High-sensitivity Mass Spectrometry Research Articles

Crucial Parameters for Immunopeptidome Characterization: A Systematic Evaluation.

Immunopeptidomics is the area of knowledge focused on the study of peptides assembled in the major histocompatibility complex (MHC), or human leukocyte antigen (HLA) in humans, which could activate the immune response via specific and selective T cell recognition. Advances in high-sensitivity mass spectrometry have enabled the detailed identification and quantification of the immunopeptidome, significantly impacting fields like oncology, infections, and autoimmune diseases. Current immunopeptidomics approaches primarily focus on workflows to identify immunopeptides from HLA molecules, requiring the isolation of the HLA from relevant cells or tissues. Common critical steps in these workflows, such as cell lysis, HLA immunoenrichment, and peptide isolation, significantly influence outcomes. A systematic evaluation of these steps led to the creation of an 'Immunopeptidome Score' to enhance the reproducibility and robustness of these workflows. This score, derived from LC-MS/MS datasets (ProteomeXchange identifier PXD038165), in combination with available information from public databases, aids in optimizing the immunopeptidome characterization process. The 'Immunopeptidome Score' has been applied in a systematic analysis of protein extraction, HLA immunoprecipitation, and peptide recovery yields across several tumor cell lines enabling the selection of peptides with optimal features and, therefore, the identification of potential biomarker and therapeutic targets.

Fully Integrated Recognition and Enrichment Electrospray Ionization Source for High-Sensitivity Mass Spectrometry Determination of Bioamine.

The development of a highly specific recognition electrospray ionization source presents a major challenge for achieving rapid ambient mass spectrometry (AMS) detection of trace harmful substances in complex samples. In this study, we constructed a molecular imprinting nanofiber electrospinning membrane-coated steel substrate (MINMCS) based on the electrospinning strategy. This was designed as a highly specific recognition and enrichment electrospray ionization source module for AMS, where the molecular imprinting nanofiber membrane served as an excellent extraction and enrichment layer. The prepared ionization source demonstrated a sufficient loading capacity for three bioamines (BAs): histamine (HIS), tyramine (TYR), and tryptamine (TRY). With simplified sample pretreatment, this ionization source exhibited sensitivity comparable to that of high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Moreover, the entire analysis process could be completed within 1 min with acceptable recoveries (83.21-101.80%). In brief, this study introduces a new integrated recognition and enrichment electrospray ionization source for the detection of harmful substances such as bioamines, showcasing significant commercial potential for the rapid detection of foodborne harmful compounds.

Identification of the binding proteins of organic acid metabolites by matrix thermal shift assay

Organic acid metabolites exhibit acidic properties. These metabolites serve as intermediates in major carbon metabolic pathways and are involved in several biochemical pathways, including the tricarboxylic acid (TCA) cycle and glycolysis. They also regulate cellular activity and play crucial roles in epigenetics, tumorigenesis, and cellular signal transduction. Knowledge of the binding proteins of organic acid metabolites is crucial for understanding their biological functions. However, identifying the binding proteins of these metabolites has long been a challenging task owing to the transient and weak nature of their interactions. Moreover, traditional methods are unsuitable for the structural modification of the ligands of organic acid metabolites because these metabolites have simple and similar structures. Even minor structural modifications can significantly affect protein interactions. Thermal proteome profiling (TPP) provides a promising avenue for identifying binding proteins without the need for structural modifications. This approach has been successfully applied to the identification of the binding proteins of several metabolites. In this study, we investigated the binding proteins of two TCA cycle intermediates, i.e., succinate and fumarate, and lactate, an end-product of glycolysis, using the matrix thermal shift assay (mTSA) technique. This technique involves combining single-temperature (52 ℃) TPP and dose-response curve analysis to identify ligand-binding proteins with high levels of confidence and determine the binding affinity between ligands and proteins. To this end, HeLa cells were lysed, followed by protein desalting to remove endogenous metabolites from the cell lysates. The desalted cell lysates were treated with fumarate or succinate at final concentrations of 0.004, 0.04, 0.4, and 2 mmol/L in the experimental groups or 2 mmol/L sodium chloride in the control group. Considering that the cellular concentration of lactate can be as high as 2-30 mmol/L, we then applied lactate at final concentrations of 0.2, 1, 5, 10, and 25 mmol/L in the experimental groups or 25 mmol/L sodium chloride in the control group. Using high-sensitivity mass spectrometry coupled with data-independent acquisition (DIA) quantification, we quantified 5870, 5744, and 5816 proteins in succinate, fumarate, and lactate mTSA experiments, respectively. By setting stringent cut-off values (i.e., significance of changes in protein thermal stability (p-value)<0.001 and quality of the dose-response curve fitting (square of Pearson's correlation coefficient, R2)>0.95), multiple binding proteins for these organic acid metabolites from background proteins were confidently determined. Several known binding proteins were identified, notably fumarate hydratase (FH) as a binding protein for fumarate, and α-ketoglutarate-dependent dioxygenase (FTO) as a binding protein for both fumarate and succinate. Additionally, the affinity data for the interactions between these metabolites and their binding proteins were obtained, which closely matched those reported in the literature. Interestingly, ornithine aminotransferase (OAT), which is involved in amino acid biosynthesis, and 3-mercaptopyruvate sulfurtransferase (MPST), which acts as an antioxidant in cells, were identified as lactate-binding proteins. Subsequently, an orthogonal assay technique developed in our laboratory, the solvent-induced precipitation (SIP) technique, was used to validate the mTSA results. SIP identified OAT as the top target candidate, validating the mTSA-based finding that OAT is a novel lactate-binding protein. Although MPST was not identified as a lactate-binding protein by SIP, statistical analysis of MPST in the mTSA experiments with 10 or 25 mmol/L lactate revealed that MPST is a lactate-binding protein with a high level of confidence. Peptide-level empirical Bayes t-tests combined with Fisher's exact test also supported the conclusion that MPST is a lactate-binding protein. Lactate is structurally similar to pyruvate, the known binding protein of MPST. Therefore, assuming that lactate could potentially occupy the binding site of pyruvate on MPST. Overall, the novel binding proteins identified for lactate suggest their potential involvement in amino acid synthesis and redox balance regulation.

Novel formylpeptide receptor 1/2 agonist limits hypertension-induced cardiovascular damage.

Formylpeptide receptors (FPRs) play a critical role in the regulation of inflammation, an important driver of hypertension-induced end-organ damage. We have previously reported that the biased FPR small-molecule agonist, compound17b (Cmpd17b), is cardioprotective against acute, severe inflammatory insults. Here, we reveal the first compelling evidence of the therapeutic potential of this novel FPR agonist against a longer-term, sustained inflammatory insult, i.e. hypertension-induced end-organ damage. The parallels between the murine and human hypertensive proteome were also investigated. The hypertensive response to angiotensin II (Ang II, 0.7 mg/kg/day, s.c.) was attenuated by Cmpd17b (50 mg/kg/day, i.p.). Impairments in cardiac and vascular function assessed via echocardiography were improved by Cmpd17b in hypertensive mice. This functional improvement was accompanied by reduced cardiac and aortic fibrosis and vascular calcification. Cmpd17b also attenuated Ang II-induced increased cardiac mitochondrial complex 2 respiration. Proteomic profiling of cardiac and aortic tissues and cells, using label-free nano-liquid chromatography with high-sensitivity mass spectrometry, detected and quantified ∼6000 proteins. We report hypertension-impacted protein clusters associated with dysregulation of inflammatory, mitochondrial, and calcium responses, as well as modified networks associated with cardiovascular remodelling, contractility, and structural/cytoskeletal organization. Cmpd17b attenuated hypertension-induced dysregulation of multiple proteins in mice, and of these, ∼110 proteins were identified as similarly dysregulated in humans suffering from adverse aortic remodelling and cardiac hypertrophy. We have demonstrated, for the first time, that the FPR agonist Cmpd17b powerfully limits hypertension-induced end-organ damage, consistent with proteome networks, supporting development of pro-resolution FPR-based therapeutics for treatment of systemic hypertension complications.

Patients with Neurodegenerative Proteinopathies Exhibit Altered Tryptophan Metabolism in the Serum and Cerebrospinal Fluid.

Some pathological conditions affecting the human body can also disrupt metabolic pathways and thus alter the overall metabolic profile. Knowledge of metabolic disturbances in specific diseases could thus enable the differential diagnosis of otherwise similar conditions. This work therefore aimed to comprehensively characterize changes in tryptophan metabolism in selected neurodegenerative diseases. Levels of 18 tryptophan-related neuroactive substances were determined by high throughput and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry in time-linked blood serum and cerebrospinal fluid samples from 100 age-matched participants belonging to five cohorts: healthy volunteers (n = 21) and patients with Lewy body disease (Parkinson's disease and dementia with Lewy bodies; n = 31), four-repeat tauopathy (progressive supranuclear palsy and corticobasal syndrome; n = 10), multiple system atrophy (n = 13), and Alzheimer's disease (n = 25). Although these conditions have different pathologies and clinical symptoms, the discovery of new biomarkers is still important. The most statistically significant differences (with p-values of ≤0.05 to ≤0.0001) between the study cohorts were observed for three tryptophan metabolites: l-kynurenine in cerebrospinal fluid and 3-hydroxy-l-kynurenine and 5-hydroxy-l-tryptophan in blood serum. This led to the discovery of distinctive correlation patterns between the profiled cerebrospinal fluid and serum metabolites that could provide a basis for the differential diagnosis of neurodegenerative tauopathies and synucleinopathies. However, further large-scale studies are needed to determine the direct involvement of these metabolites in the studied neuropathologies, their response to medication, and their potential therapeutic relevance.

Genetic Algorithm Optimized Printed Circuit Board Ion Funnel Tandem Subambient Pressure Ionization with Nanoelectrospray (SPIN) for High Sensitivity Mass Spectrometry.

The SPIN tandem ion funnel (IF) structure allows for highly sensitive mass spectrometry due to reduced ion losses in the interface region and during transmission; however, IF has an inherent mass discrimination problem, which can greatly restrain the ion transmission efficiency (TE) and therefore requires certain optimization methods. Conventional optimization methods ignore the combined effects of multiple IF characteristic parameters (electrical and dimensional parameters) and are unable to achieve efficient ion transmission over a wide mass range, thus requiring significant tuning time. In this paper, a genetic algorithm (GA)-optimized printed circuit board ion funnel (PCBIF) was designed, fabricated, preliminarily evaluated, and integrated into the SPIN interface to address the ion loss that can occur when mass spectrometers transfer ions at subambient pressure. Simulation studies have showed clearly that the effective automated GA can increase the PCBIF optimization, design, and the ion TE (finding the optimal characteristic parameters within 4 h and achieving 96% ion TE for ions with m/z between 50 and 700). Preliminary tests on built SPIN-PCBIF-MS can lead to an LOD of 0.01 nM and also indirectly suggest the effectiveness of the GA-optimized PCBIF. The proposed GA method helps to guide the design of IF and can also be used for other multivariate mass analyzers or ion transmission devices.

Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics.

There is a growing demand to develop high-throughput and high-sensitivity mass spectrometry methods for single-cell proteomics. The commonly used isobaric labeling-based multiplexed single-cell proteomics approach suffers from distorted protein quantification due to co-isolated interfering ions during MS/MS fragmentation, also known as ratio compression. We reasoned that the use of MS3-based quantification could mitigate ratio compression and provide better quantification. However, previous studies indicated reduced proteome coverages in the MS3 method, likely due to long duty cycle time and ion losses during multilevel ion selection and fragmentation. Herein, we described an improved MS acquisition method for MS3-based single-cell proteomics by employing a linear ion trap to measure reporter ions. We demonstrated that linear ion trap can increase the proteome coverages for single-cell-level peptides with even higher gain obtained via the MS3 method. The optimized real-time search MS3 method was further applied to study the immune activation of single macrophages. Among a total of 126 single cells studied, over 1200 and 1000 proteins were quantifiable when at least 50 and 75% nonmissing data were required, respectively. Our evaluation also revealed several limitations of the low-resolution ion trap detector for multiplexed single-cell proteomics and suggested experimental solutions to minimize their impacts on single-cell analysis.

Shikonin triggers GSDME-mediated pyroptosis in tumours by regulating autophagy via the ROS–MAPK14/p38α axis

BackgroundShikonin (SK), a botanical drug extracted from Lithospermum erythrorhizon, has been shown to inhibit tumour growth through apoptosis and necrosis. However, whether SK induces pyroptosis in cancer cells is still unknown. PurposeThis study aims to investigated the mechanisms of SK-induced pyroptosis in tumour cells and mice. MethodsIn vivo and in vitro methods were used in this study. Cell deaths were analysed by LDH and CCK-8 assay and western blotting. To investigated the signalling pathway of SK-induced pyroptosis, various genes expressions were supressed by shRNA or inhibitors. High-sensitivity mass spectrometry assay was used to identified potential factors that regulate GSDME-mediated pyroptosis. Finally, a mouse model was used to investigate the effect of SK administration on tumour growth in vivo. ResultsThe activation of BAX/caspase-3 signalling was essential for GSDME-mediated pyroptosis by SK. Mechanistically, the intracellular reactive oxygen species (ROS) generation induced by SK treatment initiated GSDME-dependant pyroptosis. SK stimulation induced protective autophagy in a ROS-dependant manner, and repressed autophagy significantly enhanced SK-induced pyroptosis. Moreover, MAPK14/p38α, a ROS sensor, modulated SK-induced autophagy and ultimately affected GSDME-dependant pyroptosis. ConclusionHere, for the first time we demonstrated that SK treatment induced GSDME-dependant pyroptosis in tumour cells. Our results demonstrated that SK initiates ROS signalling to drive pyroptosis in cancer cells.

Use of tandem carbon microfiber columns for on-line fractionation strategy of reducing ion suppression effects in electrospray ionization-mass spectrometry

As we all know, the complexity and diversity of complex sample are confronting with challenge of high-sensitive mass spectrometry analysis, especially direct mass spectrometry. The work proposed a two-dimensional carbon microfiber fractionation (2DμCFs) system for the reduction of ion suppression effects in electrospray ionization mass spectrometry (ESI-MS). The 2DμCFs system can on-line fractionated the complex sample into strong-polar, medium-polar and weak-polar fractions for sequential MS analysis. Direct analysis brings about the strong ion suppression effect up to 85%, but the fractionated analysis of 2DμCFs system can distinctly reduce the ion suppression effect to less than 43%, even close to none. And the fractionated analysis not only decrease the number of analytes of direct analysis, but also narrows down the polarity range of analytes within the droplets of ESI, contributing to the homogeneous distribution to reduce the ion suppression effect. As an example, the 2DμCFs system coupled with tandem mass spectrometry (MS/MS) was applied for fractionated analysis of Radix Puerariae extract in 4.5 min. Compared with direct MS/MS, the 2DμCFs-MS/MS shows the lower ion suppression and the more ionic species (m/z). In addition, and most of ionic species detected by the 2DμCFs-MS/MS, are the same as those by HPLC MS/MS. Furthermore, the 2DμCFs-MS/MS exhibit the good analysis repeatability of real sample with the RSDs less than 10.32% (intra-day), 7.12% (inter-day) and 14.28% (inter-batch of CFs and ACFs). The carbon fibers (CFs) and active carbon fibers (ACFs) columns, as the key parts, are conducive to achieve on-line fractionation of compounds based on the difference of polarity. The 2DμCFs system has the merits of on-line, speediness, low-pressure and recycle. More importantly, such fast and high-throughput method is advantageous for comprehensive screening of complex samples in drug, clinical, environment and plant.

Suzuki C-C Coupling in Paper Spray Ionization: Microsynthesis of Biaryls and High-Sensitivity MS Detection of Aryl Bromides.

Suzuki-Miyaura cross-coupling is one of the most powerful strategies for constructing biaryl compounds. However, classic Suzuki-Miyaura coupling suffers from hour-scale reaction time and competitive protodeboronation. To address these problems, a mild nonaqueous potassium trimethylsilanolate (TMSOK)-assisted Suzuki-Miyaura coupling strategy was designed for the microsynthesis of biaryls in paper spray ionization (PSI). Due to the acceleration power facilitated by microdroplet chemistry in reactive PSI, the microsynthesis of biaryls by reactive PSI was accomplished within minutes with comparable yields to the bulk, showing good substrate applicability from 32 Suzuki-Miyaura reactions of aryl bromides and aryl boronic acid/borates bearing different substituents. Based on the above TMSOK-assisted Suzuki-Miyaura coupling strategy, we further developed a high-sensitivity and selective PSI mass spectrometry (MS) method for quantitative analysis of aryl bromides, a class of environmentally persistent organic pollutants that cannot be directly detected by ambient mass spectrometry due to their low ionization efficiency. In situ derivatization of aryl bromides was achieved with aryl borates bearing quaternary ammonium groups in PSI. The proposed PSI-MS method shows good linearity over the 0.01-10 μmol L-1 range with low detection limits of 1.8-4.8 nmol L-1 as well as good applicability to the rapid determination of six aryl bromides in three environmental water samples. The proposed PSI-MS method also shows good applicability to brominated flame retardants (polybrominated diphenyls/diphenyl esters). Overall, this study provides a simple, rapid, low-cost, high-sensitivity, and high-selectivity strategy for trace aryl bromides and other brominated pollutants in real samples with minimal/no sample pretreatment.

Nuclear Condensation of CDYL Links Histone Crotonylation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease.

Emerging evidence indicates that epigenetic modulation of gene expression plays a key role in the progression of autosomal dominant polycystic kidney disease (ADPKD). However, the molecular basis for how the altered epigenome modulates transcriptional responses, and thereby disease progression in ADPKD, remains largely unknown. Kidneys from control and ADPKD mice were examined for the expression of CDYL and histone acylations. CDYL expression and its correlation with disease severity were analyzed in a cohort of patients with ADPKD. Cdyl transgenic mice were crossed with Pkd1 knockout mice to explore CDYL's role in ADPKD progression. Integrated cistromic and transcriptomic analyses were performed to identify direct CDYL target genes. High-sensitivity mass spectrometry analyses were undertaken to characterize CDYL-regulated histone lysine crotonylations (Kcr). Biochemical analysis and zebrafish models were used for investigating CDYL phase separation. CDYL was downregulated in ADPKD kidneys, accompanied by an increase of histone Kcr. Genetic overexpression of Cdyl reduced histone Kcr and slowed cyst growth. We identified CDYL-regulated cyst-associated genes, whose downregulation depended on CDYL-mediated suppression of histone Kcr. CDYL assembled nuclear condensates through liquid-liquid phase separation in cultured kidney epithelial cells and in normal kidney tissues. The phase-separating capacity of CDYL was required for efficient suppression of locus-specific histone Kcr, of expression of its target genes, and of cyst growth. These results elucidate a mechanism by which CDYL nuclear condensation links histone Kcr to transcriptional responses and cystogenesis in ADPKD.

Lipidomics in Understanding Pathophysiology and Pharmacologic Effects in Inflammatory Diseases: Considerations for Drug Development.

The lipidome has a broad range of biological and signaling functions, including serving as a structural scaffold for membranes and initiating and resolving inflammation. To investigate the biological activity of phospholipids and their bioactive metabolites, precise analytical techniques are necessary to identify specific lipids and quantify their levels. Simultaneous quantification of a set of lipids can be achieved using high sensitivity mass spectrometry (MS) techniques, whose technological advancements have significantly improved over the last decade. This has unlocked the power of metabolomics/lipidomics allowing the dynamic characterization of metabolic systems. Lipidomics is a subset of metabolomics for multianalyte identification and quantification of endogenous lipids and their metabolites. Lipidomics-based technology has the potential to drive novel biomarker discovery and therapeutic development programs; however, appropriate standards have not been established for the field. Standardization would improve lipidomic analyses and accelerate the development of innovative therapies. This review aims to summarize considerations for lipidomic study designs including instrumentation, sample stabilization, data validation, and data analysis. In addition, this review highlights how lipidomics can be applied to biomarker discovery and drug mechanism dissection in various inflammatory diseases including cardiovascular disease, neurodegeneration, lung disease, and autoimmune disease.

Metabolite and transcriptome analyses revealed the modulation of fructo-oligosaccharide on ileum metabolism of Taiping chickens.

The metabolic markers and differentially expressed genes (DEGs) related to fructo-oligosaccharide (FOS) were screened, and the response of FOS to the ileum metabolic pathway of Taiping chickens was analysed. Prebiotic are widely used in agricultural breeding for care and maintenance of animal health, especially FOS. Metabonomics evaluation of ileum of Taiping chicken ultra-performance liquid chromatography-quadruple time of-flight high-sensitivity mass spectrometry showed that 93 differentially altered metabolites were identified and divided into eight categories, of which organic acids and derivatives was the most important one. Transcriptomic analysis showed that DEGs were mainly enriched in drug metabolism-cytochrome p450, metabolism of xenobiotics by cytochrome p450, retinol metabolism and fat digestion and absorption. Integrated analysis of metabolite profiles and gene expression revealed that the significantly up-regulated GSTT1 was significantly correlated with most of the different lipid metabolites, suggesting that GSTT1may play an important role in FOS regulation of lipid metabolism. The results of this study suggest that supplementation of FOS can have a positive effect on gut metabolites, which may contribute to the overall health with indigenous chickens. Insight into the responses of intestinal prebiotics of Taiping chicken is helpful to understand the role of prebiotics in maintaining intestinal microflora balance and improving immune response and productivity of poultry from the molecular and metabolic levels.

The 370-Da Inhibitor of the Sodium Pump in the Plasma of Haemodialysis Patients

Background: Previous studies have shown that a molecule of mass 370 Da that inhibits the sodium pump can be extracted from human placentas and from the concentrated plasma or ultrafiltrate of volume-expanded patients. Aim: This study aimed to study the abundance of the 370-Da molecule and its changes across dialysis in a population of patients with renal failure treated by haemodialysis. Methods: Four millilitres of pre- and post-dialysis blood samples (2 mL plasma) were taken from patients receiving intermittent haemodialysis and analysed by high-performance liquid chromatography coupled to high sensitivity mass spectrometry. Results: In over half of the study population, the 370-Da molecule was present in abundance that exceeded the limit of quantitation. Most patients experienced a marked fall in the abundance of the molecule over a haemodiafiltration session, though exceptions were seen in 2 individuals, both of whom showed clear evidence for the presence of 2 structural isomers of the 370-Da molecule. Conclusions: Advanced renal failure is frequently accompanied by an increased abundance of a 370-Da inhibitor of the sodium pump and that abundance is strongly impacted by haemodialysis. The technique described here could readily be applied to other clinical situations where sodium pump inhibition might be anticipated, such as hypertension, pregnancy, and foetal medicine, and thereby lead to a better understanding of the physiology and pathophysiology of these conditions.

Cell affinity screening combined with nanoLC-MS/MS based peptidomics for identifying cancer cell binding peptides from Bufo Bufo gargarizans

Animal venoms contain many peptides with high specificity and selectivity against their protein targets, a characteristic which makes venoms an invaluable source of potential drugs. High-sensitivity mass spectrometry (MS)- based peptidomic platform has evolved as a predominant method for natural peptide drug discovery due to its strength for direct and rapid identification of peptides and peptide-associated post-translational modifications (PTMs). In this study, we used cell-affinity assays combined with nanoLC-MS/MS based peptidomics to identify cancer cell binding peptides (CBPs) from Bufo Bufo gargarizans. We identified 76 potential cell binding peptides and 237 non-affinity peptides in venom extracts from Asiatic toads, and some were verified with MS-parallel reaction monitoring (PRM) mode. These peptides were further analyzed and internalized within human cells and some demonstrated anti-tumor properties in vitro. These specific peptides might be used as templates for peptide-based drug design or optimization.

Quantitative proteomic analysis of aberrant expressed lysine acetylation in gastrointestinal stromal tumors

BackgroundGastrointestinal stromal tumor (GIST) is a common digestive tract tumor with high rate of metastasis and recurrence. Currently, we understand the genome, transcriptome and proteome in GIST. However, posttranscriptional modification features in GIST remain unclear. In the present study, we aimed to construct a complete profile of acetylome in GIST.MethodsFive common protein modifications, including acetylation, succinylation, crotonylation, 2-hydroxyisobutyrylation, and malonylation were tested among GIST subgroups and significantly differentially- expressed lysine acetylation was found. The acetylated peptides labeled with Tandem Mass Tag (TMT)under high sensitive mass spectrometry, and some proteins with acetylation sites were identified. Subsequently, these proteins and peptides were classified into high/moderate (H/M) risk and low (L) risk groups according to the modified NIH classification standard. Furthermore, cell components, molecular function, biological processes, KEGG pathways and protein interaction networks were analyzed.ResultsA total of 2904 acetylation sites from 1319 proteins were identified, of which quantitative information of 2548 sites from 1169 proteins was obtained. Finally, the differentially-expressed lysine acetylation sites were assessed and we found that 42 acetylated sites of 38 proteins were upregulated in the H/M risk group compared with the L risk group, while 48 acetylated sites of 44 proteins were downregulated, of which Ki67 K1063Ac and FCHSD2 K24Ac were the two acetylated proteins that were most changed.ConclusionsOur novel findings provide further understanding of acetylome in GIST and might demonstrate the possibility in the acetylation targeted diagnosis and therapy of GIST.

A Predictive Instrument for Sensitive and Expedited Measurement of Ultra-Barrier Permeation

The reliable operation of flexible display devices poses a significant engineering challenge regarding the metrology of high barriers against water vapor. No reliable results have been reported in the range of 10 –6 g∙(m 2 ∙d) −1 , and there is no standard ultra-barrier for calibration. To detect trace amount of water vapor permeation through an ultra-barrier with extremely high sensitivity and a greatly reduced test period, a predictive instrument was developed by integrating permeation models into high-sensitivity mass spectrometry measurement based on dynamic accumulation, detection, and evacuation of the permeant. Detection reliability was ensured by means of calibration using a standard polymer sample. After calibration, the lower detection limit for water vapor permeation is in the range of 10 –7 g∙(m 2 ∙d) −1 , which satisfies the ultra-barrier requirement. Predictive permeation models were developed and evaluated using experimental data so that the steady-state permeation rate can be forecasted from non-steady-state results, thus enabling effective measurement of ultra-barrier permeation within a significantly shorter test period.

Resolving whether inhalation of depleted uranium contributed to Gulf War Illness using high-sensitivity mass spectrometry

Of the hypothesized causes of Gulf War Illness (GWI), a chronic multi-symptom illness afflicting approximately 25% of military personnel deployed to the 1991 Gulf War, exposure to depleted uranium (DU) munitions has attracted international concern. Past research has not tested the potential association of GWI with inhaled DU nor used isotope mass spectrometry of sufficient sensitivity to rigorously assess prior DU exposure. We applied a standard biokinetic model to predict the urinary concentration and uranium isotopic ratios for a range of inhalation exposures. We then applied sensitive mass spectrometry capable of detecting the predicted urinary DU to 154 individuals of a population-representative sample of U.S. veterans in whom GWI had been determined by standard case definitions and DU inhalation exposures obtained by medical history. We found no difference in the 238U/235U ratio in veterans meeting the standard case definitions of GWI versus control veterans, no differences by levels of DU inhalation exposure, and no 236U associated with DU was detected. These findings show that even the highest likely levels of DU inhalation played no role in the development of GWI, leaving exposure to aerosolized organophosphate compounds (pesticides and sarin nerve agent) as the most likely cause(s) of GWI.

Ubiquitylome analysis reveals a central role for the ubiquitin-proteasome system in plant innate immunity.

Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 min with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.

Recent Developments and Applications of Quantitative Proteomics Strategies for High-Throughput Biomolecular Analyses in Cancer Research.

Innovations in medical technology and dedicated focus from the scientific community have inspired numerous treatment strategies for benign and invasive cancers. While these improvements often lend themselves to more positive prognoses and greater patient longevity, means for early detection and severity stratification have failed to keep pace. Detection and validation of cancer-specific biomarkers hinges on the ability to identify subtype-specific phenotypic and proteomic alterations and the systematic screening of diverse patient groups. For this reason, clinical and scientific research settings rely on high throughput and high sensitivity mass spectrometry methods to discover and quantify unique molecular perturbations in cancer patients. Discussed within is an overview of quantitative proteomics strategies and a summary of recent applications that enable revealing potential biomarkers and treatment targets in prostate, ovarian, breast, and pancreatic cancer in a high throughput manner.