Uncovering missing glycans and unexpected fragments with pGlycoNovo for site-specific glycosylation analysis across species.

Precision mapping of site-specific glycans using mass spectrometry is vital in glycoproteomics. However, the diversity of glycan compositions across species often exceeds database capacity, hindering the identification of rare glycans. Here, we introduce pGlycoNovo, a software within the pGlyco3 software environment, which employs a glycan first-based full-range Y-ion dynamic searching strategy. pGlycoNovo enables de novo identification of intact glycopeptides with rare glycans by considering all possible monosaccharide combinations, expanding the glycan search space to 16~1000 times compared to non-open search methods, while maintaining accuracy, sensitivity and speed. Reanalysis of SARS Covid-2 spike protein glycosylation data revealed 230 additional site-specific N-glycans and 30 previously unreported O-glycans. pGlycoNovo demonstrated high complementarity to six other tools and superior search speed. It enables characterization of site-specific N-glycosylation across five evolutionarily distant species, contributing to a dataset of 32,549 site-specific glycans on 4602 proteins, including 2409 site-specific rare glycans, and uncovering unexpected glycan fragments.

Glyco-signatures in patients with advanced lung cancer during anti-PD-1/PD-L1 immunotherapy.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1/programmed cell death ligand-1 (PD-1/PD-L1) have significantly prolonged the survival of advanced/metastatic patients with lung cancer. However, only a small proportion of patients can benefit from ICIs, and clinical management of the treatment process remains challenging. Glycosylation has added a new dimension to advance our understanding of tumor immunity and immunotherapy. To systematically characterize anti-PD-1/PD-L1 immunotherapy-related changes in serum glycoproteins, a series of serum samples from 12 patients with metastatic lung squamous cell carcinoma (SCC) and lung adenocarcinoma (ADC), collected before and during ICIs treatment, are firstly analyzed with mass-spectrometry-based label-free quantification method. Second, a stratification analysis is performed among anti-PD-1/PD-L1 responders and non-responders, with serum levels of glycopeptides correlated with treatment response. In addition, in an independent validation cohort, a large-scale site-specific profiling strategy based on chemical labeling is employed to confirm the unusual characteristics of IgG N-glycosylation associated with anti-PD-1/PD-L1 treatment. Unbiased label-free quantitative glycoproteomics reveals serum levels' alterations related to anti-PD-1/PD-L1 treatment in 27 out of 337 quantified glycopeptides. The intact glycopeptide EEQFN 177STYR (H3N4) corresponding to IgG4 is significantly increased during anti-PD-1/PD-L1 treatment (FC=2.65, P=0.0083) and has the highest increase in anti-PD-1/PD-L1 responders (FC=5.84, P=0.0190). Quantitative glycoproteomics based on protein purification and chemical labeling confirms this observation. Furthermore, obvious associations between the two intact glycopeptides (EEQFN 177STYR (H3N4) of IgG4, EEQYN 227STFR (H3N4F1) of IgG3) and response to treatment are observed, which may play a guiding role in cancer immunotherapy. Our findings could benefit future clinical disease management.

Integrated Platform for Large-Scale Quantitative Profiling of Phosphotyrosine Signaling Complexes Based on Cofractionation/Mass Spectrometry and Complex-Centric Algorithm.

The scarcity and dynamic nature of phosphotyrosine (pTyr)-modified proteins pose a challenge for researching protein complexes with pTyr modification, which are assembled through multiple protein-protein interactions. We developed an integrated complex-centric platform for large-scale quantitative profiling of pTyr signaling complexes based on cofractionation/mass spectrometry (CoFrac-MS) and a complex-centric algorithm. We initially constructed a trifunctional probe based on pTyr superbinder (SH2-S) for specifically binding and isolation of intact pTyr protein complexes. Then, the CoFrac-MS strategy was employed for the identification of pTyr protein complexes by integrating ion exchange chromatography in conjunction with data independent acquisition mass spectrometry. Furthermore, we developed a novel complex-centric algorithm for quantifying protein complexes based on the protein complex elution curve. Utilizing this algorithm, we effectively quantified 216 putative protein complexes. We further screened 21 regulated pTyr protein complexes related to the epidermal growth factor signal. Our study engenders a comprehensive framework for the intricate examination of pTyr protein complexes and presents, for the foremost occasion, a quantitative landscape delineating the composition of pTyr protein complexes in HeLa cells.

CPT1A loss disrupts BCAA metabolism to confer therapeutic vulnerability in TP53-mutated liver cancer.

Driver genomic mutations in tumors define specific molecular subtypes that display distinct malignancy competence, therapeutic resistance and clinical outcome. Although TP53 mutation has been identified as the most common mutation in hepatocellular carcinoma (HCC), current understanding on the biological traits and therapeutic strategies of this subtype has been largely unknown. Here, we reveal that fatty acid ß oxidation (FAO) is remarkable repressed in TP53 mutant HCC and which links to poor prognosis in HCC patients. We further demonstrate that carnitine palmitoyltransferase 1 (CPT1A), the rate-limiting enzyme of FAO, is universally downregulated in liver tumor tissues, and which correlates with poor prognosis in HCC and promotes HCC progression in the de novo liver tumor and xenograft tumor models. Mechanically, hepatic Cpt1a loss disrupts lipid metabolism and acetyl-CoA production. Such reduction in acetyl-CoA reduced histone acetylation and epigenetically reprograms branched-chain amino acids (BCAA) catabolism, and leads to the accumulation of cellular BCAAs and hyperactivation of mTOR signaling. Importantly, we reveal that genetic ablation of CPT1A renders TP53 mutant liver cancer mTOR-addicted and sensitivity to mTOR inhibitor AZD-8055 treatment. Consistently, Cpt1a loss in HCC directs tumor cell therapeutic response to AZD-8055. CONCLUSION: Our results show genetic evidence for CPT1A as a metabolic tumor suppressor in HCC and provide a therapeutic approach for TP53 mutant HCC patients.

Picoliter Single-Cell Reactor for Proteome Profiling by In Situ Cell Lysis, Protein Immobilization, Digestion, and Droplet Transfer.

Global profiling of single-cell proteomes can reveal cellular heterogeneity, thus benefiting precision medicine. However, current mass spectrometry (MS)-based single-cell proteomic sample processing still faces technical challenges associated with processing efficiency and protein recovery. Herein, we present an innovative sample processing platform based on a picoliter single-cell reactor (picoSCR) for single-cell proteome profiling, which involves in situ protein immobilization and sample transfer. PicoSCR helped minimize surface adsorptive losses by downscaling the processing volume to 400 pL with a contact area of less than 0.4 mm2. Besides, picoSCR reached highly efficient cell lysis and digestion within 30 min, benefiting from optimal reagent and high reactant concentrations. Using the picoSCR-nanoLC-MS system, over 1400 proteins were identified from an individual HeLa cell using data-dependent acquisition mode. Proteins with copy number below 1000 were identified, demonstrating this system with a detection limit of 1.7 zmol. Furthermore, we profiled the proteome of circulating tumor cells (CTCs). Data are available via ProteomeXchange with the identifier PXD051468. Proteins associated with epithelial-mesenchymal transition and neutrophil extracellular traps formation (which are both related to tumor metastasis) were observed in all CTCs. The cellular heterogeneity was revealed by differences in signaling pathways within individual cells. These results highlighted the potential of the picoSCR platform to help discover new biomarkers and explore differences in biological processes between cells.

Global analysis of N-myristoylation and its heterogeneity by combining N-terminomics and nanographite fluoride-based solid-phase extraction.

N-myristoylation is one of the most widespread and important lipidation in eukaryotes and some prokaryotes, which is formed by covalently attaching various fatty acids (predominantly myristic acid C14:0) to the N-terminal glycine of proteins. Disorder of N-myristoylation is critically implicated in numerous physiological and pathological processes. Here, we presented a method for purification and comprehensive characterization of endogenous, intact N-glycine lipid-acylated peptides, which combined the negative selection method for N-terminome and the nanographite fluoride-based solid-phase extraction method (NeS-nGF SPE). After optimizing experimental conditions, we conducted the first global profiling of the endogenous and heterogeneous modification states for N-terminal glycine, pinpointing the precise sites and their associated lipid moieties. Totally, we obtained 76 N-glycine lipid-acylated peptides, including 51 peptides with myristate (C14:0), 10 with myristoleate (C14:1), 6 with tetradecadienoicate (C14:2), 5 with laurate (C12:0) and 4 with lauroleate (C12:1). Therefore, our proteomic methodology could significantly facilitate precise and in-depth analysis of the endogenous N-myristoylome and its heterogeneity.

Immunomagnetic capture and traceless release of native tumor-derived exosomes from human plasma for exploring interaction with recipient cells by aptamer-functionalized nanoflowers.

BACKGROUND: Tumor-derived exosomes (TEXs) play an important role in the development process of cancer, which can transport a large number of carcinogenic molecules to normal cells, and subsequently promote tumor metastasis. However, TEXs that were utilized in most of previous researches were obtained from the cell medium of tumor cell lines, which cannot reflect the physiological state of primary cells in vivo. Isolation of native TEXs from human plasma with intact function is contributed to exploring the interaction between TEXs and recipient cells for understanding their true biological functions. RESULTS: We developed a strategy that involves both capture and release processes to obtain native TEXs from plasma of cancer patients. An MoS2-based immunomagnetic probe (Fe3O4@MoS2-Au-Aptamer, named as FMAA) with the advantages of high surface area, magnetic response and abundant affinity sites was designed and synthesized to capture TEXs through recognizing high-expression tumor-associated antigens of EpCAM. With the assistance of complementary sequences of EpCAM, TEXs were released with non-destruction and no residual labels. According to NTA analysis, 107-108 TEXs were recovered from per mL plasma of breast cancer patients. The interaction between native TEXs and normal epithelial cells confirms TEXs could induce significant activation of autophagy of recipient cells with co-culture for 12 h. Proteomics analysis demonstrated a total of 637 proteins inside epithelial cells had dynamic expression with the stimulation of TEXs and 5 proteins in the pathway of autophagy had elevated expression level. SIGNIFICANCE: This work not only obtains native TEXs from human plasma with non-destruction and no residual labels, but also explores the interaction between TEXs and recipient cells for understanding their true biological functions, which will accelerate the application of TEXs in the field of biomarkers and therapeutic drugs.

Elucidation of N-/O-glycosylation and site-specific mapping of sialic acid linkage isomers of SARS-CoV-2 human receptor angiotensin-converting enzyme 2.

Human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for cellular entry of SARS-CoV-2 into human host cells. hACE2 is heavily glycosylated and glycans on the receptor may play a role in viral binding. Thus, comprehensive characterization of hACE2 glycosylation could aid our understanding of interactions between the receptor and SARS-CoV-2 spike (S) protein, as well as provide a basis for the development of therapeutic drugs targeting this crucial interaction. Herein, 138 N-glycan compositions were identified, most of which are complex-type N-glycans, from seven N-glycosites of hACE2. Among them, 67% contain at least one sialic acid residue. At the level of glycopeptides, the overall quantification of sialylated glycan isomers observed on the sites N322 and N546 have a higher degree of NeuAc (α2-3)Gal (over 80.3%) than that of other N-glycosites (35.6-71.0%). In terms of O-glycans, 69 glycan compositions from 12 O-glycosites were identified, and especially, the C-terminus of hACE2 is heavily O-glycosylated. The terminal sialic acid linkage type of H1N1S1 and H1N1S2 are covered highly with α2,3-sialic acid. These findings could aid the investigation of the interaction between SARS-CoV-2 and human host cells.

Rapid and High-Sensitive Phosphoproteomics Elucidated the Spatial Dynamics of the Mouse Brain.

Recent developments in phosphoproteomics have enabled signaling studies where over 10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in sample size, reproducibility, and robustness, hampering experiments that involve low-input samples such as rare cells and fine-needle aspiration biopsies. To address these challenges, we introduced a simple and rapid phosphorylation enrichment method (miniPhos) that uses a minimal amount of the sample to get enough information to decipher biological significance. The miniPhos approach completed the sample pretreatment within 4 h and high effectively collected the phosphopeptides in a single-enrichment format with an optimized enrichment process and miniaturized system. This resulted in an average of 22,000 phosphorylation peptides quantified from 100 µg of proteins and even confidently localized over 4500 phosphosites from as little as 10 µg of peptides. Further application was carried out on different layers of mouse brain micro-sections; our miniPhos method provided quantitative information on protein abundance and phosphosite regulation for the most relevant neurodegenerative diseases, cancers, and signaling pathways in the mouse brain. Surprisingly, the phosphoproteome exhibited more spatial variations than the proteome in the mouse brain. Overall, spatial dynamics of phosphosites are integrated with proteins to gain insights into crosstalk of cellular regulation at different layers, thereby facilitating a more comprehensive understanding of mouse brain development and activity.

Multi-comparative Thermal Proteome Profiling Uncovers New O-GlcNAc Proteins in a System-wide Method.

Among diverse protein post-translational modifications, O-GlcNAcylation, a simple but essential monosaccharide modification, plays crucial roles in cellular processes and is closely related to various diseases. Despite its ubiquity in cells, properties of low stoichiometry and reversibility are hard nuts to crack in system-wide research of O-GlcNAc. Herein, we developed a novel method employing multi-comparative thermal proteome profiling for O-GlcNAc transferase (OGT) substrate discovery. Melting curves of proteins under different treatments were profiled and compared with high reproducibility and consistency. Consequently, proteins with significantly shifted stabilities caused by OGT and uridine-5'-diphosphate N-acetylglucosamine were screened out from which new O-GlcNAcylated proteins were uncovered.

pGlycoQuant with a deep residual network for quantitative glycoproteomics at intact glycopeptide level.

Large-scale intact glycopeptide identification has been advanced by software tools. However, tools for quantitative analysis remain lagging behind, which hinders exploring the differential site-specific glycosylation. Here, we report pGlycoQuant, a generic tool for both primary and tandem mass spectrometry-based intact glycopeptide quantitation. pGlycoQuant advances in glycopeptide matching through applying a deep learning model that reduces missing values by 19-89% compared with Byologic, MSFragger-Glyco, Skyline, and Proteome Discoverer, as well as a Match In Run algorithm for more glycopeptide coverage, greatly expanding the quantitative function of several widely used search engines, including pGlyco 2.0, pGlyco3, Byonic and MSFragger-Glyco. Further application of pGlycoQuant to the N-glycoproteomic study in three different metastatic HCC cell lines quantifies 6435 intact N-glycopeptides and, together with in vitro molecular biology experiments, illustrates site 979-core fucosylation of L1CAM as a potential regulator of HCC metastasis. We expected further applications of the freely available pGlycoQuant in glycoproteomic studies.

AMPK induces degradation of the transcriptional repressor PROX1 impairing branched amino acid metabolism and tumourigenesis.

Tumour cell metabolic plasticity is essential for tumour progression and therapeutic responses, yet the underlying mechanisms remain poorly understood. Here, we identify Prospero-related homeobox 1 (PROX1) as a crucial factor for tumour metabolic plasticity. Notably, PROX1 is reduced by glucose starvation or AMP-activated protein kinase (AMPK) activation and is elevated in liver kinase B1 (LKB1)-deficient tumours. Furthermore, the Ser79 phosphorylation of PROX1 by AMPK enhances the recruitment of CUL4-DDB1 ubiquitin ligase to promote PROX1 degradation. Downregulation of PROX1 activates branched-chain amino acids (BCAA) degradation through mediating epigenetic modifications and inhibits mammalian target-of-rapamycin (mTOR) signalling. Importantly, PROX1 deficiency or Ser79 phosphorylation in liver tumour shows therapeutic resistance to metformin. Clinically, the AMPK-PROX1 axis in human cancers is important for patient clinical outcomes. Collectively, our results demonstrate that deficiency of the LKB1-AMPK axis in cancers reactivates PROX1 to sustain intracellular BCAA pools, resulting in enhanced mTOR signalling, and facilitating tumourigenesis and aggressiveness.

All-in-One Strategy for Downstream Molecular Profiling of Tumor-Derived Exosomes.

In light of the significance of exosomes in cancer diagnosis and treatment, it is important to understand the components and functions of exosomes. Herein, an all-in-one strategy has been proposed for comprehensive characterization of exosomal proteins based on nanoporous TiO2 clusters acting as both an extractor for exosome isolation and a nanoreactor for downstream molecular profiling. With the improved hydrophilicity and inherent properties of TiO2, exosomes can be captured by a versatile nanodevice through the specific binding and hydrophilicity interaction synergistically. The strong concerted effect between exosomes and nanodevices ensured high efficiency and specificity of exosome isolation with high recovery and low contaminations. Meanwhile, highly efficient downstream proteomic analysis of the purified exosomes was also enabled by the nanoporous TiO2 clusters. Benefiting from the porous structure of the nanodevice, the lysed exosomal proteins are highly concentrated in the nanopore to achieve high-efficiency in situ proteolytic digestion. Therefore, the unique features of the TiO2 clusters ensured that all the complex steps about isolation and analysis of exosomes were completed efficiently in one simple nanodevice. The concept was first proved with exosomes from cell culture medium, where a high number of identified total proteins and protein groups in exosomes were obtained. Taking advantage of these attractive merits, the first example of the integrated platform has been successfully applied to the analysis of exosomes in complex real-case samples. Not only 196 differential protein biomarker candidates were discovered, but also many more significant cellular components and functions related to gastric cancer were found. These results suggest that the nanoporous TiO2 cluster-based all-in-one strategy can serve as a simple, cost-effective, and integrated platform to facilitate comprehensive analysis of exosomes. Such an approach will provide a valuable tool for the study of exosome markers and their functions.

Nascent Glycoproteome Reveals That N-Linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2.

Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) cell line. Besides, hydrophilic interaction liquid chromatography (HILIC) was adopted for the enrichment of glycosylated peptides. Glycoproteomic analysis revealed the abundance of glycopeptides from LAMP2, NICA, and CEIP2 was significantly changed during NGI-1 treatment. Moreover, the alterations of LAMP2 site-specific intact N-glycopeptides were comprehensively assessed. NGI-1 treatment also led to the inhibition of Cathepsin D maturation and the induction of autophagy. In summary, we provided evidence that NGI-1 repressed the expression of glycosylated LAMP2 accompanied with the occurrence of lysosomal defects and autophagy.

Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake.

Protein fatty acylation regulates numerous cell signaling pathways. Polyunsaturated fatty acids (PUFAs) exert a plethora of physiological effects, including cell signaling regulation, with underlying mechanisms to be fully understood. Herein, we report that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) regulate PI3K-AKT signaling by modifying PDK1 and AKT2. DHA-administered mice exhibit altered phosphorylation of proteins in signaling pathways. Methylene bridge-containing DHA/EPA acylate δ1 carbon of tryptophan 448/543 in PDK1 and tryptophan 414 in AKT2 via free radical pathway, recruit both the proteins to the cytoplasmic membrane, and activate PI3K signaling and glucose uptake in a tryptophan acylation-dependent but insulin-independent manner in cultured cells and in mice. DHA/EPA deplete cytosolic PDK1 and AKT2 and induce insulin resistance. Akt2 knockout in mice abrogates DHA/EPA-induced PI3K-AKT signaling. Our results identify PUFA's methylene bridge tryptophan acylation, a protein fatty acylation that regulates cell signaling and may underlie multifaceted effects of methylene-bridge-containing PUFAs.

Fast Discrimination of Sialylated N-Glycan Linkage Isomers with One-Step Derivatization by Microfluidic Capillary Electrophoresis-Mass Spectrometry.

Linkage isomers (α-2,3- or α-2,6-linkage) of sialylated N-glycans are involved in the emergence and progression of some diseases, so they are of great significance for diagnosing and monitoring diseases. However, the qualitative and quantitative analysis of sialylated N-glycan linkage isomers remains challenging due to their low abundance and limited isomeric separation techniques. Herein, we developed a novel strategy integrating one-step sialic acid derivatization, positive charge-sensitive separation and highly sensitive detection based on microfluidic capillary electrophoresis-mass spectrometry (MCE-MS) for fast and specific analysis of α-2,3- and α-2,6-linked sialylated N-glycan isomers. A kind of easily charged long-chain amino compound was screened first for one-step sialic acid derivatization so that only α-2,3- and α-2,6-linked isomers can be quickly and efficiently separated within 10 min by MCE due to the difference in structural conformation, whose separation mechanism was further theoretically supported by molecular dynamic simulation. In addition, different sialylated N-glycans were separated in order according to the number of sialic acids, so that a migration time-based prediction of the number of sialic acids was achieved. Finally, the sialylated N-glycome of human serum was profiled within 10 min and 6 of the 52 detected sialylated N-glycans could be potential diagnostic biomarkers of cervical cancer (CC), whose α-2,3- and α-2,6-linked isomers were distinguished by α-2,3Neuraminidase S.

Circulating proteomic panels for risk stratification of intracranial aneurysm and its rupture.

The prevalence of intracranial aneurysm (IA) is increasing, and the consequences of its rupture are severe. This study aimed to reveal specific, sensitive, and non-invasive biomarkers for diagnosis and classification of ruptured and unruptured IA, to benefit the development of novel treatment strategies and therapeutics altering the course of the disease. We first assembled an extensive candidate biomarker bank of IA, comprising up to 717 proteins, based on altered proteins discovered in the current tissue and serum proteomic analysis, as well as from previous studies. Mass spectrometry assays for hundreds of biomarkers were efficiently designed using our proposed deep learning-based method, termed DeepPRM. A total of 113 potential markers were further quantitated in serum cohort I (n = 212) & II (n = 32). Combined with a machine-learning-based pipeline, we built two sets of biomarker combinations (P6 & P8) to accurately distinguish IA from healthy controls (accuracy: 87.50%) or classify IA rupture patients (accuracy: 91.67%) upon evaluation in the external validation set (n = 32). This extensive circulating biomarker development study provides valuable knowledge about IA biomarkers.

Nascent Proteome and Glycoproteome Reveal the Inhibition Role of ALG1 in Hepatocellular Carcinoma Cell Migration.

Asparagine-linked glycosylation protein 1 homolog (ALG1) participates in the initial stage of protein N-glycosylation and N-glycosylation has been implicated in the process of hepatocellular carcinoma (HCC) progression. However, whether ALG1 plays a role in human HCC remains unknown. In this study, the expression profile of ALG1 in tumorous and corresponding adjacent non-tumor tissues was analyzed. The relationship of ALG1 expression with clinical features and prognosis of HCC patients was also evaluated using immuno-histochemical method. Here we found ALG1 decreased in HCC tissues compared with adjacent normal liver tissues, which predicted an unfavorable prognosis. Combined with RNA interference, nascent proteome and glycoproteome were determined systematically in Huh7 cell line. Bioinformatics analysis indicated that the differentially expressed proteins participating in the response of ALG1 knockdown were most significantly associated with cell-cell adhesion. Functional studies confirmed that knockdown of ALG1 reduced cell adhesion capacity, and promoted cell migration. Furthermore, down-regulation of H8N2 (on N-glycosite N651) and H5N4S2F1 (on N-glycosite N692) from N-cadherin was identified as a feature of ALG1 knockdown. Our findings revealed that ALG1 controlled the expression of glycosylated N-cadherin and played a role in HCC migration, with implications for prognosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-022-00050-5.

Plasma exosomal proteomic studies of corneal epithelial injury in diabetic and non-diabetic group.

OBJECTIVE: Diabetic Keratopathy (DK) is one of the significant complications of type II diabetes (T2DM) with pathogenesis not yet clarified. Since hyperglycemia is able to change the protein components contained in plasma exosomes, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as feasible to analyze the expression of plasma exosomal proteins in patients with T2DM and non-diabetic patients respectively, find critical biological markers, and explore the mechanism of DK as well as potential therapeutic targets. METHOD: Blood and clinical information of corneal epithelial injury in a diabetic group (the study group) and a non-diabetic group (the control group), who were patients admitted to the Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine from July 2020 to November 2020, were collected. The qEV size exclusion method was adopted to separate exosomes from plasma. The exosomes were then identified through transmission electron microscopy (TEM), nanoparticle tracking analyzer (NTA), and Western blot. The plasma exosomes of the study group and the control group were quantitatively analyzed by proteomics. A bioinformatics method is utilized to screen differential proteins and the expression of the differential proteins was verified by Western blot. RESULT: TEM indicated that the exosomes had a double-concave disc-like appearance, with a size of about 100 nm, and Western blot expressed as CD63 and TSG101. The plasma exosomes of the study group and the control group were analyzed by quantitative proteomics with a total number of 952 proteins detected of which 245 proteins existed in the ExoCarta exosomal protein database. Through adoption of P-value to screen credible differential proteins, the heat map displayed 28 differential proteins, 7 upregulated proteins, and 21 downregulated proteins; the volcano map displayed 7 upregulated proteins and 22 downregulated proteins; the PPI interaction map displayed 12 upregulated proteins and 18 downregulated proteins. Through GO enrichment analysis, it was identified that the differential protein participated in the main biological processes and was involved in regulating the cell's stimulation response to insulin, the insulin receptor signaling pathway, and the activity of glycosylphosphatidylinositol phospholipase D as well as anti-oxidation. The enriched cell components include main components such as exosomes, blood particles, and cytoplasm. KEGG enrichment analysis indicated that the target protein FLOT2 was mainly concentrated in insulin-related signaling pathways. Western blot indicated that the expression of FLOT2 in the study group was lower compared with the control group while the expression of Exo70 was higher. CONCLUSION: Proteomic analysis of the study group and the control group displayed a variety of proteins in plasma exosomes. The downregulated protein FLOT2 in the study group was closely related to the occurrence, development, and complication of DK in T2DM patients. The expression status of plasma FLOT2 protein in T2DM patients is expected to be a biomarker for diagnosing and monitoring of DK.

GproDIA enables data-independent acquisition glycoproteomics with comprehensive statistical control.

Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data independent acquisition (DIA) is an emerging technology with deep proteome coverage and accurate quantitative capability in proteomics studies, but is still in the early stage of development in the field of glycoproteomics. We propose GproDIA, a framework for the proteome-wide characterization of intact glycopeptides from DIA data with comprehensive statistical control by a 2-dimentional false discovery rate approach and a glycoform inference algorithm, enabling accurate identification of intact glycopeptides using wide isolation windows. We further utilize a semi-empirical spectrum prediction strategy to expand the coverage of spectral libraries of glycopeptides. We benchmark our method for N-glycopeptide profiling on DIA data of yeast and human serum samples, demonstrating that DIA with GproDIA outperforms the data-dependent acquisition-based methods for glycoproteomics in terms of capacity and data completeness of identification, as well as accuracy and precision of quantification. We expect that this work can provide a powerful tool for glycoproteomic studies.