Alterations of mannosylated glycopatterns recognized by Hippeastrum hybrid lectin in saliva of patients with lung cancer.

OBJECTIVES: Lung cancer (LC) is the malignant tumor with the highest mortality rate worldwide, and precise early diagnosis can improve patient prognosis. The purpose of this study was to investigate whether alterations in the glycopatterns recognized by the Hippeastrum hybrid lectin (HHL) in salivary proteins are associated with the development of LC. MATERIALS AND METHODS: First, we collected saliva samples from LC (15 lung adenocarcinoma (ADC); 15 squamous cell carcinoma (SCC); 15 small cell lung cancer (SCLC)) and 15 benign pulmonary disease (BPD) for high-throughput detection of abundance levels of HHL-recognized glycopatterns using protein microarrays, and then validated the pooled samples from each group with lectin blotting analysis. Finally, the N-glycan profiles of salivary glycoproteins isolated from the pooled samples using HHL-magnetic particle conjugates were characterized separately using MALDI-TOF/TOF-MS. RESULTS: The results showed that the abundance level of glycopatterns recognized by HHL in salivary proteins was elevated in LC compared to BPD. The proportion of mannosylated N-glycans was notably higher in ADC (31.7%), SCC (39.0%), and SCLC (46.6%) compared to BPD (23.3%). CONCLUSIONS: The altered salivary glycopatterns such as oligomannose, Manα1-3Man, or Manα1-6Man N-glycans recognized by HHL might serve as potential biomarkers for the diagnosis of LC patients. CLINICAL RELEVANCE: This study provides crucial information for studying changes in salivary to differentiate between BPD and LC and facilitate the discovery of biomarkers for LC diagnosis based on precise alterations of mannosylated N-glycans in saliva.

Measurement constrained emission estimates of alkylated polycyclic aromatic hydrocarbons in the Canadian Athabasca oil sands region.

Alkylated polycyclic aromatic hydrocarbons (APAH) are important contaminants of crude oil production and exhibit similar toxicity to their parent compounds. This study developed an emission inventory of APAH in a major oil sands development region of Alberta, Canada, and validated the inventory with ambient concentration measurements through dispersion modeling. The initial estimate of regional total annual emissions of 21 APAH species was 362 tonnes/year in the last decade, of which 309 and 53 tonnes/year were in particle-bound and gas-phase APAH, respectively. Fugitive dust from oil sands mining activities is the primary source of particle-bound APAH, emitting 274 tonnes/year. Other major sources of APAH include point sources (31), tailings ponds (21), anthropogenic fuel consumption from mine fleet (17), and local transportation (13). The group of species with highest emissions was C1-C4 alkylnaphthalenes (53%), followed by C1-C4 alkylphenanthrenes/anthracenes (19%), C1-C4 fluorenes (13%), and C1-C4 fluoranthenes/pyrenes and C1-C4 benz[a]anthracenes/chrysene/triphenylenes (7% each). CALPUFF dispersion modeling was performed using the APAH emissions as model input. The model-predicted annual average ambient APAH concentrations at 17 monitoring sites were 1%-52% (19% on average) lower than the measurements. Inverse dispersion modeling was then applied to adjust APAH emissions higher by 19% for each of the 21 APAH species, which resulted in a revised estimate of APAH emissions to 431 tonnes/year. With the revised emissions as model input, model bias in the predicted ambient concentration was reduced from -19% to -8%. The model results showed the highest concentrations of APAH were near tailings ponds and open mining faces and downwind areas, with total APAH concentrations being higher than 50 ng/m3.

Characterization of N-glycosylation and its functional role in SIDT1-Mediated RNA uptake.

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications.

Identification of a Non-Invasive Urinary Exosomal Biomarker for Diabetic Nephropathy Using Data-Independent Acquisition Proteomics.

Diabetic nephropathy (DN), as the one of most common complications of diabetes, is generally diagnosed based on a longstanding duration, albuminuria, and decreased kidney function. Some patients with the comorbidities of diabetes and other primary renal diseases have similar clinical features to DN, which is defined as non-diabetic renal disease (NDRD). It is necessary to distinguish between DN and NDRD, considering they differ in their pathological characteristics, treatment regimes, and prognosis. Renal biopsy provides a gold standard; however, it is difficult for this to be conducted in all patients. Therefore, it is necessary to discover non-invasive biomarkers that can distinguish between DN and NDRD. In this research, the urinary exosomes were isolated from the midstream morning urine based on ultracentrifugation combined with 0.22 µm membrane filtration. Data-independent acquisition-based quantitative proteomics were used to define the proteome profile of urinary exosomes from DN (n = 12) and NDRD (n = 15) patients diagnosed with renal biopsy and Type 2 diabetes mellitus (T2DM) patients without renal damage (n = 9), as well as healthy people (n = 12). In each sample, 3372 ± 722.1 proteins were identified on average. We isolated 371 urinary exosome proteins that were significantly and differentially expressed between DN and NDRD patients, and bioinformatic analysis revealed them to be mainly enriched in the immune and metabolic pathways. The use of least absolute shrinkage and selection operator (LASSO) logistic regression further identified phytanoyl-CoA dioxygenase domain containing 1 (PHYHD1) as the differential diagnostic biomarker, the efficacy of which was verified with another cohort including eight DN patients, five NDRD patients, seven T2DM patients, and nine healthy people. Additionally, a concentration above 1.203 µg/L was established for DN based on the ELISA method. Furthermore, of the 19 significantly different expressed urinary exosome proteins selected by using the protein-protein interaction network and LASSO logistic regression, 13 of them were significantly related to clinical indicators that could reflect the level of renal function and hyperglycemic management.

Contributions of the oil sands sources to the ambient concentrations and deposition of particulate elements in the Canadian Athabasca oil sands region.

In this study, model sensitivity tests were conducted to investigate the relative contributions between emission sources of oil sands (OS) activities and other sources to the ambient concentrations and deposition of 29 particulate elements in the Athabasca oil sands region (AOSR) of Canada. Element emission sources from a recently developed emission database were grouped into three source sectors for elements in PM2.5 (OS-Industrial, OS-Dust, and Non-OS) and two source sectors for elements in PM2.5-10 (OS-All and Non-OS). The OS-Dust and OS-Industrial sectors (combined as one sector for PM2.5-10; OS-All) included element sources linked to dust and other industrial activities from the OS activities, respectively, whereas the Non-OS sector included remaining sources in the region, unrelated to the OS activities. The OS-Industrial, OS-Dust, and Non-OS emissions (tonnes/year) of all elements in PM2.5 were 326, 1430, and 562, respectively. The OS-All and Non-OS emissions (tonnes/year) of all elements in PM2.5-10 were 5890 and 2900, respectively. The element concentrations were simulated by the CALPUFF dispersion model. The sum of the domain averaged annual mean concentrations of all elements in PM2.5 and PM2.5-10 from all sources were 57.3 ng/m3 and 30.4 ng/m3, respectively. Except for Co (PM2.5 and PM2.5-10), Sb (PM2.5-10), and Sn (PM2.5-10), major proportions (≥ 59 %) of the ambient concentrations of the individual elements were linked to the OS source sector. Overall, the OS sector was responsible for 78 % and 68 % of the sum of the mean ambient concentrations of all elements in PM2.5 and PM2.5-10, respectively, which are close to the corresponding emission contributions (76 % and 67 %, respectively). Likewise, the bulk proportion (∼74 %) of the sum of the total atmospheric deposition of all elements was also associated with the OS sources. Carcinogenic and non-carcinogenic risks associated with inhalation exposure to airborne elements were below the recommended threshold risk levels.

A subpopulation of CD146+ macrophages enhances antitumor immunity by activating the NLRP3 inflammasome.

As one of the main tumor-infiltrating immune cell types, tumor-associated macrophages (TAMs) determine the efficacy of immunotherapy. However, limited knowledge about their phenotypically and functionally heterogeneous nature restricts their application in tumor immunotherapy. In this study, we identified a subpopulation of CD146+ TAMs that exerted antitumor activity in both human samples and animal models. CD146 expression in TAMs was negatively controlled by STAT3 signaling. Reducing this population of TAMs promoted tumor development by facilitating myeloid-derived suppressor cell recruitment via activation of JNK signaling. Interestingly, CD146 was involved in the NLRP3 inflammasome-mediated activation of macrophages in the tumor microenvironment, partially by inhibiting transmembrane protein 176B (TMEM176B), an immunoregulatory cation channel. Treatment with a TMEM176B inhibitor enhanced the antitumor activity of CD146+ TAMs. These data reveal a crucial antitumor role of CD146+ TAMs and highlight the promising immunotherapeutic approach of inhibiting CD146 and TMEM176B.

Atmospheric deposition mapping of particulate elements in the Canadian Athabasca oil sands region.

This study used a deposition modeling framework to generate gridded dry, wet, and total (dry + wet) deposition fluxes of 27 particulate elements over the Canadian Athabasca oil sands region and its surrounding areas for the years 2016-2017. The framework employed the element concentrations from the CALPUFF dispersion model outputs that were bias-corrected against measured concentrations, modeled dry deposition velocities, precipitation analysis data, and literature values of element-specific fine mode fractions and scavenging ratios by rain and snow. The annual total deposition (mg/m2/year) of all elements (EM) across the domain ranged from 4.49 to 5450 and the mean and median deposition were 60.9 and 31.0, respectively. Total EM deposition decreased rapidly within a short distance from the oil sands mining area. Annual mean total deposition (mg/m2/year) of EM was 717 in Zone 1 (within 30 km from a reference point, representing the center of the oil sands mining area), 115 in Zone 2 (30-100 km from the reference point), and 35.4 in Zone 3 (beyond 100 km from the reference point). The deposition of individual elements was primarily governed by their respective concentrations and among all elements the annual mean total deposition (µg/m2/year) over the domain varied five orders of magnitude ranging from 0.758 (Ag) to 20,000 (Si). Annual mean dry and wet deposition (mg/m2/year) of EM over the domain were 15.7 and 45.2, respectively. Aside from S, which has relatively lower precipitation scavenging efficiencies, wet deposition was the dominant deposition type in the region contributing from 51% (Pb) to 86% (Ca) of the respective total deposition. Total EM deposition over the domain in the warm season (66.2 mg/m2/year) was slightly higher than that in the cold season (55.6 mg/m2/year). Deposition of individual elements in Zone 1 were generally lower than their deposition at other sites across North America.

Emissions database development and dispersion model predictions of airborne particulate elements in the Canadian Athabasca oil sands region.

This study developed an emission inventory for 29 elements in PM2.5 and PM2.5-10 covering an area of approximately 300 by 420 km2 in the Athabasca Oil Sands Region in northern Alberta, Canada. Emission sources were aggregated into nine categories, of which the Oil Sands (OS) Sources emitted the most, followed by the Non-OS Dust sources for both fine and coarse elements over the study area. The top six fine particulate elements include Si, Ca, Al, Fe, S, and K (933, 442, 323, 269, 116, and 103 tonnes/year, respectively), the sum of which accounted for 20.5% of the total PM2.5 emissions. The top five coarse elements include Si, Ca, Al, Fe, and K (3713, 1815, 1198, 1073, and 404 tonnes/year), and their sum accounted for 29% of the total PM2.5-10 emissions. Using this emission inventory as input, the CALPUFF dispersion model simulated reasonable element concentrations in both PM2.5 and PM2.5-10 when compared to measurements collected at three sites during 2016-2017. Modeled PM10 concentrations of all elements were very close to the measurements at an industrial site with the highest ambient concentration, overestimated by 65% at another industrial site with moderate ambient concentration, and underestimated by 27% at a remote site with very low ambient concentration. Model-measurement differences of annual average concentrations were within 20% for Si, Ca, Al, Fe, Ti, Mn, and Cu in PM2.5, and were 20-50% for K, S, and Zn in PM2.5 at two sites located within the OS surface mineable area. Model-measurement differences were larger, but still within a factor of two for elements in PM2.5-10 at these two sites and for elements in both PM2.5 and PM2.5-10 at a background site.

Mapping Proteome and Lipidome Changes in Early-Onset Non-Alcoholic Fatty Liver Disease Using Hepatic 3D Spheroids.

Non-alcoholic fatty liver disease affects one-fourth of the world's population. Central to the disease progression is lipid accumulation in the liver, followed by inflammation, fibrosis and cirrhosis. The underlying mechanism behind the early stages of the disease is poorly understood. We have exposed human hepatic HepG2/C3A cells-based spheroids to 65 µM oleic acid and 45 µM palmitic acid and employed proteomics and lipidomics analysis to investigate their effect on hepatocytes. The treatment successfully induced in vivo hallmarks of NAFLD, as evidenced by intracellular lipid accumulation and increased ATP levels. Quantitative lipidome analysis revealed an increase in ceramides, LPC and saturated triglycerides and a decrease in the ratio of PC/PE, similar to the changes observed in patients' liver biopsies. The proteomics analysis combined with qPCR showed increased epithelial to mesenchymal transition (EMT) signalling. Activation of EMT was further validated by transcriptomics in TGF-ß treated spheroids, where an increase in mesenchymal cell markers (N-cadherin and collagen expression) was found. Our study demonstrates that this model system thus closely echoes several of the clinical features of non-alcoholic fatty liver disease and can be used to investigate the underlying molecular changes occurring in the condition.

Structure of Arabidopsis SOQ1 lumenal region unveils C-terminal domain essential for negative regulation of photoprotective qH.

Non-photochemical quenching (NPQ) plays an important role for phototrophs in decreasing photo-oxidative damage. qH is a sustained form of NPQ and depends on the plastid lipocalin (LCNP). A thylakoid membrane-anchored protein SUPPRESSOR OF QUENCHING1 (SOQ1) prevents qH formation by inhibiting LCNP. SOQ1 suppresses qH with its lumen-located thioredoxin (Trx)-like and NHL domains. Here we report structural data, genetic modification and biochemical characterization of Arabidopsis SOQ1 lumenal domains. Our results show that the Trx-like and NHL domains are associated together, with the cysteine motif located at their interface. Residue E859, required for SOQ1 function, is pivotal for maintaining the Trx-NHL association. Importantly, the C-terminal region of SOQ1 forms an independent ß-stranded domain that has structural homology to the N-terminal domain of bacterial disulfide bond protein D and is essential for negative regulation of qH. Furthermore, SOQ1 is susceptible to cleavage at the loops connecting the neighbouring lumenal domains both in vitro and in vivo, which could be a regulatory process for its suppression function of qH.

Plasma proteome profiling combined with clinical and genetic features reveals the pathophysiological characteristics of ß-thalassemia.

The phenotype of ß-thalassemia underlies multigene interactions, making clinical stratification complicated. An increasing number of genetic modifiers affecting the disease severity have been identified, but are still unable to meet the demand of precision diagnosis. Here, we systematically conducted a comparative plasma proteomic profiling on patients with ß-thalassemia and healthy controls. Among 246 dysregulated proteins, 13 core protein signatures with excellent biomarker potential are proposed. The combination of proteome and patients' clinical data revealed patients with codons 41/42 -TTCT mutations have an elevated risk of higher iron burden, dysplasia, and osteoporosis than patients with other genotypes. Notably, 85 proteins correlating to fetal hemoglobin (Hb F) were identified, among which the abundance of 27 proteins may affect the transfusion burden in patients with ß-thalassemia. The current study thus provides protein signatures as potential diagnostic biomarkers or therapeutic clues for ß-thalassemia.

Alternations of N-glycans recognized by Phaseolus vulgaris leucoagglutinin in the saliva of patients with breast cancer.

Breast cancer is the most frequently diagnosed cancer in most countries. Early diagnosis of breast disease is necessary for its prognosis and treatment. Altered protein glycosylation has been shown to be expressed in precursor lesions of breast cancer, making them powerful early diagnostic biomarkers. The present study validated alterations of the N-glycan profiles of their salivary glycoproteins isolated by the Phaseolus vulgaris leucoagglutinin (PHA-E+L)-magnetic particle conjugates from 141 female subjects (66 healthy volunteers (HV), and 75 patients with breast disease including breast benign cyst (BB) or breast cancer in stage I/II (BC-I/II)) were analyzed and annotated by MALDI-TOF/TOF-MS. The results showed that there were 11, 20, 16, and 17 N-glycans recognized by PHA-E+L identified and annotated from the pooled salivary samples of HV, BB, BC-I, and BC-II, respectively. There were 3 N-glycans peaks (m/z 2459.8799, 2507.9139, and 2954.0547), 2 N-glycans peaks (m/z 1957.7265 and 2794.0427), and 2 N-glycans peaks (m/z 1866.6608 and 2240.8056) recognized by PHA-E+L that existed only in BB, BC-I, and BC-II, respectively. The present study compared the alternations of N-glycans from the salivary proteins isolated by PHA-E+L-magnetic particle conjugates among HV, BB, BC-I, and BC-II, which could provide information on N-glycans during the development of breast cancer in saliva to promote the study of its biomarkers.

LncRNA-encoded microproteins: A new form of cargo in cell culture-derived and circulating extracellular vesicles.

Advancements in omics-based technologies over the past few years have led to the discovery of numerous biologically relevant peptides encoded by small open reading frames (smORFs) embedded in long noncoding RNA (lncRNA) transcripts (referred to as microproteins here) in a variety of species. However, the mechanisms and modes of action that underlie the roles of microproteins have yet to be fully characterized. Herein, we provide the first experimental evidence of abundant microproteins in extracellular vesicles (EVs) derived from glioma cancer cells, indicating that the EV-mediated transfer of microproteins may represent a novel mechanism for intercellular communication. Intriguingly, when examining human plasma, 48, 11 and 3 microproteins were identified from purified EVs, whole plasma and EV-free plasma, respectively, suggesting that circulating microproteins are primarily enriched in EVs. Most importantly, the preliminary data showed that the expression profile of EV microproteins in glioma patient diverged from the health donors, suggesting that the circulating microproteins in EVs might have potential diagnostic application in identifying patients with glioma.

Comprehensive analysis of glycosphingolipid glycans by lectin microarrays and MALDI-TOF mass spectrometry.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates present on the cell membrane; they play significant roles in many bioprocesses such as cell adhesion, embryonic development, signal transduction and carcinogenesis. Analyzing such amphiphilic molecules is a major challenge in the field of glycosphingolipidomics. We provide a step-by-step protocol that uses a lectin microarray to analyze GSL glycans from cultured cells. The procedure describes (i) extraction of GSLs from cell pellets, (ii) N-monodeacylation using sphingolipid ceramide N-deacylase digestion to form lyso-GSLs, (iii) fluorescence labeling at the newly exposed amine group, (iv) preparation of a lectin microarray, (v) GSL-glycan analysis by a lectin microarray, (vi) complementary mass spectrometry analysis and (vii) data acquisition and analysis. This method is high-throughput, low cost and easy to conduct, and it provides detailed information about glycan linkages. This protocol takes ~10 d.

Deeply Mining a Universe of Peptides Encoded by Long Noncoding RNAs.

Many small ORFs embedded in long noncoding RNA (lncRNA) transcripts have been shown to encode biologically functional polypeptides (small ORF-encoded polypeptides [SEPs]) in different organisms. Despite some novel SEPs have been found, the identification is still hampered by their poor predictability, diminutive size, and low relative abundance. Here, we take advantage of NONCODE, a repository containing the most complete collection and annotation of lncRNA transcripts from different species, to build a novel database that attempts to maximize a collection of SEPs from human and mouse lncRNA transcripts. In order to further improve SEP discovery, we implemented two effective and complementary polypeptide enrichment strategies using 30-kDa molecular weight cutoff filter and C8 solid-phase extraction column. These combined strategies enabled us to discover 353 SEPs from eight human cell lines and 409 SEPs from three mouse cell lines and eight mouse tissues. Importantly, 19 of them were then verified through in vitro expression, immunoblotting, parallel reaction monitoring, and synthetic peptides. Subsequent bioinformatics analysis revealed that some of the physical and chemical properties of these novel SEPs, including amino acid composition and codon usage, are different from those commonly found in canonical proteins. Intriguingly, nearly 65% of the identified SEPs were found to be initiated with non-AUG start codons. The 762 novel SEPs probably represent the largest number of SEPs detected by MS reported to date. These novel SEPs might not only provide new clues for the annotation of noncoding elements in the genome but also serve as a valuable resource for functional study.

Proteomics reveals the function reverse of MPSSS-treated prostate cancer-associated fibroblasts to suppress PC-3 cell viability via the FoxO pathway.

Prostate cancer-associated fibroblasts (prostate CAFs) are essential components of the tumor microenvironment and can promote tumor progression through their immunosuppressive functions. MPSSS, a novel polysaccharide purified from Lentinus edodes, has been reported to have anti-tumor activity. MPSSS could also inhibit the immunosuppressive function of prostate CAFs, which has been demonstrated through that the secretome of MPSSS-treated prostate CAFs could inhibit the proliferation of T cells. However, how the secretome of MPSSS-treated prostate CAFs influence prostate cancer progression is still unclear. Interestingly, we found that the low molecular weight (3-100kD) secretome of prostate CAFs (lmwCAFS) could promote the growth of PC-3 cells, while that of MPSSS-treated prostate CAFs (MT-lmwCAFS) could inhibit their growth. We carried out comparative secretomic analysis of lmwCAFS and MT-lmwCAFS to identify functional molecules that inhibit the growth of PC-3 cells, and proteomic analysis of lmwCAFS-treated PC-3 cells and MT-lmwCAFS-treated PC-3 cells to investigate the underlying molecular mechanism. These analyses suggest that TGF-ß3 from MT-lmwCAFS may inhibit the growth of PC-3 cells. The validated experiments revealed that TGF-ß3 from MT-lmwCAFS activated p21 expression in PC-3 cells by regulating the FoxO pathway thereby inducing G0/G1 cell cycle arrest of PC-3 cells. Overall, our data demonstrated that MPSSS reversed the ability of prostate CAFs to suppress the cell viability of PC-3 cells, which might provide a potential therapeutic strategy to prevent prostate cancer progression.

Oleic Acid and Eicosapentaenoic Acid Reverse Palmitic Acid-induced Insulin Resistance in Human HepG2 Cells via the Reactive Oxygen Species/JUN Pathway.

Oleic acid (OA), a monounsaturated fatty acid (MUFA), has previously been shown to reverse saturated fatty acid palmitic acid (PA)-induced hepatic insulin resistance (IR). However, its underlying molecular mechanism is unclear. In addition, previous studies have shown that eicosapentaenoic acid (EPA), a ω-3 polyunsaturated fatty acid (PUFA), reverses PA-induced muscle IR, but whether EPA plays the same role in hepatic IR and its possible mechanism involved need to be further clarified. Here, we confirmed that EPA reversed PA-induced IR in HepG2 cells and compared the proteomic changes in HepG2 cells after treatment with different free fatty acids (FFAs). A total of 234 proteins were determined to be differentially expressed after PA+OA treatment. Their functions were mainly related to responses to stress and endogenous stimuli, lipid metabolic process, and protein binding. For PA+EPA treatment, the PA-induced expression changes of 1326 proteins could be reversed by EPA, 415 of which were mitochondrial proteins, with most of the functional proteins involved in oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle. Mechanistic studies revealed that the protein encoded by JUN and reactive oxygen species (ROS) play a role in OA- and EPA-reversed PA-induced IR, respectively. EPA and OA alleviated PA-induced abnormal adenosine triphosphate (ATP) production, ROS generation, and calcium (Ca2+) content. Importantly, H2O2-activated production of ROS increased the protein expression of JUN, further resulting in IR in HepG2 cells. Taken together, we demonstrate that ROS/JUN is a common response pathway employed by HepG2 cells toward FFA-regulated IR.

Sequential Precipitation and Delipidation Enables Efficient Enrichment of Low-Molecular Weight Proteins and Peptides from Human Plasma.

Low-molecular weight proteins and peptides (LMWPs, <30 kDa) in human plasma serve as potential biomarkers or drug targets and are endowed with desirable traits for biological and clinical studies. However, the identification of LMWPs from plasma is retarded by high-abundance proteins, high-molecular weight proteins, and lipids. Here, we present a sequential precipitation and delipidation (SPD) method for the efficient enrichment of LMWPs based on methyl-tert-butyl ether/methanol/water systems. The enriched LMWP sample was analyzed by single-shot liquid chromatography-tandem mass spectrometry employing both HCD and EThcD without tryptic digestion, and 725 peptides were identified on average. The LMWP sample was also digested and analyzed using a bottom-up proteomics pipeline, and 289 proteins were identified, of which 129 (44.6%) proteins were less than 30 kDa and lipoprotein-associated proteins were significantly enriched. Additionally, 25 neuropeptides and 19 long noncoding RNA-encoded polypeptides were identified. Taken together, the SPD method shows good sensitivity and reproducibility when compared with other enrichment methods and has great potential for clinical biomarker discovery and application.

Proteomic profiling of MIN6 cell-derived exosomes.

Exosomes have been widely used in research on the early clinical diagnosis, prognosis and treatment of various cancers due to their features of small size (30-120 nm), non-immunogenicity and ability to cross biological barriers. However, few studies have investigated exosomes involved in metabolic diseases. Early studies have found that adipose tissue can be a source of exosomes regulating metabolism, but the related functions of exosomes secreted by other tissues in the regulation of metabolic diseases have not been determined. In addition, islets were found to be able to secrete miRNA via exosomes, suggesting that islet exosomes may be among the sources of exosomes involved in the regulation of metabolic diseases and that the relevant protein profiles have not been characterized to date. Therefore, identifying the protein contents of pancreatic ß cell-derived exosomes would benefit further research investigating the protein functions and mechanisms associated with diabetes-related metabolic diseases. SIGNIFICANCE: Exosomes are emerging tools for investigating metabolic diseases in recent years, but little research has been done. In our work, functional identification of MIN6 cell-derived exosomal proteins and comparative analysis of islet ß cell exosomal protein data from different cell lines or from different species revealed that exosomes secreted by islet ß cells may be involved in the regulation of glucose metabolism. These results may suggest that intercellular communication induced by exosome transfer among tissues may account for the major reason of diabetic metabolic disorder. In addition, these results may provide a theoretical basis for the study of the physiological and pathological functions of islet ß cell exosomes for the future studies.

Abnormal Galactosylated-Glycans recognized by Bandeiraea Simplicifolia Lectin I in saliva of patients with breast Cancer.

Currently, the definitive diagnosis in breast cancer requires biopsy and histopathology, such the most effective markers are tissue-based. However, the advantages of saliva in collection and storage make it possible for assessing human pathology and contributing to the development of cancer-related biomarkers for clinical application. The present study validated alteration of salivary protein glycopatterns recognized by Bandeiraea simplicifolia lectin I (BS-I) in the saliva of patients with breast diseases using saliva microarrays, and the N/O-glycan profiles of their salivary glycoproteins isolated by the BS-I-magnetic particle conjugates from 259 female subjects (66 healthy volunteers (HV), 65 benign breast cyst or tumor patients (BB), 66 patients with breast cancer in stage I (BC-I) and 62 patients with breast cancer in stage II (BC-II)) were analyzed by MALDI-TOF/TOF-MS. The results showed that the expression level of galactosylated glycans recognized by BS-I was significantly increased in patients with breast cancer compared with HV (p < 0.05). Totally, there were 11/10, 10/19, 7/24 and 7/9 galactosylated N-/O-linked glycans were identified and annotated from the pooled salivary samples of HV, BB, BC-I and BC-II, respectively. One galactosylated N-glycan peak (m/z 2773.977), and 4 galactosylated O-glycan peaks (m/z 868.295, 882.243, 884.270 and 1030.348) were found only in BC-I. These findings could provide pivotal information on galactosylated N/O-linked glycans related to breast cancer, and promote the study of biomarkers for early-stage breast cancer based on precise alterations of galactosylated N/O-glycans in saliva.