Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of small extracellular vesicles from C2C12 myoblasts identify specific PTM patterns in ligand-receptor interactions.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available.In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC-MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided.In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.

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.

Proteomics protocol for obtaining extracellular vesicle from human plasma using asymmetrical flow field-flow fractionation technology.

Plasma extracellular vesicles (EVs) represent a potential resource for biomarkers of multiple diseases. Here, we present a protocol for obtaining EVs from human plasma using asymmetrical flow field-flow fractionation technology. We describe steps for using tandem mass tags to perform comparative proteomic studies of a large clinical cohort. We then detail targeted quantitative analysis of differential proteins based on a parallel reaction monitoring technique. For complete details on the use and execution of this protocol, please refer to Wu et al. (2020)1 and Li et al. (2023).2.

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.

Differential proteomic patterns of plasma extracellular vesicles show potential to discriminate ß-thalassemia subtypes.

The observed specificity of ß-thalassemia-subtype phenotypes makes new diagnostic strategies that complement current screening methods necessary to determine each subtype and facilitate therapeutic regimens for different patients. Here, we performed quantitative proteomics of plasma-derived extracellular vesicles (EVs) of ß-thalassemia major (TM) patients, ß-thalassemia intermedia (TI) patients, and healthy controls to explore subgroup characteristics and potential biomarkers. Plasma quantitative proteomics among the same cohorts were analyzed in parallel to compare the biomarker potential of both specimens. EV proteomics showed significantly more abnormalities in immunity and lipid metabolism in TI and TM, respectively. The differential proteomic patterns of EVs were consistent with but more striking than those of plasma. Notably, we also found EV proteins to have a superior performance for discriminating ß-thalassemia subtypes. These findings allowed us to propose a diagnostic model consisting of five proteins in EVs with subtyping potential, demonstrating the ability of plasma-derived EVs for the diagnosis of ß-thalassemia patients.

Comprehensive Proteomics Analysis Identifies CD38-Mediated NAD+ Decline Orchestrating Renal Fibrosis in Pediatric Patients With Obstructive Nephropathy.

Obstructive nephropathy is one of the leading causes of kidney injury and renal fibrosis in pediatric patients. Although considerable advances have been made in understanding the pathophysiology of obstructive nephropathy, most of them were based on animal experiments and a comprehensive understanding of obstructive nephropathy in pediatric patients at the molecular level remains limited. Here, we performed a comparative proteomics analysis of obstructed kidneys from pediatric patients with ureteropelvic junction obstruction and healthy kidney tissues. Intriguingly, the proteomics revealed extensive metabolic reprogramming in kidneys from individuals with ureteropelvic junction obstruction. Moreover, we uncovered the dysregulation of NAD+ metabolism and NAD+-related metabolic pathways, including mitochondrial dysfunction, the Krebs cycle, and tryptophan metabolism, which led to decreased NAD+ levels in obstructed kidneys. Importantly, the major NADase CD38 was strongly induced in human and experimental obstructive nephropathy. Genetic deletion or pharmacological inhibition of CD38 as well as NAD+ supplementation significantly recovered NAD+ levels in obstructed kidneys and reduced obstruction-induced renal fibrosis, partially through the mechanisms of blunting the recruitment of immune cells and NF-κB signaling. Thus, our work not only provides an enriched resource for future investigations of obstructive nephropathy but also establishes CD38-mediated NAD+ decline as a potential therapeutic target for obstruction-induced renal fibrosis.

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.

Semiconducting mineral induced photochemical conversion of PAHs in aquatic environment: Mechanism study and fate prediction.

Semiconducting minerals (such as iron sulfides) are highly abundant in surface water, but their influences on the natural photochemical process of contaminants are still unknown. By simulating the natural water environment under solar irradiation, this work comprehensively investigated the photochemical processes of anthracene (a typical Polycyclic Aromatic Hydrocarbons) in both freshwater and seawater. The results show that the natural pyrite (NP) significantly promotes the degradation of anthracene under solar illumination via 1) NP induced photocatalytic degradation of anthracene, and 2) Fenton reaction due to the NP induced photocatalytic generation of H2O2. The material characterization and theoretical calculation reveal that the natural impurity in NP enlarges its band gap, which limits the utilization of solar spectra to shorter wavelength. The contribution of generated reactive intermediates on anthracene degradation follows the order of 1O2 >OH > O2- in freshwater and O2- >1O2 >OH in seawater. The photochemically generated H2O2 is a vital source for OH generation (from Fenton reaction). The steady-state concentration of OH, 1O2 and O2- in freshwater were monitored as 3.0 × 10-15 M, 1.1 × 10-13 M, and 4.5 × 10-14 M, respectively. However, the OH concentration in seawater can be negligible due to the quenching effects by halides, and the 1O2 and O2- concentrations are higher than that in freshwater. An anthracene degradation kinetic model was built based on the experimentally determined reactive intermediates concentration and its second order rate constant with anthracene. Moreover, the anthracene degradation pathway was proposed based on intermediates analysis and DFT calculation, and its toxicity evolution during the photochemical process was assessed by quantitative structure-activity relationship (QSAR) based prediction. This finding suggests that the natural semiconducting minerals can affect the fate and environmental risks of contaminants in natural water.

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.

[Preface for special issue on multi-omics frontier technologies].

With wide applications of genomics, transcriptomics, proteomics and metabolomics in the post-genome era, functional explanation has become the central task in life science research, and multi-omics data integrative analysis has become an indispensable strategy for uncovering the underlying biological mechanism. This special issue aimed to introduce the related research advances and applications in multi-omics by inviting the domestic experts. In total, 28 papers have been collected in this issue, for researcher's reference in multi-omics.

[Advances in the methods of phosphopeptide enrichment and separation in phosphoproteomic research].

The systematic and in-depth study of phosphoproteome rely on highly reproducible and specific phosphopeptide enrichment methods. At present, a variety of enrichment methods have been developed based on different principles, and these methods often display different selectivity and specificity. It is therefore very important to select the most suitable enrichment method according to different research purposes. This review summarized the phosphopeptide enrichment based on affinity chromatography, immunoprecipitation, chemical derivatization, chromatography and other newly developed methods. The advantages and disadvantages of these methods, as well as the related optimization and improvement strategies, were discussed in detail. In addition, we also briefly summarized the progress of the combination of phosphopeptide enrichment and fractionation methods developed in recent years.

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.

SARS-CoV-2 ORF8 reshapes the ER through forming mixed disulfides with ER oxidoreductases.

The replication machinery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is closely associated with the endoplasmic reticulum (ER) in host cells. Activation of the unfolded protein response (UPR) is a strategy hijacked by coronavirus to facilitate its replication and suppress host innate immunity. Here, we have found that SARS-CoV-2 ORF8 protein accumulates in the ER and escapes the degradation system by forming mixed disulfide complexes with ER oxidoreductases. ORF8 induces the activation of three UPR pathways through targeting key UPR components, remodels ER morphology and accelerates protein trafficking. Moreover, small molecule reducing agents release ORF8 from the mixed disulfide complexes and facilitate its degradation, therefore mitigate ER stress. Our study reveals a unique mechanism by which SARS-CoV-2 ORF8 escapes degradation by host cells and regulates ER reshaping. Targeting ORF8-involved mixed disulfide complexes could be a new strategy to alleviate SARS-CoV-2 induced ER stress and related diseases.

CrrAB regulates PagP-mediated glycerophosphoglycerol palmitoylation in the outer membrane of Klebsiella pneumoniae.

The outer membrane (OM) of Gram-negative bacteria is an evolving antibiotic barrier composed of a glycerophospholipid (GP) inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The two-component regulatory system CrrAB has only recently been reported to confer high-level polymyxin resistance and virulence in Klebsiella pneumoniae. Mutations in crrB have been shown to lead to the modification of the lipid A moiety of LPS through CrrAB activation. However, functions of CrrAB activation in the regulation of other lipids are unclear. Work here demonstrates that CrrAB activation not only stimulates LPS modification but also regulates synthesis of acyl-glycerophosphoglycerols (acyl-PGs), a lipid species with undefined functions and biosynthesis. Among all possible modulators of acyl-PG identified from proteomic data, we found expression of lipid A palmitoyltransferase (PagP) was significantly upregulated in the crrB mutant. Furthermore, comparative lipidomics showed that most of the increasing acyl-PG activated by CrrAB was decreased after pagP knockout with CRISPR-Cas9. These results suggest that PagP also transfers a palmitate chain from GPs to PGs, generating acyl-PGs. Further investigation revealed that PagP mainly regulates the GP contents within the OM, leading to an increased ratio of acyl-PG to PG species and improving OM hydrophobicity, which may contribute to resistance against certain cationic antimicrobial peptides resistance upon LPS modification. Taken together, this work suggests that CrrAB regulates the palmitoylation of PGs and lipid A within the OM through upregulated PagP, which functions together to form an outer membrane barrier critical for bacterial survival.

Comprehensive Evaluation of Different TiO2-Based Phosphopeptide Enrichment and Fractionation Methods for Phosphoproteomics.

Protein phosphorylation is an essential post-translational modification that regulates multiple cellular processes. Due to their low stoichiometry and ionization efficiency, it is critical to efficiently enrich phosphopeptides for phosphoproteomics. Several phosphopeptide enrichment methods have been reported; however, few studies have comprehensively compared different TiO2-based phosphopeptide enrichment methods using complex proteomic samples. Here, we compared four TiO2-based phosphopeptide enrichment methods that used four non-phosphopeptide excluders (glutamic acid, lactic acid, glycolic acid, and DHB). We found that these four TiO2-based phosphopeptide enrichment methods had different enrichment specificities and that phosphopeptides enriched by the four methods had different physicochemical characteristics. More importantly, we discovered that phosphopeptides had a higher deamidation ratio than peptides from cell lysate and that phosphopeptides enriched using the glutamic acid method had a higher deamidation ratio than the other three methods. We then compared two phosphopeptide fractionation methods: ammonia- or TEA-based high pH reversed-phase (HpH-RP). We found that fewer phosphopeptides, especially multi-phosphorylated peptides, were identified using the ammonia-based method than using the TEA-based method. Therefore, the TEA-based HpH-RP fractionation method performed better than the ammonia method. In conclusion, we comprehensively evaluated different TiO2-based phosphopeptide enrichment and fractionation methods, providing a basis for selecting the proper protocols for comprehensive phosphoproteomics.

Salivary Glycopatterns as Potential Non-Invasive Biomarkers for Diagnosing and Reflecting Severity and Prognosis of Diabetic Nephropathy.

Discriminating between diabetic nephropathy (DN) and non-diabetic renal disease (NDRD) can help provide more specific treatments. However, there are no ideal biomarkers for their differentiation. Thus, the aim of this study was to identify biomarkers for diagnosing and predicting the progression of DN by investigating different salivary glycopatterns. Lectin microarrays were used to screen different glycopatterns in patients with DN or NDRD. The results were validated by lectin blotting. Logistic regression and artificial neural network analyses were used to construct diagnostic models and were validated in in another cohort. Pearson's correlation analysis, Cox regression, and Kaplan-Meier survival curves were used to analyse the correlation between lectins, and disease severity and progression. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analyses were used to identify corresponding glycoproteins and predict their function. Both the logistic regression model and the artificial neural network model achieved high diagnostic accuracy. The levels of Aleuria aurantia lectin (AAL), Lycopersicon esculentum lectin (LEL), Lens culinaris lectin (LCA), Vicia villosa lectin (VVA), and Narcissus pseudonarcissus lectin (NPA) were significantly correlated with the clinical and pathological parameters related to DN severity. A high level of LCA and a low level of LEL were associated with a higher risk of progression to end-stage renal disease. Glycopatterns in the saliva could be a non-invasive tool for distinguishing between DN and NDRD. The AAL, LEL, LCA, VVA, and NPA levels could reflect the severity of DN, and the LEL and LCA levels could indicate the prognosis of DN.