Mass Spectrometry-based Shotgun Research Articles

Prognostic significance of plasma molecular lipid species in coronary artery disease patients: A prospective cohort study

Abstract Background Circulating plasma lipids are vital indicators for quantifying coronary artery disease (CAD) risk, yet traditional biomarkers like LDL, HDL, and triglycerides often exhibit limited predictive capacity. Advancements in lipidomics have identified novel molecular constituents with the potential to improve prognostication in CAD patients. However, the prognostic value of lipidomics for long-term mortality has not yet been investigated. Objective In our study, which was part of the European Collaborative Project on Inflammation and Vascular Wall Remodeling in Atherosclerosis (ATHEROREMO), we aimed to investigate the predictive value of specific molecular lipid species for long-term mortality. Methods We utilized data from the 581 CAD patients enrolled in the ATHEROREMO clinical imaging study, who were admitted for either diagnostic coronary angiography or percutaneous coronary intervention. We measured a series of serum lipid species, comprising ceramides (Cer) and cholesteryl esters (CE), by mass spectrometry-based shotgun lipidomics. Long-term all-cause mortality follow-up was conducted in 2022 via the Civil Registry. We used the Kaplan-Meier method and multivariable Cox-proportional hazards regression (correction for age, sex, cardiac risk factors, statin use, hypercholesterolemia, and angiography indication) to assess survival distributions and associations with all-cause mortality. Results Mean age was 61.5 years (IQR: 53.5-61.5), 75% were male. A total of 55% patients were diagnosed with acute coronary syndrome (ACS) and 45% with stable angina pectoris (SAP). During a median follow-up of 11 years (IQR: 10.1-13.4) 179 patients died. Five molecular lipid species (two cholesteryl esters, one ceramide and two ceramide ratios) correlated with mortality (see Table). As an example, Figure 1 demonstrates a clear survival difference between patients with Cer(d18:1/16:0)/Cer(d18:1/24:0) ratio below versus above the median. Findings were consistent for ACS and SAP. Conclusion Our study demonstrates the prognostic value of specific cholesteryl esters, ceramides and ceramide ratios for 11-year mortality in patients with CAD. To the best of our knowledge, this is the longest follow-up study so far within this domain.Table.Lipid molecule and Mortality 11-yFig.1 Kaplan Meier Ceramide ratio

Using established biorepositories for emerging research questions: a feasibility study

BackgroundProteomics and metabolomics offer substantial potential for advancing kidney transplant research by providing versatile opportunities for gaining insights into the biomolecular processes occurring in donors, recipients, and grafts. To achieve this, adequate quality and numbers of biological samples are required. Whilst access to donor samples is facilitated by initiatives such as the QUOD biobank, an adequately powered biobank allowing exploration of recipient-related aspects in long-term transplant outcomes is missing. Rich, yet unverified resources of recipient material are the serum repositories present in the immunological laboratories of kidney transplant centers that prospectively collect recipient sera for immunological monitoring. However, it is yet unsure whether these samples are also suitable for -omics applications, since such clinical samples are collected and stored by individual centers using non-uniform protocols and undergo an undocumented number of freeze–thaw cycles. Whilst these handling and storage aspects may affect individual proteins and metabolites, it was reasoned that incidental handling/storage artifacts will have a limited effect on a theoretical network (pathway) analysis. To test the potential of such long-term stored clinical serum samples for pathway profiling, we submitted these samples to discovery proteomics and metabolomics.MethodsA mass spectrometry-based shotgun discovery approach was used to obtain an overview of proteins and metabolites in clinical serum samples from the immunological laboratories of the Dutch PROCARE consortium. Parallel analyses were performed with material from the strictly protocolized QUOD biobank.ResultsFollowing metabolomics, more than 800 compounds could be identified in both sample groups, of which 163 endogenous metabolites were found in samples from both biorepositories. Proteomics yielded more than 600 proteins in both groups. Despite the higher prevalence of fragments in the clinical, non-uniformly collected samples compared to the biobanked ones (42.5% vs 26.5% of their proteomes, respectively), these fragments could still be connected to their parent proteins. Next, the proteomic and metabolomic profiles were successfully mapped onto theoretical pathways through integrated pathway analysis, which showed significant enrichment of 79 pathways.ConclusionsThis feasibility study demonstrated that long-term stored serum samples from clinical biorepositories can be used for qualitative proteomic and metabolomic pathway analysis, a notion with far-reaching implications for all biomedical, long-term outcome-dependent research questions and studies focusing on rare events.

Minimal Change Disease: Pathogenetic Insights from Glomerular Proteomics.

The mechanism underlying podocyte dysfunction in minimal change disease (MCD) remains unknown. This study aimed to shed light on the potential pathophysiology of MCD using glomerular proteomic analysis. Shotgun proteomics using label-free quantitative mass spectrometry was performed on formalin-fixed, paraffin-embedded (FFPE) renal biopsies from two groups of samples: control (CTR) and MCD. Glomeruli were excised from FFPE renal biopsies using laser capture microdissection (LCM), and a single-pot solid-phase-enhanced sample preparation (SP3) digestion method was used to improve yield and protein identifications. Principal component analysis (PCA) revealed a distinct separation between the CTR and MCD groups. Forty-eight proteins with different abundance between the two groups (p-value ≤ 0.05 and |FC| ≥ 1.5) were identified. These may represent differences in podocyte structure, as well as changes in endothelial or mesangial cells and extracellular matrix, and some were indeed found in several of these structures. However, most differentially expressed proteins were linked to the podocyte cytoskeleton and its dynamics. Some of these proteins are known to be involved in focal adhesion (NID1 and ITGA3) or slit diaphragm signaling (ANXA2, TJP1 and MYO1C), while others are structural components of the actin and microtubule cytoskeleton of podocytes (ACTR3 and NES). This study suggests the potential of mass spectrometry-based shotgun proteomic analysis with LCM glomeruli to yield valuable insights into the pathogenesis of podocytopathies like MCD. The most significantly dysregulated proteins in MCD could be attributable to cytoskeleton dysfunction or may be a compensatory response to cytoskeleton malfunction caused by various triggers.

Exploring the cellulolytic activity of environmental mycobacteria

Although studies on non-tuberculous mycobacteria have increased in recent years because they cause a considerable proportion of infections, their cellulolytic system is still poorly studied. This study presents a characterization of the cellulolytic activities of environmental mycobacterial isolates derived from soil and water samples from the central region of Argentina, aimed to evaluate the conservation of the mechanism for the degradation of cellulose in this group of bacteria. The molecular and genomic identification revealed identity with Mycolicibacterium septicum. The endoglucanase and total cellulase activities were assessed both qualitatively and quantitatively and the optimal enzymatic conditions were characterized. A specific protein of around 56 kDa with cellulolytic activity was detected in a zymogram. Protein sequences possibly arising from a cellulase were identified by mass spectrometry-based shotgun proteomics. Results showed that M. septicum encodes for cellulose- and hemicellulose-related degrading enzymes, including at least an active β-1,4 endoglucanase enzyme that could be useful to improve its survival in the environment. Given the important health issues related to mycobacteria, the results of the present study may contribute to the knowledge of their cellulolytic system, which could be important for their ability to survive in many different types of environments.

An altered plasma lipidome-phenome network characterizes heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a multifactorial, multisystemic syndrome that involves alterations in lipid metabolism. This study aimed to test whether distinct plasma lipid profiles or lipid entities or both are associated with clinical and functional echocardiographic parameters in HFpEF. We examined the human plasma lipidome in HFpEF patients (n=18) with left ventricular ejection fraction ≥50% and N-terminal pro-brain natriuretic peptide (NT-proBNP) >125pg/mL and control subjects (n=12) using mass spectrometry-based shotgun lipidomics. The cohort included 8 women and 22 men with average age of 67.8±8.6 SD. The control and disease groups were not significantly different with respect to age, body mass index, systolic and diastolic blood pressure, and waist-to-hip ratio. The disease group experienced more fatigue (P<0.001), had more often coronary artery disease (P=0.04), and received more medications (beta-blockers, P<0.001). The disease group had significantly different levels of HFpEF-relevant parameters, including NT-proBNP (P<0.001), left ventricular mass index (P=0.005), left atrial volume index (P=0.001), and left ventricular filling index (P<0.001), and lower left ventricular end-diastolic diameter (P=0.014), with no difference in left ventricular ejection fraction. Significant differences in lipid profiles between HFpEF patients and controls could not be detected, including no significant differences in abundance of circulating lipids binned by carbon chain length or by double bonds, nor at the level of individual lipid species. However, there was a striking correlation between selected lipids with smoking status that was independent of disease status, as well as between specific lipids and hyperlipidaemia [with corresponding significance of either false discovery rate (FDR) <0.1 or FDR<0.01]. In an exploratory network analysis of correlations, we observed significantly stronger correlations within the HFpEF group between individual lipids from the cholesterol ester and phosphatidylcholine (PC) classes and clinical/echocardiographic parameters such as left atrial volume index, left ventricular end-diastolic diameters, and heart rate (FDR<0.1). In contrast, the control group showed significantly stronger negative correlations (FDR<0.1) between individual species from the PC and sphingomyelin classes and left ventricular mass index or systolic blood pressure. We did not find significant direct associations between plasma lipidomic parameters and HFpEF and therefore could not conclude that any specific lipids are biomarkers of HFpEF. The validation in larger cohort is needed to confidently conclude the absence of first-order associations.

Quantifying yeast lipidomics by high-performance thin-layer chromatography (HPTLC) and comparison to mass spectrometry-based shotgun lipidomics.

Lipidomic analysis in diverse biological settings has become a frequent tool to increase our understanding of the processes of life. Cellular lipids play important roles not only as being the main components of cellular membranes, but also in the regulation of cell homeostasis as lipid signaling molecules. Yeast has been harnessed for biomedical research based on its good conservation of genetics and fundamental cell organisation principles and molecular pathways. Further application in so-called humanised yeast models have been developed which take advantage of yeast as providing the basics of a living cell with full control over heterologous expression. Here we present evidence that high-performance thin-layer chromatography (HPTLC) represents an effective alternative to replace cost intensive mass spectrometry-based lipidomic analyses. We provide statistical comparison of identical samples by both methods, which support the use of HPTLC for quantitative analysis of the main yeast lipid classes.

Cationic amphiphilic drugs induce accumulation of cytolytic lysoglycerophospholipids in the lysosomes of cancer cells and block their recycling into common membrane glycerophospholipids.

Lysosomes are acidic organelles responsible for lipid catabolism, and their functions can be disrupted by cationic amphiphilic drugs that neutralize lumenal pH and thereby inhibit most lysosomal hydrolases. These drugs can also induce lysosomal membrane permeabilization and cancer cell death, but the underlying mechanism remains elusive. Here, we uncover that the cationic amphiphilic drugs induce a substantial accumulation of cytolytic lysoglycerophospholipids within the lysosomes of cancer cells, and thereby prevent the recycling of lysoglycerophospholipids to produce common membrane glycerophospholipids. Using quantitative mass spectrometry-based shotgun lipidomics, we demonstrate that structurally diverse cationic amphiphilic drugs, along with other types of lysosomal pH-neutralizing reagents, elevate the amounts of lysoglycerophospholipids in MCF7 breast carcinoma cells. Lysoglycerophospholipids constitute ∼11 mol% of total glycerophospholipids in lysosomes purified from MCF7 cells, compared with ∼1 mol% in the cell lysates. Treatment with cationic amphiphilic drug siramesine further elevates the lysosomal lysoglycerophospholipid content to ∼24 mol% of total glycerophospholipids. Exogenously added traceable lysophosphatidylcholine is rapidly acylated to form diacylphosphatidylcholine, but siramesine treatment sequesters the lysophosphatidylcholine in the lysosomes and prevents it from undergoing acylation. These findings shed light on the unexplored role of lysosomes in the recycling of lysoglycerophospholipids and uncover the mechanism of action of promising anticancer agents.

Proteomic patterns in glomerular research, a laser capture microdissection and liquid chromatography-tandem mass spectrometry approach

Abstract Introduction: Molecular techniques have the potential to shed light on glomerular diseases that conventional renal pathology may be unable to reveal. The aim of this study was to investigate whether proteomic patterns of glomeruli obtained from kidney biopsies can differentiate between minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS) and control groups (CTR). Methods: 18 formalin-fixed, paraffin-embedded (FFPE) renal biopsies comprising three groups of samples (CTR=3, MCD=6, FSGS=9) were subjected to label-free quantitative mass spectrometry. Glomeruli were excised from FFPE renal biopsies by laser capture microdissection (LCM) and, to increase both yield and protein identifications, single-pot solid-phase-enhanced sample preparation (SP3) digest method was applied. The samples were analyzed by mass spectrometry based shotgun proteomics. Results: The proteome profiling resulted in the identification of a total of 723 proteins. Multivariate analysis provided several proteins important in the separation of the three groups. Pattern Hunter analysis revealed moderate and high correlation of proteins against CTR-FSGS-MCD or CTR-MCD-FSGS patterns. The most significant pathways involved were associated with nephrin family and cytoskeleton interactions, as well as laminin/extracellular matrix related proteins. Univariate analysis revealed 58 significant different proteins among the three groups. Signaling pathways of these proteins were also associated with nephrin family interactions and cytoskeleton organization. Conclusions: This study demonstrates that mass spectrometry-based shotgun proteomic analysis of LCM glomeruli yields reproducible and quantitative data capable of discriminating between different disease conditions. Differentially expressed proteins provide insights into pathogenesis of glomerular disease.

A Sphingolipidomic Profiling Approach for Comparing X-ray-Exposed and Unexposed HepG2 Cells.

An analytical method based on tandem mass spectrometry-shotgun is presently proposed to obtain sphingolipidomic profiles useful for the characterization of lipid extract from X-ray-exposed and unexposed hepatocellular carcinoma cells (HepG2). To obtain a targeted lipidic profile from a specific biological system, the best extraction method must be identified before instrumental analysis. Accordingly, four different classic lipid extraction protocols were compared in terms of efficiency, specificity, and reproducibility. The performance of each procedure was evaluated using the Fourier-transform infrared spectroscopic technique; subsequently, the quality of extracts was estimated using electrospray ionization tandem mass spectrometry. The selected procedure based on chloroform/methanol/water was successfully used in mass spectrometry-based shotgun sphingolipidomics, allowing for evaluation of the response of cells to X-ray irradiation, the most common anticancer therapy. Using a relative quantitative approach, the changes in the sphingolipid profiles of irradiated cell extracts were demonstrated, confirming that lipidomic technologies are also useful tools for studying the key sphingolipid role in regulating cancer growth during radiotherapy.

PepGM: A probabilistic graphical model for taxonomic inference of viral proteome samples with associated confidence scores.

Inferring taxonomy in mass spectrometry-based shotgun proteomics is a complex task. In multi-species or viral samples of unknown taxonomic origin, the presence of proteins and corresponding taxa must be inferred from a list of identified peptides which is often complicated by protein homology: many proteins do not only share peptides within a taxon but also between taxa. However, correct taxonomic inference is crucial when identifying different viral strains with high sequence homology-considering, e.g., the different epidemiological characteristics of the various strains of SARS-CoV-2. Additionally, many viruses mutate frequently, further complicating the correct identification of viral proteomic samples. We present PepGM, a probabilistic graphical model for the taxonomic assignment of virus proteomic samples with strain-level resolution and associated confidence scores. PepGM combines the results of a standard proteomic database search algorithm with belief propagation to calculate the marginal distributions, and thus confidence scores, for potential taxonomic assignments. We demonstrate the performance of PepGM using several publicly available virus proteomic datasets, showing its strain-level resolution performance. In two out of eight cases, the taxonomic assignments were only correct on the species level, which PepGM clearly indicates by lower confidence scores. PepGM is written in Python and embedded into a Snakemake workflow. It is available on https://github.com/BAMeScience/PepGM.

A Proteomics Data Mining Strategy for the Identification of Quinoa Grain Proteins with Potential Immunonutritional Bioactivities.

Quinoa proteins are attracting global interest for their wide amino acid profile and as a promising source for the development of biomedical treatments, including those against immune-mediated diseases. However, information about the bioactivity of quinoa proteins is scarce. In this study, a quinoa grain proteome map obtained by label-free mass spectrometry-based shotgun proteomics was investigated for the identification of quinoa grain proteins with potential immunonutritional bioactivities, including those related to cancer. After carefully examining the sequence similarities of the 1211 identified quinoa grain proteins against already described bioactive proteins from other plant organisms, 71, 48, and 3 of them were classified as antimicrobial peptides (AMPs), oxidative stress induced peptides (OSIPs), and serine-type protease inhibitors (STPIs), respectively, suggesting their potential as immunomodulatory, anti-inflammatory, and anticancer agents. In addition, data interpretation using Venn diagrams, heat maps, and scatterplots revealed proteome similarities and differences with respect to the AMPs, OSIPs, and STPIs, and the most relevant bioactive proteins in the predominant commercial quinoa grains (i.e., black, red, white (from Peru), and royal (white from Bolivia)). The presented proteomics data mining strategy allows easy screening for potentially relevant quinoa grain proteins and commercial classes for immunonutrition, as a basis for future bioactivity testing.

Comparative proteomics analysis of transforming growth factor-beta1-overexpressed human dental pulp stem cell-derived secretome on CD44-mediated fibroblast activation via canonical smad signal pathway

ABSTRACT Purpose The aim of this study investigates whether the secretome collected from human dental pulp stem cells (hDPSCs) transfected with transforming growth factor-beta1 (TGF-β1) is related to CD44 expression of fibroblasts and canonical smad signaling pathway via proteomic analyzes. Materials and Methods In order to obtain secretome, hDPSCs were conditioned with serum-free alpha-MEM in an incubator containing 37°C, 5% CO2, and humidity for 18–24 h. Proteins in control and TGF-β1 secretome were analyzed by tandem mass spectrometry-based shotgun proteomic method. Bioinformatic evaluations were completed via Ingenuity Pathway Analysis (IPA, QIAGEN) software. CD44 expressions in fibroblasts were evaluated by real time-PCR, western blot, and immunofluorescent staining. The relationship of canonical smad pathway and CD44 was analyzed by western blot and LC-MS/MS. Cell cycle, proliferation and wound healing tests were performed in the secretome groups. Results Venn diagram was showed 174 common proteins were identified from each group. In the control secretome 140 unique proteins were identified and 66 entries were exclusive for TGF-β1 secretome. CD44 gene and protein expressions were increased in fibroblasts treated with TGF-β1 secretome. Relationship between targeted protein data showed that activation of the canonical TGF-β1/Smad pathway was up-regulated CD44 expression in fibroblasts. The canonical smad pathway-mediated upregulation of CD44 may increase the mitotic activity, proliferation, and wound healing potential in fibroblasts. Conclusion While TGF-β1-transfected hDPSC secretome may be a potential therapeutic candidate in regenerative connective tissue therapies as it induces fibroblast activation, anti-TGF-β1-based therapies would be considered in histopathological conditions such as pulmonary fibrosis or hepatic fibrosis.

Transforming growth factor β1-enriched secretome up-regulate osteogenic differentiation of dental pulp stem cells, and a potential therapeutic for gingival wound healing: A comparative proteomics study

Objectives: Current study aimed at comparing the human dental pulp-derived stem cell (hDPSC) secretome (Control secretome) and transforming growth factor beta1 (TGF-β1)-transfected hDPSC secretome (TGF-β1 Secretome), which have the potential to be therapeutic in terms of regenerative dentistry, in terms of osteogenesis, adipogenesis and gingival wound healing with proteomic analyses. Materials and Methods: pCMV-TGF-β1 plasmid was transfected into hDPSCs by electroporation. hDPSC and TGF-β1 transfected hDPSC secretomes were collected for LC-MS/MS. Protein contents in control secretome and TGF-β1 secretome were analyzed by tandem mass spectrometry-based shotgun proteomic method. Bioinformatic evaluations for canonical pathways, upstream regulators and networks were completed via Ingenuity Pathway Analysis (IPA, QIAGEN) software. Surface marker expressions between groups, treated secretome were measured by flow cytometry. To support the proteomic data morphologically, we performed osteogenic-adipogenic differentiation in hDPSCs treated with control secretome and TGF-β1 secretome, and scratch wound healing assay in gingival fibroblasts. Statistical analyses were performed by GraphPad Prism 8.02. Results: Venn diagram classification showed us 174 common proteins were identified from each group. In the control secretome 140 unique proteins were identified and 66 entries were exclusive for TGF-β1 secretome. TGF-β1 secretome was found to have therapeutic effect on MSC-specific immunophenotypes. TGF-β1 secretome was determined to up-regulate osteogenesis-related molecules and pathways while down-regulating adipogenesis-related pathways. Analysis of canonical pathways showed that TGF-β1 secretome is associated with the wound healing pathway. Conclusion: Our study provided the first evidence that proteins identified in TGF-β1-transfected hDPSC secretomes are potential regulators of osteogenic/adipogenic differentiation and fibroblast wound healing. Clinical Significance: Based on these results, TGF-β1 secretome may have a therapeutic effect in repairing osteoporosis-related bone injuries, wound healing of oral mucosa and gingival tissue. TGF-β1 secretome may be a potential cell-free therapeutic in orthopedics and regenerative dentistry.

Longitudinal Proteomic Analysis of Plasma across Healthy Pregnancies Reveals Indicators of Gestational Age

Longitudinal changes in the blood proteome during gestation relate to fetal development and maternal homeostasis. Charting the maternal blood proteome in normal pregnancies is critical for establishing a baseline reference when assessing complications and disease. Using mass spectrometry-based shotgun proteomics, we surveyed the maternal plasma proteome across uncomplicated pregnancies. Results indicate a significant rise in proteins that govern placentation and are vital to the development and health of the fetus. Importantly, we uncovered proteome signatures that strongly correlated with gestational age. Fold increases and correlations between the plasma concentrations of ADAM12 (ρ = 0.973), PSG1 (ρ = 0.936), and/or CSH1/2 (ρ = 0.928) with gestational age were validated with ELISA. Proteomic and validation analyses demonstrate that the maternal plasma concentration of ADAM12, either independently or in combination with either PSG1 or CSH1/2, correlates with gestational age within ±8 days throughout pregnancy. These findings suggest that the gestational age in healthy pregnancies may be determined by referencing the concentration of select plasma proteins.

Mass Spectrometry-Based Shotgun Glycomics Using Labeled Glycan Libraries.

Mass spectrometry-based shotgun glycomics (MS-SG) is a rapid, sensitive, label-, and immobilization-free approach for the discovery of natural ligands of glycan-binding proteins (GBPs). To perform MS-SG, natural libraries of glycans derived from glycoconjugates in cells or tissues are screened against a target GBP using catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS). Because glycan concentrations are challenging to determine, ligand affinities cannot be directly measured. In principle, relative affinities can be ranked by combining CaR-ESI-MS data with relative concentrations established by hydrophilic interaction liquid chromatography (HILIC) performed on the fluorophore-labeled glycan library. To validate this approach, as well as the feasibility of performing CaR-ESI-MS directly on labeled glycans, libraries of labeled N-glycans extracted from the human monocytic U937 cells or intestinal tissues were labeled with 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), or procainamide (proA). The libraries were screened against plant and human GBPs with known specificities for α2-3- and α2-6-linked sialosides and quantified by HILIC. Dramatic differences, in some cases, were found for affinity rankings obtained with libraries labeled with different fluorophores, as well as those produced using the combined unlabeled/labeled library approach. The origin of these differences could be explained by differential glycan labeling efficiencies, the impact of specific labels on glycan affinities for the GBPs, and the relative efficiency of release of ligands from GBPs in CaR-ESI-MS. Overall, the results of this study suggest that the 2-AB(CaR-ESI-MS)/2-AB(HILIC) combination provides the most reliable description of the binding specificities of GBPs for N-glycans and is recommended for MS-SG applications.

Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering.

SUMMARYSingle-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge of current methods is their inability to identify and provide accurate quantitative information for low-abundance proteins. Herein, we describe an ion-mobility-enhanced mass spectrometry acquisition and peptide identification method, transferring identification based on FAIMS filtering (TIFF), to improve the sensitivity and accuracy of label-free scProteomics. TIFF extends the ion accumulation times for peptide ions by filtering out singly charged ions. The peptide identities are assigned by a three-dimensional MS1 feature matching approach (retention time, accurate mass, and FAIMS compensation voltage). The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells, with >1,100 proteins consistently identified. As a demonstration, we applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during lipopolysaccharide stimulation and identified time-dependent proteome changes. A record of this paper’s transparent peer review process is included in the supplemental information.

Lysosomal Changes in Mitosis.

The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphingomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.

Maternal Metals/Metalloid Blood Levels Are Associated With Lipidomic Profiles Among Pregnant Women in Puerto Rico.

Background/Aim: The association between heavy metal exposure and adverse birth outcomes is well-established. However, there is a paucity of research identifying biomarker profiles that may improve the early detection of heavy metal-induced adverse birth outcomes. Because lipids are abundant in our body and associated with important signaling pathways, we assessed associations between maternal metals/metalloid blood levels with lipidomic profiles among 83 pregnant women in the Puerto Rico PROTECT birth cohort.Methods: We measured 10 metals/metalloid blood levels during 24–28 weeks of pregnancy. Prenatal plasma lipidomic profiles were identified by liquid chromatography–mass spectrometry-based shotgun lipidomics. We derived sums for each lipid class and sums for each lipid sub-class (saturated, monounsaturated, polyunsaturated), which were then regressed on metals/metalloid. False discovery rate (FDR) adjusted p-values (q-values) were used to account for multiple comparisons.Results: A total of 587 unique lipids from 19 lipid classes were profiled. When controlling for multiple comparisons, we observed that maternal exposure to manganese and zinc were negatively associated with plasmenyl-phosphatidylethanolamine (PLPE), particularly those containing polyunsaturated fatty acid (PUFA) chains. In contrast to manganese and zinc, arsenic and mercury were positively associated with PLPE and plasmenyl-phosphatidylcholine (PLPC).Conclusion: Certain metals were significantly associated with lipids that are responsible for the biophysical properties of the cell membrane and antioxidant defense in lipid peroxidation. This study highlighted lipid-metal associations and we anticipate that this study will open up new avenues for developing diagnostic tools.

Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of the longissimus thoracis of yaks

Yaks (Bos mutus) live in the Qinghai–Tibet plateau. The quality of yak meat is unique due to its genetic and physiological characteristics. Identification of the proteome of yak muscle could help to reveal its meat-quality properties. The common proteome, phosphoproteome, and N-glycoproteome of yak longissimus thoracis (YLT) were analyzed by liquid chromatography-tandem mass spectrometry-based shotgun analysis. A total of 1812 common proteins, 1303 phosphoproteins (3918 phosphorylation sites), and 204 N-glycoproteins (285 N-glycosylation sites) were identified in YLT. The common proteins in YLT were involved mainly in myofibril structure and energy metabolism; phosphoproteins were associated primarily with myofibril organization, regulation of energy metabolism, and signaling; N-glycoproteins were engaged mainly in extracellular-matrix organization, cellular immunity, and organismal homeostasis. We reported, for the first time, the “panorama” of the YLT proteome, specifically the N-glycoproteome of YLT. Our results provide essential information for understanding post mortem physiology (rigor mortis and aging) and the quality of yak meat.

Arsenic trioxide inhibits the activity of SphK1 by decreasing the level of phosphatidylserine and phosphatidic acid in the human gastric cancer cell line MGC-803

Sphingosine kinase 1 (SphK1) is an important synthetase during the synthesis of sphingosine-1-phosphate (S1P) from sphingosine (Sph). Previous studies demonstrated that arsenic trioxide (As2O3) could reduce the level of S1P in human gastric cancer cell line MGC-803, indicating that As2O3 may inhibit the activity of SphK1. In this study, the effect of As2O3 on the SphK1 activation pathway was investigated. Western blot and quantitative real-time PCR analysis were used to evaluate the changes in protein and mRNA levels. The multi-dimensional mass spectrometry-based shotgun lipidomics method (MDMS-SL) was used for the quantitative detection of phosphatidylserine (PS) and phosphatidic acid (PA). The results revealed that As2O3 did not affect the protein and mRNA expression of SphK1 in the MGC-803 cells. However, As2O3 increased the levels of p-ERK1/2 and CIB1 in the SphK1 activation pathway and decreased the levels of PS and PA in the MGC-803 cells. The outcomes suggested that As2O3 may enhance the activity of SphK1 by increasing the levels of p-ERK1/2 and CIB1 and decrease the activity of SphK1 by decreasing the levels of PS and PA. It was suggested that the inhibition effect is stronger and resulting in an overall decrease in the activity of SphK1.