Application of SWATH Mass Spectrometry and Machine Learning in the Diagnosis of Inflammatory Bowel Disease Based on the Stool Proteome.

Inflammatory bowel disease (IBD) flare-ups exhibit symptoms that are similar to other diseases and conditions, making diagnosis and treatment complicated. Currently, the gold standard for diagnosing and monitoring IBD is colonoscopy and biopsy, which are invasive and uncomfortable procedures, and the fecal calprotectin test, which is not sufficiently accurate. Therefore, it is necessary to develop an alternative method. In this study, our aim was to provide proof of concept for the application of Sequential Window Acquisition of All Theoretical Mass Spectra-Mass spectrometry (SWATH-MS) and machine learning to develop a non-invasive and accurate predictive model using the stool proteome to distinguish between active IBD patients and symptomatic non-IBD patients. Proteome profiles of 123 samples were obtained and data processing procedures were optimized to select an appropriate pipeline. The differentially abundant analysis identified 48 proteins. Utilizing correlation-based feature selection (Cfs), 7 proteins were selected for proceeding steps. To identify the most appropriate predictive machine learning model, five of the most popular methods, including support vector machines (SVMs), random forests, logistic regression, naive Bayes, and k-nearest neighbors (KNN), were assessed. The generated model was validated by implementing the algorithm on 45 prospective unseen datasets; the results showed a sensitivity of 96% and a specificity of 76%, indicating its performance. In conclusion, this study illustrates the effectiveness of utilizing the stool proteome obtained through SWATH-MS in accurately diagnosing active IBD via a machine learning model.

Assessing the precision of a detergent-assisted cartridge precipitation workflow for non-targeted quantitative proteomics.

Detergent-based workflows incorporating sodium dodecyl sulfate (SDS) necessitate additional steps for detergent removal ahead of mass spectrometry (MS). These steps may lead to variable protein recovery, inconsistent enzyme digestion efficiency, and unreliable MS signals. To validate a detergent-based workflow for quantitative proteomics, we herein evaluate the precision of a bottom-up sample preparation strategy incorporating cartridge-based protein precipitation with organic solvent to deplete SDS. The variance of data-independent acquisition (SWATH-MS) data was isolated from sample preparation error by modelling the variance as a function of peptide signal intensity. Our SDS-assisted cartridge workflow yield a coefficient of variance (CV) of 13%-14%. By comparison, conventional (detergent-free) in-solution digestion increased the CV to 50%; in-gel digestion provided lower CVs between 14% and 20%. By filtering peptides predicting to display lower precision, we further enhance the validity of data in global comparative proteomics. These results demonstrate the detergent-based precipitation workflow is a reliable approach for in depth, label-free quantitative proteome analysis.

Short and Long-Term Outcomes in Hirschsprung Disease: Are the Syndrome-Associated Patients Really Doing Worse?

INTRODUCTION: Given the lack of data to help caregivers in the follow-up of Hirschsprung disease (HD), this study aimed to compare the functional outcomes of isolated Hirschsprung disease (I-HD) to syndrome-associated Hirschsprung disease (SA-HD) at 1, 3, 5, and 10 years. METHODS: A retrospective chart review of patients diagnosed with HD between January 1990 and May 2021 at our pediatric center was performed to collect data on patient characteristics, investigations, and treatments. Ninety-five patients were identified, of whom 76 were included in the study. SA-HD is defined as a syndrome known to be associated with HD or cognitive impairment. RESULTS: Patient characteristics were comparable between groups ( P > 0.05). There were 52 patients with I-HD and 24 with SA-HD. The patients median age was 9 days at diagnosis and 1.5 month at surgery. SA-HD patients became bowel continent at a significantly older age (mean age 8.43 vs 4.94 years, P = 0.0471) and received more bowel continence medications. At 5 years, SA-HD patients requiring ≥2 medications for bowel continence represented 54.5% versus 11.1% of I-HD patients ( P = 0.009). Lastly, SA-HD patients had urinary incontinence at a significantly older age ( P = 0.0136, 5 years). CONCLUSION: Clinicians should be aware that SA-HD patients are more prone to bladder dysfunction and became bowel continent at an older age than I-HD patients. They need more and prolonged bowel management medications, and other important complications need to be addressed in patient care. These results should prompt a longer follow-up period for these patients, especially in SA-HD.

Proteomics Profiling of Stool Samples from Preterm Neonates with SWATH/DIA Mass Spectrometry for Predicting Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a life-threatening condition for premature infants in neonatal intensive care units. Finding indicators that can predict NEC development before symptoms appear would provide more time to apply targeted interventions. In this study, stools from 132 very-low-birth-weight (VLBW) infants were collected daily in the context of a multi-center prospective study aimed at investigating the potential of fecal biomarkers for NEC prediction using proteomics technology. Eight of the VLBW infants received a stage-3 NEC diagnosis. Stools collected from the NEC infants up to 10 days before their diagnosis were available for seven of them. Their samples were matched with those from seven pairs of non-NEC controls. The samples were processed for liquid chromatography-tandem mass spectrometry analysis using SWATH/DIA acquisition and cross-compatible proteomic software to perform label-free quantification. ROC curve and principal component analyses were used to explore discriminating information and to evaluate candidate protein markers. A series of 36 proteins showed the most efficient capacity with a signature that predicted all seven NEC infants at least a week in advance. Overall, our study demonstrates that multiplexed proteomic signature detection constitutes a promising approach for the early detection of NEC development in premature infants.

Design and Structure-Activity Relationship of a Potent Furin Inhibitor Derived from Influenza Hemagglutinin.

Furin plays an important role in various pathological states, especially in bacterial and viral infections. A detailed understanding of the structural requirements for inhibitors targeting this enzyme is crucial to develop new therapeutic strategies in infectious diseases, including an urgent unmet need for SARS-CoV-2 infection. Previously, we have identified a potent furin inhibitor, peptide Ac-RARRRKKRT-NH 2 (CF1), based on the highly pathogenic avian influenza hemagglutinin. The goal of this study was to determine how its N-terminal part (the P8-P5 positions) affects its activity profile. To do so, the positional-scanning libraries of individual peptides modified at the selected positions with natural amino acids were generated. Subsequently, the best substitutions were combined together and/or replaced by unnatural residues to expand our investigations. The results reveal that the affinity of CF1 can be improved (2-2.5-fold) by substituting its P5 position with the small hydrophobic residues (Ile or Val) or a basic Lys.

Mast Cell Degranulation Increases Mouse Mast Cell Protease 4-Dependent Vasopressor Responses to Big Endothelin-1 But Not Angiotensin I.

Mouse mast cell protease 4 (mMCP-4), the murine functional analog to the human chymase, is a serine protease synthesized and stored in mast cell secretory granules. Our previous studies reported physiologic and pathologic roles for mMCP-4 in the maturation and synthesis of the vasoactive peptide endothelin-1 (ET-1) from its precursor, big ET-1. The aim of this study was to investigate the impact of mast cell degranulation or stabilization on mMCP-4-dependent pressor responses after the administration of big ET-1 or angiotensin I (Ang I). In anesthetized mice, mast cell degranulation induced by compound 48/80 (C48/80) or stabilization by cromolyn enhanced or repressed, respectively, the dose-dependent vasopressor responses to big ET-1 in wild-type (WT) mice but not in mMCP-4 knockout mice in a chymase inhibitor (TY-51469)-sensitive fashion. In addition, mMCP-4-dependent hydrolysis of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was depleted or enhanced in peritoneal mast cells isolated from mice pretreated with C48/80 or cromolyn, respectively. Furthermore, C48/80 or cromolyn markedly increased or abolished, respectively, ET-1 (1-31) conversion from exogenous big ET-1 in WT mice peritoneal fluid-isolated mast cells, in vitro. Finally, the vasopressor responses to Ang I were unaffected by mast cell activation or stabilization, whereas those induced by the angiotensin-converting enzyme-resistant Ang I analog, [Pro11, D-Ala12] Ang I, were potentiated by C48/80. Altogether, the present study shows that mast cell activation enhances the mMCP-4-dependent vasoactive properties of big ET-1 but not Ang I in the mouse model. SIGNIFICANCE STATEMENT: The current work demonstrates a significant role for mast cell stability in the cardiovascular pharmacology of big endothelin-1 but not angiotensin I in the murine systemic circulation.

Investigating protein thiol chemistry associated with dehydroascorbate, homocysteine and glutathione using mass spectrometry.

RATIONALE: Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigae S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry. METHODS: Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) and Grx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS. RESULTS: ESI-MS analysis reveals that DHA mediates disulfide formation and S-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1_ HHb and Prdx2. The GS-S-protein adducts over time dominate the ESI-MS spectrum profile. CONCLUSIONS: Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.

Dehydroascorbic acid S-Thiolation of peptides and proteins: Role of homocysteine and glutathione.

Ascorbic acid (vitamin C) plays a significant role in the prevention of oxidative stress. In this process, ascorbate is oxidized to dehydroascorbate (DHA). We have investigated the impact of DHA on peptide/protein intramolecular disulfide formation as well as S-glutathionylation and S-homocysteinylation. S-glutathionylation of peptides/proteins is a reversible, potential regulatory mechanism in oxidative stress. Although the exact role of protein S-homocysteinylation is unknown, it has been proposed to be of importance in pathobiological processes such as onset of cardiovascular disease. Using an in vitro model system, we demonstrate that DHA causes disulfide bond formation within the active site of recombinant human glutaredoxin (Grx-1). DHA also facilities the formation of S-glutathionylation and S-homocysteinylation of a model peptide (AcFHACAAK) as well as Grx-1. We discuss the possible mechanisms of peptide/protein S-thiolation, which can occur either via thiol exchange or a thiohemiketal intermediate. A thiohemiketal DHA-peptide adduct was detected by mass spectrometry and its location on the peptide/protein cysteinyl thiol group was unambiguously confirmed by tandem mass spectrometry. This demonstrates that peptide/protein S-thiolation mediated by DHA is not limited to thiol exchange reactions but also takes place directly via the formation of a thiohemiketal peptide intermediate. Finally, we investigated a potential reducing role of glutathione (GSH) in the presence of S-homocysteinylated peptide/protein adducts. S-homocysteinylated AcFHACAAK, human hemoglobin α-chain and Grx-1 were incubated with GSH. Both peptide and proteins were reduced, and homocysteine replaced with GS-adducts by thiol exchange, as a function of time.

Mouse Mast Cell Protease 4 Deletion Protects Heart Function and Survival After Permanent Myocardial Infarction.

Chymase, a mast cell serine protease involved in the generation of multiple cardiovascular factors, such as angiotensin II and endothelin-1 (ET-1), is elevated and participates in tissue degeneration after permanent myocardial infarction (PMI). Anesthetized 4-month old male wild-type (WT) C57BL/6J mice and mouse mast cell protease-4 knockout (mMCP-4 KO) congeners were subjected to ligation of the left anterior descending (LAD) coronary artery. A group of mice was then subjected to Kaplan-Meier 28-day survival analysis. In another group of mice, 18F-fluorodeoxyglucose positron emission tomography (PET) was performed to evaluate heart function and the infarcted zone 3 days post-PMI surgery. Cardiac morphology following PMI was evaluated on formalin-fixed heart slices and glycoproteomic analysis was performed using mass spectrometry. Finally, cardiac and lung tissue content of immunoreactive ET-1 was determined. PMI caused 60% mortality in WT mice, due to left ventricular wall rupture, and 7% in mMCP-4 KO mice. Cardiac PET analysis revealed a significant reduction in left ventricular volume (systolic and diastolic) and preserved the ejection fraction in mMCP-4 KO compared to WT animals. The infarcted area, apoptotic signaling and wall remodeling were significantly decreased in mMCP-4 KO mice compared to their WT congeners, while collagen deposition was increased. Glycoproteomic analysis showed an increase in apolipoprotein A1, an established chymase substrate in mMCP-4 KO mice compared to WT mice post-PMI. ET-1 levels were increased in the lungs of WT, but not mMCP-4 KO mice, 24 h post-PMI. Thus, the genetic deletion of mMCP-4 improved survival and heart function post-PMI.

Peak AAA fatty acid homolog contaminants present in the dietary supplement l-Tryptophan associated with the onset of eosinophilia-myalgia syndrome.

The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as case-associated contaminants. Recently the final and most statistically significant contaminant, "Peak AAA" was structurally characterized. The "compound" was actually shown to be two structural isomers resulting from condensation reactions of L-Trp with fatty acids derived from the bacterial cell membrane. They were identified as the indole C-2 anteiso (AAA1-343) and linear (AAA2-343) aliphatic chain isomers. Based on those findings, we utilized a combination of on-line HPLC-electrospray ionization mass spectrometry (LC-MS), as well as both precursor and product ion tandem mass spectrometry (MS/MS) to facilitate identification of a homologous family of condensation products related to AAA1-343 and AAA2-343. We structurally characterized eight new AAA1-XXX/AAA2-XXX contaminants, where XXX represents the integer molecular ions of all the related homologs, differing by aliphatic chain length and isomer configuration. The contaminants were derived from the following fatty acids of the bacterial cell membrane, 5-methylheptanoic acid (anteiso-C8:0) for AAA1-315; n-octanoic acid (n-C8:0) for AAA2-315; 6-methyloctanoic acid (anteiso-C9:0) for AAA1-329; n-nonanoic acid (n-C9:0) for AAA2-329; 10-methyldodecanoic acid (anteiso-C13:0) for AAA1-385; n-tridecanoic acid (n-C13:0) for AAA2-385; 11-methyltridecanoic acid (anteiso-C14:0) for AAA1-399; and n-tetradecanoic acid (n-C14:0) for AAA2-399. The concentration levels for these contaminants were estimated to be 0.1-7.9 µg / 500 mg of an individual SD L-Trp tablet or capsule The structural similarity of these homologs to case-related contaminants of Spanish Toxic Oil Syndrome (TOS) is discussed.

Structure determination of disease associated peak AAA from l-Tryptophan implicated in the eosinophilia-myalgia syndrome.

The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere was caused by the ingestion of l-Tryptophan (L-Trp) solely manufactured by the Japanese company Showa Denko K.K. (SD). Six compounds present in the SD L-Trp were reported to be case-associated contaminants. However, "one" of these compounds, Peak AAA has remained structurally uncharacterized, despite the fact that it was described as "the only statistically significant (p=0.0014) contaminant". Here, we employ on-line microcapillary-high performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS) to determine that Peak AAA is in fact two structurally related isomers. Peak AAA1 and Peak AAA2 differed in LC retention times, and were determined by accurate mass-LC-MS to both have a protonated molecular ion (MH+) of mass 343.239Da (Da), corresponding to a molecular formula of C21H30N2O2, and possessing eight degrees of unsaturation (DoU) for the non-protonated molecule. By comparing the LC-MS and LC-MS-MS retention times and spectra with authentic synthetic standards, Peak AAA1 was identified as the intermolecular condensation product of L-Trp with anteiso 7-methylnonanoic acid, to afford (S)-2-amino-3-(2-((S,E)-7-methylnon-1-en-1-yl)-1H-indol-3-yl)propanoic acid. Peak AAA2 was determined to be a condensation product of L-Trp with decanoic acid, which produced (S)-2-amino-3-(2-((E)-dec-1-en-1-yl)-1H-indol-3-yl)propanoic acid.

Multi-omic network-based interrogation of rat liver metabolism following gastric bypass surgery featuring SWATH proteomics.

Morbidly obese patients often elect for Roux-en-Y gastric bypass (RYGB), a form of bariatric surgery that triggers a remarkable 30% reduction in excess body weight and reversal of insulin resistance for those who are type II diabetic. A more complete understanding of the underlying molecular mechanisms that drive the complex metabolic reprogramming post-RYGB could lead to innovative non-invasive therapeutics that mimic the beneficial effects of the surgery, namely weight loss, achievement of glycemic control, or reversal of non-alcoholic steatohepatitis (NASH). To facilitate these discoveries, we hereby demonstrate the first multi-omic interrogation of a rodent RYGB model to reveal tissue-specific pathway modules implicated in the control of body weight regulation and energy homeostasis. In this study, we focus on and evaluate liver metabolism three months following RYGB in rats using both SWATH proteomics, a burgeoning label free approach using high resolution mass spectrometry to quantify protein levels in biological samples, as well as MRM metabolomics. The SWATH analysis enabled the quantification of 1378 proteins in liver tissue extracts, of which we report the significant down-regulation of Thrsp and Acot13 in RYGB as putative targets of lipid metabolism for weight loss. Furthermore, we develop a computational graph-based metabolic network module detection algorithm for the discovery of non-canonical pathways, or sub-networks, enriched with significantly elevated or depleted metabolites and proteins in RYGB-treated rat livers. The analysis revealed a network connection between the depleted protein Baat and the depleted metabolite taurine, corroborating the clinical observation that taurine-conjugated bile acid levels are perturbed post-RYGB.

Molecular Consequences of Proprotein Convertase 1/3 (PC1/3) Inhibition in Macrophages for Application to Cancer Immunotherapy: A Proteomic Study.

Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macrophages and regulated as a function of the Toll-like receptor immune response. Here, we investigated the intracellular impact of the down-regulation of the proprotein convertase 1/3 in NR8383 macrophages and confirmed the results on macrophages from PC1/3 deficient mice. A complete proteomic study of secretomes and intracellular proteins was undertaken and revealed that inhibition of proprotein convertase 1/3 orient macrophages toward an M1 activated phenotype. This phenotype is characterized by filopodial extensions, Toll-like receptor 4 MyD88-dependent signaling, calcium entry augmentation and the secretion of pro-inflammatory factors. In response to endotoxin/lipopolysaccharide, these intracellular modifications increased, and the secreted factors attracted naïve T helper lymphocytes to promote the cytotoxic response. Importantly, the application of these factors onto breast and ovarian cancer cells resulted in a decrease viability or resistance. Under inhibitory conditions using interleukin 10, PC1/3-knockdown macrophages continued to secrete inflammatory factors. These data indicate that targeted inhibition of proprotein convertase 1/3 could represent a novel type of immune therapy to reactivate intra-tumoral macrophages.

Chymase inhibitor-sensitive synthesis of endothelin-1 (1-31) by recombinant mouse mast cell protease 4 and human chymase.

Important structural differences imply that human and mouse mast cell chymases may differ with respect to their enzymatic properties. We compared in this study the catalytic efficiencies of recombinant human chymase (rCMA1) and its functional murine homologue recombinant mouse mast cell protease-4 (rmMCP-4) toward a fluorogenic chymase substrate (Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin (AMC) and by their ability to convert Big-endothelin (ET)-1 into ET-1 (1-31) using a LC/MS/MS system. Activities toward a fluorogenic substrate (Suc-Leu-Leu-Val-Tyr-AMC) and Big ET-1 were also measured in extracts from mouse peritoneal mast cells, LUVA human mast cell-like cells and human aortas. The specificity of these activities was assessed with the chymase inhibitor TY-51469 (2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonyl-phenyl]thiazole-4-carboxylic acid). For similar affinities, rmMCP-4 showed a higher activity toward the fluorogenic substrate and a higher ability to process Big ET-1 as compared to recombinant CMA1 (chymase activity (kcat/KM in µM(-1)s(-1)): 2.29 × 10(-4)vs. 6.41 × 10(-6); ET-1 (1-31) production: 2.19 × 10(-3)vs. 6.57 × 10(-5)), and both of these activities of mouse and human chymase were sensitive to TY-51469. Furthermore, extracts from mouse peritoneal mast cells, LUVA cells and human aorta homogenates contained processing activities toward the fluorogenic chymase substrate as well as Big ET-1, all of which were sensitive to TY-51469. Finally, the pressor responses to Big ET-1 but not to ET-1 were significantly reduced in conscious and free moving mMCP-4 KO mice when compared to wild type congeners. Our results suggest that both mouse and human chymases have potent ET-1 (1-31)-producing abilities, with the murine isoform being more efficient.

Optimization of furin inhibitors to protect against the activation of influenza hemagglutinin H5 and Shiga toxin.

Proprotein convertases (PCs) are crucial in the processing and entry of viral or bacterial protein precursors and confer increased infectivity of pathogens bearing a PC activation site, which results in increased symptom severity and lethality. Previously, we developed a nanomolar peptide inhibitor of PCs to prevent PC activation of infectious agents. Herein, we describe a peptidomimetic approach that increases the stability of this inhibitor for use in vivo to prevent systemic infections and cellular damage, such as that caused by influenza H5N1 and Shiga toxin. The addition of azaß(3)-amino acids to both termini of the peptide successfully prevented influenza hemagglutinin 5 fusogenicity and Shiga toxin Vero toxicity in cell-based assays. The results from a cell-based model using stable shRNA-induced proprotein convertase knockdown indicate that only furin is the major proprotein convertase required for HA5 cleavage.

Modification of peptide and protein cysteine thiol groups by conjugation with a degradation product of ascorbate.

Ascorbate is an important water-soluble antioxidant, which when oxidized by reactive oxygen species is converted into dehydroascorbate (DHA). If not rapidly reduced back to ascorbate, DHA decomposes to a reactive 5-carbon compound (DHA*, +130 Da) that can modify reduced cysteinyl residues in peptides and proteins in vitro. The formation of cysteine adducts by DHA* was characterized by mass spectrometry using reduced insulin B-chain, α-lactalbumin, and hemoglobin. Mass spectrometry of DHA* modified insulin B-chain revealed the presence of one and two DHA* adducts. Enzymatic cleavage and tandem mass spectrometry of modified peptides allowed unambiguous localization of DHA* to the two cysteine residues in positions 7 and 19 of the insulin B-chain. Incubations of DHA with α-lactalbumin revealed that approximately 25% of the protein population was in a reduced state and could be modified by DHA*. The adduct was assigned to the N-terminally located cysteinyl residue in position 6. Incubation of hemoglobin with DHA followed by pepsin digestion and electrospray ionization tandem mass spectrometry (ESI-MSMS) of the peptide mixture allowed for the identification of three modified peptides. Tandem mass spectrometry of the modified peptides, two from the hemoglobin A-chain with identical mass and one from the hemoglobin B-chain, gave a complete series of y-type fragment ions, which were assigned to the cysteine containing peptides (100)LLSHCL(105) (A-chain), (101)LSHCLL(106) (A-chain), and (111)VCVLAHHFGKE(121) (B-chain). Although the DHA* adduct was lost from the peptides derived from α-lactalbumin and hemoglobin before fragmentation of the peptide bond, carbamidomethylation of the proteins prior to incubation with DHA abolished the formation of DHA*-protein adducts and confirmed that the target was indeed the cysteine thiol group. Future studies are focused on the modification of proteins by DHA* in cells and in vivo.

Proprotein convertase 1/3 (PC1/3) in the rat alveolar macrophage cell line NR8383: localization, trafficking and effects on cytokine secretion.

The proprotein convertase 1/3 (PC1/3) is an important post-translational processing enzyme for the activation of precursor proteins within the regulated secretory pathway. Well characterized for its role in the neural and endocrine systems, we recently reported an unconventional role of PC1/3 as a modulator of the Toll-like receptor innate immune response. There are only a few reports that have studied PC1/3 expression in macrophages, and more investigation is needed to better characterize its function. These studies would greatly benefit from model cell lines. Our study aims to identify and characterize PC1/3 in a relevant model macrophage cell line and to determine the links between PC1/3 and innate immune cellular responses. We describe the rat alveolar cell line, NR8383, as expressing PC1/3 and the most common Toll-like receptors. In NR8383 cells, PC1/3 is localized at the Trans-Golgi network and traffics to lysosome related vesicles upon lipopolysaccharide stimulation. Moreover, we report the co-localization of PC1/3 and Toll-like receptor 4 upon lipopolysaccharide stimulation. Down regulation of PC1/3 by shRNA produce a similar phenotype in NR8383 to what we previously reported in isolated peritoneal macrophages. PC1/3 shRNA induced changes in the cellular organization and expression of the specific trafficking regulator RAB GTPase. As a consequence, NR8383 down-regulated for PC1/3, present an abnormal cytokine secretion profile. We conclude that the NR8383 cell line represents a good model to study PC1/3 in macrophages and we present PC1/3 as an important regulator of vesicle trafficking and secretion in macrophages.

Proteomic analyses of serous and endometrioid epithelial ovarian cancers - cases studies - molecular insights of a possible histological etiology of serous ovarian cancer.

PURPOSE: Epithelial ovarian carcinogenesis may occur de novo on the surface of ovarian mesothelial epithelial cells or from cells originating in other organs. Foreign Müllerian cell intrusion into the ovarian environment has been hypothesized to explain the latter scenario. In this study, MALDI MS profiling technology was used to provide molecular insights regarding these potentially different mechanisms. EXPERIMENTAL DESIGN: Using MALDI MS profiling, the molecular disease signatures were established in their anatomical context. MALDI MS profiling was used on serous and endometrioid cancer biopsies to investigate cases of epithelial ovarian cancer. We then applied bioinformatic methods and identification strategies on the LC-MS/MS analyses of extracts from digested formalin-fixed, paraffin-embedded tissues. Extracts from selected regions (i.e. serous ovarian adenocarcinoma, fallopian tube serous adenocarcinoma, endometrioid ovarian cancer, benign endometrium, and benign ovarian tissues) were performed, and peptide digests were subjected to LC-MS/MS analysis. RESULTS: Comparison of the proteins identified from benign endometrium or three ovarian cancer types (i.e. serous ovarian adenocarcinoma, endometrioid ovarian adenocarcinoma, and serous fallopian tube adenocarcinoma) provided new evidence of a possible correlation between the fallopian tubes and serous ovarian adenocarcinoma. Here, we propose a workflow consisting of the comparison of multiple tissues in their anatomical context in an individual patient. CONCLUSION AND CLINICAL RELEVANCE: The present study provides new insights into the molecular similarities between these two tissues and an assessment of highly specific markers for an individualized patient diagnosis and care.

Need satisfaction, work-school interference and school dropout: an application of self-determination theory.

BACKGROUND: In many parts of the world, it is common for secondary school students to be involved in part-time employment. Research shows that working can have a negative impact on school engagement. However, the majority of studies have focused on the amount of time that students spend working rather than on the quality of work experience and its influence on school engagement. AIMS: This study explored the relation of part-time work and school experiences to dropout intentions among secondary school and junior college students. The study was conceptualized from a self-determination theory perspective (Deci & Ryan, 2000). SAMPLE: Participants were 3,248 students from rural and suburban schools in the greater region of Montreal, Canada. METHOD: Questionnaires were used to assess the number of hours worked, the extent to which work interfered with or facilitated school functioning, autonomy, competence, and relatedness experienced in the work and school domains. School performance and school dropout intentions were also assessed. RESULTS: A curvilinear relation between work hours and dropout intentions was found, reflecting that part-time work began to be associated with higher dropout intentions only when students worked more than 7 hr per week. Analyses also showed that work-school interference was related to dropout intentions, and that this variable served to mediate the relation of employer autonomy support to dropout intentions. CONCLUSIONS: These results suggest that both the quantity and the quality of students' part-time work experiences need to be considered when examining the relation of work to school engagement.

Targeted mass spectrometry imaging: specific targeting mass spectrometry imaging technologies from history to perspective.

Since its introduction during the last decade, MALDI mass spectrometry imaging (MSI) is now a routine technique in biology. Nevertheless, a missing link exists in MALDI MSI. Lipids, peptides/proteins, metabolites and drugs can easily be mapped using MALDI-MSI, but this technique has not yet been used to map the transcriptome, which includes microRNA, siRNA and other components. This latter field of research is now one of the major fields in clinical research and needs to be explored using MALDI-MSI. To investigate the transcriptome, a novel imaging technique has been developed called Tag-Mass imaging mass spectrometry. The aim of this review is to discuss this technique from its history to its place in the future of mass spectrometric imaging.