. The highest yield of TFSR was 1.04% with the ethanol concentration 58%, the material-to-liquid ratio 1:20 (v/w), the extraction time 2.6 h, and the extraction temperature 67 °C. The results of radical scavenging activities showed that ethyl acetate fraction (EAF) was the strongest by using 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2’-azino-bis (3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS) and iron ion reducing analysis. The EAF had the highest flavonoids contents. Four fractions A, B, C and D were enrichment from EAF by polyamide resin. Fraction B had the highest flavonoids content. Thirteen chemical components of flavonoids in fraction B were first identified by Ultimate 3000 Nano LC System coupled to a Q Exactive HF benchtop Orbitrap mass spectrometer (UHPLC-HRMS/MS). Among of the thirteen chemical components, isoquercetin and daidzein were identified accurately by comparing with standard samples. Radical scavenging analysis showed that isoquercetin and EAF had strong activities. Therefore, sunflower receptacles can be used as a source of natural flavonoids. TFSR as a natural radical scavenger has potential applications in pharmaceutical industry.

Identification of antioxidant peptides from cheddar cheese made with Lactobacillus helveticus

The aim of the study was to identify potential antioxidant peptides from cheese made with Lactobacillus helveticus 1.0612. The results demonstrated that the nutrient composition of cheese was not affected by the addition of Lactobacillus helveticus, and Lactobacillus helveticus maintained its viability at 7.64 log CFU/g at the end of ripening. Lactobacillus helveticus can significantly increased the antioxidant activity of cheese during ripening time (P < 0.05), and the antioxidant activity reached maximum at the 5th month of ripening time. The potential antioxidant activity water soluble extracts of cheese were fractioned and purified by ultrafiltration and size exclusion technology. The fraction with the highest antioxidant activity was subjected to Q Exactive Plus-Orbitrap mass spectrometer for the identification of peptides. 15 peptides were identified from the most active fractions, and mainly derived from β- and αs1-casein. The size of the identified peptide sequence varied from 6 to 14 amino acid residues, and the molecular weight ranges from 700 to 1300 Da. 10 peptides contained at least one hydrophobic amino acids proline residue. Peptides VLPVPQK and EDVPSER have been reported for their antioxidant activity, DKIHPF for ACE-inhibitory activity. Moreover, several new peptides (EMPFPK, KEMPFPK, SDIPNPIGSENSEK), with potential antioxidant activity, have been identified.

Mass Spectrometry-Based Analysis of Mycobacterial Single-Colony Proteome.

Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an emerging area with established protocols and promising results. Traditional proteomic studies, especially involving bacteria, have been limited to suspension cultures with large protein yields. Such studies, however, remain population centered with the uniqueness of individual responses to environmental challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony from 7H10 media, with growth supplements for optimal growth. Following protein purification and digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer. Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative power while increasing reproducibility, of "very limiting samples."

662. Identification of Clinically Relevant Microbes with the MasSpec Pen

BackgroundIn the age of antimicrobial resistance, rapid identification of infectious agents is critical for antimicrobial stewardship and effective therapy. To this end, ambient ionization mass spectrometry techniques have been applied for rapid identification of microbes directly from culture isolates. We have developed a handheld, mass spectrometry-based device, the MasSpec Pen, that permits direct molecular analysis of a biological sample in seconds (Scheme 1). Here, we employ the MasSpec Pen to identify clinically relevant microbes directly from culture isolates.Methods Staphylococcus aureus, Staphylococcus epidermidis, Group A and B Streptococcus, Kingella kingae (K.k), and Pseudomonas aeruginosa (P.a) were cultured on 5% sheep’s blood nutrient agar at 37 °C overnight. Colonies were transferred to a glass slide where they were analyzed directly with the MasSpec Pen coupled to a Q Exactive mass spectrometer (Thermo Scientific) in negative ion mode. For MasSpec Pen analysis, a 10 µL droplet of water was held in contact with the sample surface for 3 seconds and then aspirated to the mass spectrometer for analysis.Data was normalized and the molecular features resulting from the analysis solvent and nutrient medium were removed. The least absolute shrinkage and selection operator (lasso) statistical method was used to build classification models for prediction of bacterial identity. Model performance was evaluated by leave-one-out cross-validation and a validation set of samples.Scheme 1: MasSpec Pen workflow ResultsVarious small molecules were detected including metabolites and glycerophospholipid species. The mass spectral profiles for each species exhibited qualitative differences among them (Figure 1). Additionally, several quorum-sensing molecules were observed in P.a. including hydroxy-heptyl-quinoline (m/z 242.155).Lasso statistical classifiers were created to differentiate organisms at the level of Gram type, genus, and species with each model comprised of a sparse set of molecular features. Accuracies of 90% or greater were achieved for all lasso models and as high as 98% for the differentiation of Staphylococcus (Staph.) and Streptococcus (Strep.).Figure 1: Molecular profiles of species analyzed Figure 2: Statistical classification results ConclusionThese results demonstrate the potential of the MasSpec Pen as a tool for clinical analysis of infected biospecimens.DisclosuresSydney C. Povilaitis, BA, MS Pen Technologies, Inc. (Other Financial or Material Support, Patent) Livia Eberlin, PhD, MS Pen Technolpogies, Inc. (Board Member, Shareholder)

Comparison of protein and peptide fractionation approaches in protein identification and quantification from Saccharomyces cerevisiae

Shotgun proteomics is a high-throughput technology which has been developed with the aim of investigating the maximum number of proteins in cells in a given experiment. However, protein discovery and data generation vary in depth and coverage when different technical strategies are selected. In this study, three different sample preparation approaches, and peptide or protein fractionation methods, were applied to identify and quantify proteins from log-phase yeast lysate: sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), filter-aided sample preparation coupled with gas phase fractionation (FASP-GPF), and FASP - high pH reversed phase fractionation (HpH). Fractions were initially analyzed and compared using nanoflow liquid chromatography – tandem mass spectrometry (nanoLC-MS/MS) employing data dependent acquisition on a linear ion trap instrument. The number of fractions and analytical replicates was adjusted so that each experiment used a similar amount of mass spectrometric instrument time. A second set of experiments was performed, comparing FASP-GPF, SDS-PAGE and FASP-HpH using a Q Exactive Orbitrap mass spectrometer. Compared with results from the linear ion trap mass spectrometer, the use of a Q Exactive Orbitrap mass spectrometer enabled a substantial increase in protein identifications, and an even greater increase in peptide identifications. This shows that the main advantage of using the higher resolution instrument is in increased proteome coverage. A total of 1035, 1357 and 2134 proteins were separately identified by FASP-GPF, SDS-PAGE and FASP-HpH. Combining results from the Orbitrap experiments, there were a total of 2269 proteins found, with 94% of them identified using the FASP-HpH method. Therefore, the FASP-HpH method is the optimal choice among these approaches, when applied to this type of sample.

Metabolomics profiling of plasma, urine and saliva after short term training in young professional football players in Saudi Arabia

Metabolomics profiling was carried out to observe the effect of short-term intensive physical activity on the metabolome of young Saudi professional football players. Urine, plasma and saliva were collected on 2 days pre- and post-training. An Orbitrap Exactive mass spectrometer was used to analyze the samples. A reversed-phase (RP) column was used for the analysis of non-polar plasma metabolites, and a ZIC-pHILIC column was used for the analysis of plasma, saliva and urine. mzMine was used to extract the data, and the results were modelled using Simca-P 14.1 software. There was no marked variation in the metabolite profiles between pre day 1 and 2 or between post day 1 and 2 according to principal components analysis (PCA). When orthogonal partial least squares (OPLSDA) modelling was also used, and then models could be fitted based on a total number of metabolites of 75, 16 and 32 for urine, plasma and saliva using hydrophilic interaction chromatography (HILIC) and 6 for analysis of plasma with reversed-phase (RP) chromatography respectively. The present study concludes that acylcarnitine may increase post-exercise in football players suggesting that they may burn fat rather than glucose. The levels of carnitine metabolites in plasma post-exercise could provide an important indicator of fitness.

Method of extraction and proteome profiling of mycobacteria using liquid chromatography-high resolution mass spectrometry

Advances in massively parallel sequencing, of complete bacterial genomes, have led to many novel findings in the field of genomics. However, these data often lack correlation with expressed protein profiles. It has been demonstrated that even very closely related genomes, such as in mycobacteria, express drastically different phenotypes. These phenotypes often have major roles in pathogenicity. Therefore, it is just as important to have a method for examining the proteome of a bacterium as well as its genome. These studies are further complicated in mycobacteria due to the cell wall and mycolic acid. A comprehensive method for the identification and characterization of the whole mycobacterium protein profile is needed. In the present study, a simple, sensitive, and specific liquid chromatography tandem mass spectrometry method was developed for the extraction, purification and profiling the mycobacterial proteome in various species. During development, sonication and bead-beating cell lysis protocol was tested using 15% Acetonitrile and 6 M guanidine-HCl (GuHCl) as extraction solvent. Sonication lysis in 6 M GuHCl with glass beads was the preferred method for cell lysis. This method was developed using reverse phase liquid chromatography and a Q Exactive ™ Plus Orbitrap™ mass spectrometer for peptide and protein identification. Bottom-up liquid chromatography-mass spectrometry LC–MS analysis resulted in identification of greater than 2500 proteins.

Palladium-Catalyzed O - and N -Glycosylation with Glycosyl Chlorides

Despite the significant progress in carbohydrate chemistry, there remains a pressing need for efficient and practical glycosylation methods using simple glycosyl donors and with high atom economy. ...

Characteristics of Atmospheric Fine Particulate Matter (PM2.5) Induced Differentially Expressed Proteins Determined by Proteomics and Bioinformatics Analyses

ObjectiveTo screen the differentially expressed proteins (DEPs) in human bronchial epithelial cells (HBE) treated with atmospheric fine particulate matter (PM2.5). MethodsHBE cells were treated with PM2.5 samples from Shenzhen and Taiyuan for 24 h. To detect overall protein expression, the Q Exactive mass spectrometer was used. Gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and Perseus software were used to screen DEPs. ResultsOverall, 67 DEPs were screened in the Shenzhen sample-treated group, of which 46 were upregulated and 21 were downregulated. In total, 252 DEPs were screened in the Taiyuan sample-treated group, of which 134 were upregulated and 118 were downregulated. KEGG analysis demonstrated that DEPs were mainly enriched in ubiquitin-mediated proteolysis and HIF-1 signal pathways in Shenzhen PM2.5 samples-treated group. The GO analysis demonstrated that Shenzhen sample-induced DEPs were mainly involved in the biological process for absorption of various metal ions and cell components. The Taiyuan PM2.5-induced DEPs were mainly involved in biological processes of protein aggregation regulation and molecular function of oxidase activity. Additionally, three important DEPs, including ANXA2, DIABLO, and AIMP1, were screened. ConclusionOur findings provide a valuable basis for further evaluation of PM2.5-associated carcinogenesis.