Combined Mass Spectrometry Analysis Research Articles

Investigating the effects of thermal processing on bitter substances in atemoya (Annona cherimola × Annona squamosa) through sensory-guided separation

Atemoya (Annona cherimola × Annona squamosa) is a specialty crop in Taiwan. Thermal treatment induces bitterness, complicating seasonal production adjustments and surplus reduction. In this research, sensory-guided separation, metabolomics, and orthogonal partial least squares discrimination analysis (OPLS-DA) are used for identifying the bitterness in atemoya which originates from catechins, epicatechin trimers, and proanthocyanidins. Different thermal treatments (65 °C, 75 °C, and 85 °C) revealed that the glucose and fructose contents in atemoya significantly decreased, while total phenols, flavonoids, and tannins significantly increased. The concentration of 5-hydroxymethylfurfural (5-HMF) increased from 23.16 ng/g in untreated samples to 400.71 ng/g (AP-65), 1208.59 ng/g (AP-75), and 2838.51 ng/g (AP-85). However, these levels are below the 5-HMF bitterness threshold of 3780 ng/g. Combining mass spectrometry analysis with sensory evaluation, OPLS-DA revealed that atemoya treated at 65 °C, 75 °C, and 85 °C exhibited significant bitterness, with the main bitter components being proanthocyanidin dimers and trimers.

Rapid classification of coffee origin by combining mass spectrometry analysis of coffee aroma with deep learning

Mislabeling the geographical origin of coffee is a prevalent form of fraud. In this study, a rapid, nondestructive, and high-throughput method combining mass spectrometry (MS) analysis and intelligence algorithms to classify coffee origin was developed. Specifically, volatile compounds in coffee aroma were detected using self-aspiration corona discharge ionization mass spectrometry (SACDI–MS), and the acquired MS data were processed using a customized deep learning algorithm to perform origin authentication automatically. To facilitate high-throughput analysis, an air curtain sampling device was designed and coupled with SACDI–MS to prevent volatile mixing and signal overlap. An accuracy of 99.78% was achieved in the classification of coffee samples from six origins at a throughput of 1 s per sample. The proposed approach may be effective in preventing coffee fraud owing to its straightforward operation, rapidity, and high accuracy and thus benefit consumers.

VPS35 promotes gastric cancer progression through integrin/FAK/SRC signalling-mediated IL-6/STAT3 pathway activation in a YAP-dependent manner

VPS35 is a key subunit of the retromer complex responsible for recognising cytosolic retrieval signals in cargo and is involved in neurodegenerative disease and tumour progression. However, the function and molecular mechanism of VPS35 in gastric cancer (GC) remains largely unknown. Here, we demonstrated that VPS35 was significantly upregulated in GC, which was associated with poor survival. VPS35 promoted GC cell proliferation and metastasis both in vitro and in vivo. Mechanistically, VPS35 activated FAK-SRC kinases through integrin-mediated outside-in signalling, leading to the activation of YAP and subsequent IL-6 expression induction in tumour cells. What’s more, combined mass spectrometry analysis of MGC-803 cell and bioinformatic analysis, we found that phosphorylation of VPS35 was enhanced in GC cells, and phosphorylated VPS35 has enhanced interaction with ITGB3. VPS35 interacted with ITGB3 and affected the recycling of ITGB3 in GC cells. Gain- and loss-of-function experiments revealed that VPS35 promoted tumour proliferation and metastasis via the IL-6/STAT3 pathway. Interestingly, we also found that STAT3 directly bound to the VPS35 promoter and increased VPS35 transcription, thereby establishing a positive regulatory feedback loop. In addition, we demonstrated that VPS35 knockdown sensitised GC cells to 5-FU and cisplatin. These findings provide evidence that VPS35 promotes tumour proliferation and metastasis, and highlight the potential of targeting VPS35- and IL-6/STAT3-mediated tumour interactions as promising therapeutic strategies for GC.

A Novel Strategy Based on Permanent Protein Modifications Induced by Formaldehyde for Food Safety Analysis.

The illegal additions of chemicals in food products are serious incidents threatening current public safety. To date, ideal methods to determine permanent traces of prohibited chemicals in foods are still lacking. For example, formaldehyde (FA) can be added illegally as a food preservative. However, most current methods that are dependent on the direct detection of FA are not able to determine if FA has ever been added once food products are rinsed completely. Herein, we present a novel approach relying upon protein modifications induced by FA (PMIF) to examine FA in foods. We reveal the entire catalog of PMIFs in food products by combining mass spectrometry analysis with unrestrictive identification of protein modifications. Consequently, four obvious PMIFs were identified and confirmed as markers to discriminate the addition of FA in foods. Our study demonstrates that the approach based on PMIFs enables detecting the imprinted trace of FA even if the food products have been washed thoroughly. Our work presents a novel strategy for analysis of chemical additives, offering broad potential applications in protein analysis and food safety.

Cheetah-MS: a web server to model protein complexes using tandem cross-linking mass spectrometry data.

SummaryProtein–protein interactions (PPIs) are central in many biological processes but difficult to characterize, especially in complex, unfractionated samples. Chemical cross-linking combined with mass spectrometry (MS) and computational modeling is gaining recognition as a viable tool in protein interaction studies. Here, we introduce Cheetah-MS, a web server for predicting the PPIs in a complex mixture of samples. It combines the capability and sensitivity of MS to analyze complex samples with the power and resolution of protein–protein docking. It produces the quaternary structure of the PPI of interest by analyzing tandem MS/MS data (also called MS2). Combining MS analysis and modeling increases the sensitivity and, importantly, facilitates the interpretation of the results.Availability and implementationCheetah-MS is freely available as a web server at https://www.txms.org.

Impact of tumor heterogeneity and microenvironment in identifying neoantigens in a patient with ovarian cancer.

Identification of neoepitopes as tumor-specific targets remains challenging, especially for cancers with low mutational burden, such as ovarian cancer. To identify mutated human leukocyte antigen (HLA) ligands as potential targets for immunotherapy in ovarian cancer, we combined mass spectrometry analysis of the major histocompatibility complex (MHC) class I peptidomes of ovarian cancer cells with parallel sequencing of whole exome and RNA in a patient with high-grade serous ovarian cancer. Four of six predicted mutated epitopes capable of binding to HLA-A*02:01 induced peptide-specific T cell responses in blood from healthy donors. In contrast, all six peptides failed to induce autologous peptide-specific response by T cells in peripheral blood or tumor-infiltrating lymphocytes from ascites of the patient. Surprisingly, T cell responses against a low-affinity p53-mutant Y220C epitope were consistently detected in the patient with either unprimed or in vitro peptide-stimulated T cells even though the patient's primary tumor did not bear this mutation. Our results demonstrated that tumor heterogeneity and distinct immune microenvironments within a patient should be taken into consideration for identification of immunogenic neoantigens. T cell responses to a driver gene-derived p53 Y220C mutation in ovarian cancer warrant further study.

Tob2 phosphorylation regulates global mRNA turnover to reshape transcriptome and impact cell proliferation

Tob2, an anti-proliferative protein, promotes deadenylation through recruiting Caf1 deadenylase to the mRNA poly(A) tail by simultaneously interacting with both Caf1 and poly(A)-binding protein (PABP). Previously, we found that changes in Tob2 phosphorylation can alter its PABP-binding ability and deadenylation-promoting function. However, it remained unknown regarding the relevant kinase(s). Moreover, it was unclear whether Tob2 phosphorylation modulates the transcriptome and whether the phosphorylation is linked to Tob2's anti-proliferative function. In this study, we found that c-Jun amino-terminal kinase (JNK) increases phosphorylation of Tob2 at many Ser/Thr sites in the intrinsically disordered region (IDR) that contains two separate PABP-interacting PAM2 motifs. JNK-induced phosphorylation or phosphomimetic mutations at these sites weaken the Tob2–PABP interaction. In contrast, JNK-independent phosphorylation of Tob2 at serine 254 (S254) greatly enhances Tob2 interaction with PABP and its ability to promote deadenylation. We discovered that both PAM2 motifs are required for Tob2 to display these features. Combining mass spectrometry analysis, poly(A) size-distribution profiling, transcriptome-wide mRNA turnover analyses, and cell proliferation assays, we found that the phosphomimetic mutation at S254 (S254D) enhances Tob2's association with PABP, leading to accelerated deadenylation and decay of mRNAs globally. Moreover, the Tob2–S254D mutant accelerates the decay of many transcripts coding for cell cycle related proteins and enhances anti-proliferation function. Our findings reveal a novel mechanism by which Ccr4–Not complex is recruited by Tob2 to the mRNA 3′ poly(A)-PABP complex in a phosphorylation dependent manner to promote rapid deadenylation and decay across the transcriptome, eliciting transcriptome reprogramming and suppressed cell proliferation.

Delineating T cell autoreactivity in autoimmune diabetes by assessing the MHC-II-bound peptidome

Abstract Development of tissue-specific autoimmune diseases is tightly linked to major histocompatibility complex class II alleles (MHC-II). In humans, the HLA-DQ2/DQ8 variants contributes the highest genetic risk to type 1 diabetes (T1D). However, it remains unclear how the MHC-II-specific peptidome defines the outcomes of T cell self-reactivity. To address this question, we performed unbiased analyses of the MHC-II immunopeptidome of the pancreatic islets of non-obese diabetic (NOD) mice at the start of the autoimmune process. NOD mice spontaneously develop autoimmune diabetes due to the expression of the I-Ag7 MHC-II molecule, which is structurally homologous to HLA-DQ8. Combining mass spectrometry analysis and immunoassays, we identified the precise MHC-II-specific epitopes derived from diverse β-cell proteins and functionally characterized the corresponding T cell responses. The major peptides derived from insulin B chain and C-peptides and consisted of sets of varying length, usually around 14–16 residues. Their presence correlated with the initial T cell autoreactivity. Other minor epitopes elicited T cell responses at a more advanced stage, the phenomenon of epitope spreading. We also identified post translational change consisting of deamidation of C peptides- see abstract of Lichti et al. Importantly, most of the immunogenic epitopes were independently identified in the MHC-II peptidome of pancreatic lymph nodes and spleen, revealing a correlation of peripheral presentation with the tissue autoreactivity. In sum, these data demonstrate the landscape of the MHC-II epitopes being presented in a target autoimmune organ and delineate the characteristic features of a self-peptide that foster pathogenic responses.

Transcriptomics and proteomics analyses of anti-cancer mechanisms of TR35–An active fraction from Xinjiang Bactrian camel milk in esophageal carcinoma cell

The aim of the paper is to investigate the effect of the active fraction extracted from the Xinjiang Bactrian camel whey on the human cancer cells using an invitro and invivo model of human carcinoma of the esophagus. Our results demonstrated that an antitumor active fraction, TR35, isolated from Xinjiang Bactrian camel milk could significantly inhibit Eca109cell proliferation and induce its apoptosis (indicated by MTT assay, Annexin V-FITC Apoptosis Detection, and caspase-3 activity). Moreover, we found that TR35 could inhibit the growth of xenografted tumor in nude mice without loss in body weight. Furthermore, we used RNA-Seq and 2-DE combined Mass Spectrometry analysis to identify differentially expressed RNA and protein markers of apoptosis and necrosis. Compared with untreated Eca109cells, a total of 405 differentially expressed genes and 55 differentially expressed proteins were identified in TR35 treated Eca109cells. KEGG analysis uncovered signaling pathways closely associated with cancer inhibition that were enriched in the TR35-treated cells. These results might implicate that downregulation of specific proteins identified in this study may be the cause of this tumor growth inhibition. This study sheds light on the potential therapeutic advantages based on the historical anti-cancer activities of camel milk.

Transfer-RNA-mediated enhancement of ribosomal proteins S6 kinases signaling for cell proliferation

ABSTRACTWhile transfer-RNAs (tRNAs) are known to transport amino acids to ribosome, new functions are being unveiled from tRNAs and their fragments beyond protein synthesis. Here we show that phosphorylation of 90-kDa RPS6K (ribosomal proteins S6 kinase) was enhanced by tRNALeu overexpression under amino acids starvation condition. The phosphorylation of 90-kDa RPS6K was decreased by siRNA specific to tRNALeu and was independent to mTOR (mammalian target of rapamycin) signaling. Among the 90-kDa RPS6K family, RSK1 (ribosomal S6 kinase 1) and MSK2 (mitogen-and stress-activated protein kinase 2) were the major kinases phosphorylated by tRNALeu overexpression. Through SILAC (stable isotope labeling by/with amino acids in cell culture) and combined mass spectrometry analysis, we identified EBP1 (ErbB3-binding protein 1) as the tRNALeu-binding protein. We suspected that the overexpression of free tRNALeu would reinforce ErbB2/ErbB3 signaling pathway by disturbing the interaction between ErbB3 and EBP1, resulting in RSK1/MSK2 phosphorylation, improving cell proliferation and resistance to death. Analysis of samples from patients with breast cancer also indicated an association between tRNALeu overexpression and the ErbB2-positive population. Our results suggested a possible link between tRNALeu overexpression and RSK1/MSK2 activation and ErbB2/ErbB3 signaling.

A reverse signaling pathway downstream of Sema4A controls cell migration via Scrib.

Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor βPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration.

Impact of the Secretome of Human Mesenchymal Stem Cells on Brain Structure and Animal Behavior in a Rat Model of Parkinson's Disease.

Research in the last decade strongly suggests that mesenchymal stem cell (MSC)‐mediated therapeutic benefits are mainly due to their secretome, which has been proposed as a possible therapeutic tool for the treatment of Parkinson's disease (PD). Indeed, it has been shown that the MSC secretome increases neurogenesis and cell survival, and has numerous neuroprotective actions under different conditions. Additionally, using dynamic culturing conditions (through computer‐controlled bioreactors) can further modulate the MSC secretome, thereby generating a more potent neurotrophic factor cocktail (i.e., conditioned medium). In this study, we have characterized the MSC secretome by proteomic‐based analysis, investigating its therapeutic effects on the physiological recovery of a 6‐hydroxidopamine (6‐OHDA) PD rat model. For this purpose, we injected MSC secretome into the substantia nigra (SNc) and striatum (STR), characterizing the behavioral performance and determining histological parameters for injected animals versus untreated groups. We observed that the secretome potentiated the increase of dopaminergic neurons (i.e., tyrosine hydroxylase‐positive cells) and neuronal terminals in the SNc and STR, respectively, thereby supporting the recovery observed in the Parkinsonian rats’ motor performance outcomes (assessed by rotarod and staircase tests). Finally, proteomic characterization of the MSC secretome (through combined mass spectrometry analysis and Bioplex assays) revealed the presence of important neuroregulatory molecules, namely cystatin C, glia‐derived nexin, galectin‐1, pigment epithelium‐derived factor, vascular endothelial growth factor, brain‐derived neurotrophic factor, interleukin‐6, and glial cell line‐derived neurotrophic factor. Overall, we concluded that the use of human MSC secretome alone was able to partially revert the motor phenotype and the neuronal structure of 6‐OHDA PD animals. This indicates that the human MSC secretome could represent a novel therapeutic for the treatment of PD. Stem Cells Translational Medicine 2017;6:634–646

The existence of 5-hydroxymethylcytosine and 5-formylcytosine in both DNA and RNA in mammals.

We developed a novel strategy by oxidation-derivatization combined mass spectrometry analysis for the determination of 5-hydroxymethylcytosine and 5-formylcytosine in both DNA and RNA. We reported the presence of 5-formylcytosine in RNA of mammals and found that ascorbic acid and hydroquinone can increase the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine in DNA and RNA.

Autonomous metabolomics for rapid metabolite identification in global profiling.

An autonomous metabolomic workflow combining mass spectrometry analysis with tandem mass spectrometry data acquisition was designed to allow for simultaneous data processing and metabolite characterization. Although previously tandem mass spectrometry data have been generated on the fly, the experiments described herein combine this technology with the bioinformatic resources of XCMS and METLIN. As a result of this unique integration, we can analyze large profiling datasets and simultaneously obtain structural identifications. Validation of the workflow on bacterial samples allowed the profiling on the order of a thousand metabolite features with simultaneous tandem mass spectra data acquisition. The tandem mass spectrometry data acquisition enabled automatic search and matching against the METLIN tandem mass spectrometry database, shortening the current workflow from days to hours. Overall, the autonomous approach to untargeted metabolomics provides an efficient means of metabolomic profiling, and will ultimately allow the more rapid integration of comparative analyses, metabolite identification, and data analysis at a systems biology level.

Catalytic mechanism of α-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling.

Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason–Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the α-phosphate site. Phosphorus-31 NMR spectroscopy (31P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue α,β-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme–product complex structure.

Determination of Glyphosate as cross‐contaminant in a commercial herbicide by capillary electrophoresis‐electrospray ionization‐mass spectrometry

Capillary electrophoresis combined with mass spectrometry (CE-MS) was used for the rapid determination of the negatively charged herbicide, Glyphosate, in a selective dried granule (DG) formulation. The CE-MS method was required to ensure product safety from the risk of cross contamination of a selective herbicide formulation. Glyphosate separation was achieved by using a bare fused-silica capillary column, operated in the reversed-polarity mode, using ammonium formate buffer, pH 2.5. The total CE-MS analysis time was under 10 min and the limit of detection was 10 ng/mL. The CE-MS analysis of Glyphosate was simple, rapid, and selective. The method involved minimal sample handling and was proven to be ideal for cross-contamination investigations in manufacturing samples.