Differential expression of N-glycopeptides derived from serum glycoproteins in mild cognitive impairment (MCI) patients.

Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities with the aging of individuals, such as language or visual/spatial comprehension. MCI is considered a prodromal phase of more complicated neurodegenerative diseases such as Alzheimer's. Therefore, accurate diagnosis and better understanding of the disease prognosis will facilitate prevention of neurodegeneration. However, the existing diagnostic methods fail to provide precise and well-timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of the serum N-glycoproteome expression could represent an essential contributor to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using less invasive procedures. In this approach, we identified N-glycopeptides with different expressions between healthy and MCI patients from serum glycoproteins. Seven of the N-glycopeptides showed outstanding AUC values, among them the antithrombin-III Asn224 + 4-5-0-2 with an AUC value of 1.00 and a p value of 0.0004. According to proteomics and ingenuity pathway analysis (IPA), our data is in line with recent publications, and the glycoproteins carrying the identified N-sites play an important role in neurodegeneration.

The role of N-glycosylation in cancer.

Despite advances in understanding the development and progression of cancer in recent years, there remains a lack of comprehensive characterization of the cancer glycoproteome. Glycoproteins play an important role in medicine and are involved in various human disease conditions including cancer. Glycan-moieties participate in fundamental cancer processes like cell signaling, invasion, angiogenesis, and metastasis. Aberrant N-glycosylation significantly impacts cancer processes and targeted therapies in clinic. Therefore, understanding N-glycosylation in a tumor is essential for comprehending disease progression and discovering anti-cancer targets and biomarkers for therapy monitoring and diagnosis. This review presents the fundamental process of protein N-glycosylation and summarizes glycosylation changes in tumor cells, including increased terminal sialylation, N-glycan branching, and core-fucosylation. Also, the role of N-glycosylation in tumor signaling pathways, migration, and metabolism are discussed. Glycoproteins and glycopeptides as potential biomarkers for early detection of cancer based on site specificity have been introduced. Collectively, understanding and exploring the cancer glycoproteome, along with its role in medicine, implication in cancer and other human diseases, highlights the significance of N-glycosylation in tumor processes, necessitating further research for potential anti-cancer targets and biomarkers.

In-Depth Proteome Profiling of Small Extracellular Vesicles Isolated from Cancer Cell Lines and Patient Serum.

Extracellular vesicle (EV) secretion has been observed in many types of both normal and tumor cells. EVs contain a variety of distinctive cargoes, allowing tumor-derived serum proteins in EVs to act as a minimally invasive method for clinical monitoring. We have undertaken a comprehensive study of the protein content of the EVs from several cancer cell lines using direct data-independent analysis. Several thousand proteins were detected, including many classic EV markers such as CD9, CD81, CD63, TSG101, and Syndecan-1, among others. We detected many distinctive cancer-specific proteins, including several known markers used in cancer detection and monitoring. We further studied the protein content of EVs from patient serum for both normal controls and pancreatic cancer and hepatocellular carcinoma. The EVs for these studies have been isolated by various methods for comparison, including ultracentrifugation and CD9 immunoaffinity column. Typically, 500-1000 proteins were identified, where most of them overlapped with the EV proteins identified from the cell lines studied. We were able to identify many of the cell-line EV protein markers in the serum EVs, in addition to the large numbers of proteins specific to pancreatic and HCC cancers.

Evaluation of the effect of dimethyl fumarate on human bone marrow-derived mesenchymal stem cells using bottom-up proteomics.

Mesenchymal stem cells (MSCs) have potential as a viable treatment option in the field of regenerative medicine, but MSC-based therapy needs to be more efficient. Preconditioning is a method to improve MSC-based therapy, and dimethyl fumarate (DMF) - an agent that can enhance the antioxidative capacity of cells - can be considered for preconditioning of MSCs. In this study, we treated bone marrow-derived MSCs with DMF and evaluated their proteome using bottom-up proteomics. The MSCs were exposed to 10 µM DMF for 24 h, followed by lysis with an SDS solution, digestion with trypsin using an s-trap column, and analysis using nanoLC-MS/MS, which identified 2262 proteins with confidence. Bioinformatic analysis of the identified proteins revealed 47 upregulated proteins and 81 downregulated proteins upon DMF treatment. Pathway enrichment analysis suggested a possible decrease in autophagy and a decrease in the activity of the TCA cycle, while indicating a potential increase in proliferation and antioxidant activity in DMF-treated MSCs compared to untreated MSCs. Our findings suggest that DMF can enhance the proliferation of MSCs and increase their stability, and that preconditioning could improve the therapeutic efficacy of MSCs for the treatment of regenerative diseases.

Size-exclusion chromatography for the characterization of urinary extracellular vesicles.

In recent years, extracellular vesicles (EVs) have gained attention for their potential as biomarkers for the early diagnosis and treatment of various diseases. Traditionally, EV isolation has relied exclusively on ultracentrifugation. However, alternative enrichment methods such as size-exclusion chromatography (SEC) and polyethylene glycol-based precipitation have been introduced. This study utilized SEC as a characterization tool to assess the efficiency of EV isolation. Urinary EVs isolated from human urine using centrifugation (40,000 × g) were analyzed using an SEC column with a pore size of 1000 Å, an inner diameter of 7.8 mm, and a length of 300 mm. The EVs were detected sequentially using UV (280 nm) and fluorescence (λex/em = 550 nm/565 nm); the EVs were observed at approximately 6 min, while the proteins were observed at approximately 12 min. The repeated centrifugation enrichment steps resulted in an increase in EV peaks and a decrease in protein peaks. SEC analysis of the enriched EV samples confirmed that a four-cycle repetition of centrifugation is necessary for successful EV enrichment and removal of non-EV proteins from 40 mL of human urine.

Potential applications of mesenchymal stem cells and their derived exosomes in regenerative medicine.

INTRODUCTION: Regenerative medicine involves the replacement of damaged cells, tissues, or organs to restore normal function. Mesenchymal stem cells (MSCs) and exosomes secreted by MSCs have unique advantages that make them a suitable candidate in the field of regenerative medicine. AREAS COVERED: This article provides a comprehensive overview of regenerative medicine, focusing on the use of MSCs and their exosomes as potential therapies for replacing damaged cells, tissues, or organs. This article discusses the distinct advantages of both MSCs and their secreted exosomes, including their immunomodulatory effects, lack of immunogenicity, and recruitment to damaged areas. While both MSCs and exosomes have these advantages, MSCs also have the unique ability to self-renew and differentiate. This article also assesses the current challenges associated with the application of MSCs and their secreted exosomes in therapy. We have reviewed proposed solutions for improving MSC or exosome therapy, including ex-vivo preconditioning strategies, genetic modification, and encapsulation. Literature search was conducted using Google Scholar and PubMed databases. EXPERT OPINION: Providing insight into the future development of MSC and exosome-based therapies and to encourage the scientific community to focus on the identified gaps, develop appropriate guidelines, and enhance the clinical application of these therapies.

Discovery of Core-Fucosylated Glycopeptides as Diagnostic Biomarkers for Early HCC in Patients with NASH Cirrhosis Using LC-HCD-PRM-MS/MS.

Aberrant changes in site-specific core fucosylation (CF) of serum proteins contribute to cancer development and progression, which enables them as potential diagnostic markers of tumors. An optimized data-dependent acquisition (DDA) workflow involving isobaric tags for relative and absolute quantitation-labeling and enrichment of CF peptides by lens culinaris lectin was applied to identify CF of serum proteins in a test set of patients with nonalcoholic steatohepatitis (NASH)-related cirrhosis (N = 16) and hepatocellular carcinoma (HCC, N = 17), respectively. A total of 624 CF peptides from 343 proteins, with 683 CF sites, were identified in our DDA-mass spectrometry (MS) analysis. Subsequently, 19 candidate CF peptide markers were evaluated by a target parallel reaction-monitoring-MS workflow in a validation set of 58 patients, including NASH-related cirrhosis (N = 29), early-stage HCC (N = 21), and late-stage HCC (N = 8). Significant changes (p < 0.01) were observed in four CF peptides between cirrhosis and HCC, where peptide LGSFEGLVn160LTFIHLQHNR from LUM in combination with AFP showed the best diagnostic performance in discriminating HCC from cirrhosis, with an area under curve (AUC) of 0.855 compared to AFP only (AUC = 0.717). This peptide in combination with AFP also significantly improved diagnostic performance in distinguishing early HCC from cirrhosis, with an AUC of 0.839 compared to AFP only (AUC = 0.689). Validation of this novel promising biomarker panel in larger cohorts should be performed.

Mass spectrometry based biomarkers for early detection of HCC using a glycoproteomic approach.

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related mortality worldwide and 80%-90% of HCC develops in patients that have underlying cirrhosis. Better methods of surveillance are needed to increase early detection of HCC and the proportion of patients that can be offered curative therapies. Recent work in novel mass spec-based methods for glycomic and glycopeptide analysis for discovery and confirmation of markers for early detection of HCC versus cirrhosis is reviewed in this chapter. Results from recent work in these fields by several groups and the progress made in developing markers of early HCC which can outperform the current serum-based markers are described and discussed. Also, recent developments in isoform analysis of glycans and glycopeptides and in various mass spec fragmentation methods will be described and discussed.

GlycoSLASH: Concurrent Glycopeptide Identification from Multiple Related LC-MS/MS Data Sets by Using Spectral Clustering and Library Searching.

Liquid chromatography coupled with tandem mass spectrometry is commonly adopted in large-scale glycoproteomic studies involving hundreds of disease and control samples. The software for glycopeptide identification in such data (e.g., the commercial software Byonic) analyzes the individual data set and does not exploit the redundant spectra of glycopeptides presented in the related data sets. Herein, we present a novel concurrent approach for glycopeptide identification in multiple related glycoproteomic data sets by using spectral clustering and spectral library searching. The evaluation on two large-scale glycoproteomic data sets showed that the concurrent approach can identify 105%-224% more spectra as glycopeptides compared to the glycopeptide identification on individual data sets using Byonic alone. The improvement of glycopeptide identification also enabled the discovery of several potential biomarkers of protein glycosylations in hepatocellular carcinoma patients.

Matrix-assisted laser desorption/ionization-Fourier-transform ion cyclotron resonance-mass spectrometry analysis of exosomal lipids from human serum.

RATIONALE: Exosomes contain biomarkers such as proteins and lipids that help in understanding normal physiology and diseases. Lipids, in particular, are infrequently studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for biomarker discovery. In this study, MALDI was equipped with a high-resolution MS to investigate exosomal lipids from human serum. METHODS: Exosomal lipids were profiled using MALDI with Fourier-transform ion cyclotron resonance (FTICR)-MS. Four matrices (i.e., α-cyano-4-hydroxycinnamic acid [CHCA], 2,5-dihydroxybenzoic acid, sinapinic acid, and graphene oxide [GO]) and three sample preparation methods (i.e., dried droplet, thin layer, and two layer) were compared for the number of lipid species detected and the relative abundance of each lipid from human serum and human serum exosomes. RESULTS: In sum, 172 and 89 lipid species were identified from human serum and human serum exosomes, respectively, using all the methods. The highest number of exosome lipid species, 69, was detected using the CHCA matrix, whereas only 8 exosome lipid species were identified using the GO matrix. Among the identified lipid species, phosphatidylcholine was identified most frequently, probably due to the use of a positive ion mode. CONCLUSIONS: Exosomes and human serum showed comparable lipid profiles as determined using MALDI-FTICR-MS. These findings provide a new perspective on exosomal lipidomics analysis and may serve as a foundation for future lipidomics-based biomarker research using MALDI-FTICR-MS.

A guide to mass spectrometric analysis of extracellular vesicle proteins for biomarker discovery.

Exosomes (small extracellular vesicles) in living organisms play an important role in processes such as cell proliferation or intercellular communication. Recently, exosomes have been extensively investigated for biomarker discoveries for various diseases. An important aspect of exosome analysis involves the development of enrichment methods that have been introduced for successful isolation of exosomes. These methods include ultracentrifugation, size exclusion chromatography, polyethylene glycol-based precipitation, immunoaffinity-based enrichment, ultrafiltration, and asymmetric flow field-flow fractionation among others. To confirm the presence of exosomes, various characterization methods have been utilized such as Western blot analysis, atomic force microscopy, electron microscopy, optical methods, zeta potential, visual inspection, and mass spectrometry. Recent advances in high-resolution separations, high-performance mass spectrometry and comprehensive proteome databases have all contributed to the successful analysis of exosomes from patient samples. Herein we review various exosome enrichment methods, characterization methods, and recent trends of exosome investigations using mass spectrometry-based approaches for biomarker discovery.

LC-MS/MS Isomeric Profiling of N-Glycans Derived from Low-Abundant Serum Glycoproteins in Mild Cognitive Impairment Patients.

Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities, such as language or virtual/spatial comprehension. This cognitive decline is mostly observed with the aging of individuals. Recently, MCI has been considered as a prodromal phase of Alzheimer's disease (AD), with a 10-15% conversion rate. However, the existing diagnostic methods fail to provide precise and well-timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of serum N-glycan expression could represent essential contributors to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using non-invasive procedures. Herein, we undertook an LC-MS/MS glycomics approach to determine and characterize potential N-glycan markers in depleted blood serum samples from MCI patients. For the first time, we profiled the isomeric glycome of the low abundant serum glycoproteins extracted from serum samples of control and MCI patients using an LC-MS/MS analytical strategy. Additionally, the MRM validation of the identified data showed five isomeric N-glycans with the ability to discriminate between healthy and MCI patients: the sialylated N-glycans GlcNAc5,Hex6,Neu5Ac3 and GlcNAc6,Hex7,Neu5Ac4 with single AUCs of 0.92 and 0.87, respectively, and a combined AUC of 0.96; and the sialylated-fucosylated N-glycans GlcNAc4,Hex5,Fuc,Neu5Ac, GlcNAc5,Hex6,Fuc,Neu5Ac2, and GlcNAc6,Hex7,Fuc,Neu5Ac3 with single AUCs of 0.94, 0.67, and 0.88, respectively, and a combined AUC of 0.98. According to the ingenuity pathway analysis (IPA) and in line with recent publications, the identified N-glycans may play an important role in neuroinflammation. It is a process that plays a fundamental role in neuroinflammation, an important process in the progression of neurodegenerative diseases.

Sequential Method for Analysis of CTCs and Exosomes from the Same Sample of Patient Blood.

Circulating tumor cells (CTCs) and exosomes, both released from the primary tumor into peripheral blood, are a promising source of cancer biomarkers. They are detectable in the blood and carry a large diversity of biological molecules, which can be used for the diagnosis and monitoring of minimally invasive cancers. However, due to their intrinsic differences in counts, size, and molecular contents, studies have focused on only one type of vesicle. Herein, we have developed an integrated system to sequentially isolate CTCs and exosomes from a single patient blood sample for further profiling and analysis. The CTCs are isolated using a commercial filtration method and then the remaining blood is processed using multiple cycles of ultracentrifugation to isolate the exosomes. The method uses two available technologies where the eluent from CTC isolation is usually discarded and interfaces them, so that the eluent can be interfaced to exosome isolation methods. The CTCs are identified based on fluorescence staining of their surface markers, while the exosomes are analyzed using transmission electron microscopy, nanosight tracking analysis, and mass spec proteomic analysis. This analysis showed CTCs detected by their surface markers for metastatic hepatocellular carcinoma (HCC), while essentially none were detected for cirrhosis. The exosome analysis resulted in the identification of ∼500-1000 exosome proteins per sample confirmed by detection of exosome surface markers CD9, CD63, CD81, and TSG101 in addition to proteins related to cancer progression. Proteins enriched in HCC exosomes were shown to be involved in the immune response, metastasis, and proliferation.

Glycopeptides with Sialyl Lewis Antigen in Serum Haptoglobin as Candidate Biomarkers for Nonalcoholic Steatohepatitis Hepatocellular Carcinoma Using a Higher-Energy Collision-Induced Dissociation Parallel Reaction Monitoring-Mass Spectrometry Method.

Nonalcoholic steatohepatitis (NASH) is the fastest growing cause of hepatocellular carcinoma (HCC) in the United States. Changes in N-glycosylation on specific glycosites of serum proteins have been investigated as potential markers for the early detection of NASH-related HCC. Herein, we report a glycopeptide with a Sialyl Lewis structure derived from serum haptoglobin (Hp) as a potential marker for NASH related HCCs among 95 patients with NASH, including 46 cirrhosis, 32 early-stage HCC, and 17 late-stage HCC. Hp immuno-isolated from patient serum was analyzed using LC-HCD-PRM-MS/MS followed by data analysis via Skyline software. Two glycopeptides involving site N184 and four glycopeptides involving site N241 were significantly changed in patients with HCC vs NASH cirrhosis (P < 0.05). The two-marker panel using N-glycopeptide N241_A4G4F2S4 showed the best performance for HCC detection when combined with α-fetoprotein (AFP), with an improved estimated area under the curve (AUC) = 0.898 (95% CI: 0.835, 0.951), compared to the AUC of 0.790(95% CI, 0.697 0.872) using AFP alone (P = 0.048). At 90% specificity, the combination of N241_A4G4F2S4 + AFP had an improved sensitivity of 63.3%, compared to the sensitivity of 52.3% using AFP alone. When using three markers, the panel of AFP + N241_A2G2F1S2 + N241_A4G4F2S4 yielded an estimated AUC of 0.928 (95% CI: 0.877, 0.970). Our findings indicated that N241_A4G4F2S4 may play an important role in distinguishing HCC from NASH cirrhosis.

A Fucosylated Glycopeptide as a Candidate Biomarker for Early Diagnosis of NASH Hepatocellular Carcinoma Using a Stepped HCD Method and PRM Evaluation.

Aberrant specific N-glycosylation, especially the increase in fucosylation on specific peptide sites of serum proteins have been investigated as potential markers for diagnosis of nonalcoholic steatohepatitis (NASH)-related HCC. We have combined a workflow involving broad scale marker discovery in serum followed by targeted marker evaluation of these fucosylated glycopeptides. This workflow involved an LC-Stepped HCD-DDA-MS/MS method coupled with offline peptide fractionation for large-scale identification of N-glycopeptides directly from pooled serum samples (each n=10) as well as differential determination of N-glycosylation changes between disease states. We then evaluated the fucosylation level of the glycoprotein ceruloplasmin among 62 patient samples (35 cirrhosis, 27 early-stage NASH HCC) by LC-Stepped HCD-PRM-MS/MS to quantitatively analyze 18 targeted glycopeptides. Of these targets, we found the ratio of fucosylation of a tri-antennary glycopeptide from site N762, involving N762_ HexNAc(5)Hex(6)Fuc(2)NeuAc(3) (P=0.0486), increased significantly from cirrhosis to early HCC. This fucosylation ratio of a tri-antennary glycopeptide in CERU could be a potential biomarker for further validation in a larger sample set and could be a promising candidate for early detection of NASH HCC.

Large Scale Screening and Quantitative Analysis of Site-Specific N-Glycopeptides from Human Serum in Early Alzheimer's Disease Using LC-HCD-PRM-MS.

Glycopeptide analysis by mass spectrometry may provide an important opportunity in discovery of biomarkers to aid in early detection of Alzheimer's Disease (AD). In this work, we have used a NanoLC-Stepped-HCD-DDA-MS/MS platform and a NanoLC-Stepped-HCD-PRM-MS platform for large-scale screening and quantification of novel N-glycopeptide biomarkers for early detection of AD in patient serum. N-glycopeptides were retrieved from 10 µL of serum in patients with mild cognitive impairment (MCI, a prodromal phase of AD) and normal controls, respectively, after trypsin digestion, glycopeptide enrichment, fractionation, and NanoLC-Stepped-HCD-DDA-MS/MS or NanoLC-Stepped-HCD-PRM-MS analysis. Using a combination of Byonic, Byologic and Skyline softwares, we were able to accomplish both identification and label-free quantitation of site-specific N-glycopeptides between MCI and normal controls. Differential quantitation analysis by Byologic showed that 29 N-glycopeptides derived from 16 glycoproteins were significantly changed in MCI compared to normal controls. Further, HCD-PRM-MS quantitative analysis of the selected N-glycopeptide candidates confirmed that EHEGAIYPDN138TTDFQR_HexNAc(4)Hex(5)-Fuc(2)NeuAc(1) from CERU, and VCQDCPLLAPLN156DTR_HexNAc(4)Hex(5)NeuAc(2) from AHSG can significantly discriminate MCI from normal controls. These two glycopeptides had the area under the receiver operating characteristic curve (AUC) of 0.850 (95% CI, 0.66-1.0) and 0.867 (95% CI, 0.68-1.0), respectively (p<0.05). The result demonstrates that changes in the expression level of the N-glycopeptides provide potential serum biomarkers for detection of AD at a very early stage.

Column-based Technology for CD9-HPLC Immunoaffinity Isolation of Serum Extracellular Vesicles.

Serum-derived extracellular vesicles (EVs) are a promising source of biomarkers; however, major challenges in EV separation and proteomic profiling remain for isolating EVs from a small amount, that is, on the microliter scale, of human serum while minimizing the contamination of blood proteins and lipoprotein particles coeluting in EV preparations. Herein we have developed a column-based CD9-antibody-immobilized high-performance liquid chromatography immunoaffinity chromatography(CD9-HPLC-IAC) technology for EV isolation from a microliter scale of serum for downstream proteomic analysis. The CD9-HPLC-IAC method achieved EV isolation from 40 µL of serum in 30 min with a yield of 8.0 × 109 EVs, where EVs were further processed with a postcolumn cleaning step using the 50 kDa molecular weight cut-off filter for the buffer exchange, concentration, and reduction of potentially coeluting serum proteins. In total, 482 proteins were identified in EVs by using liquid chromatography tandem mass spectrometry, including the common exosomal markers such as CD63, CD81, CD82, Alix, and TSG101. The statistical analysis of EV protein content showed that the top 10 serum proteins in EVs were significantly decreased by using the CD9-HPLC-IAC method compared with the use of ultracentrifugation (p = 0.001) and size exclusion chromatography (p = 0.009), and apolipoproteins were significantly reduced 4.8-fold compared with the SEC method (p < 0.001). The result demonstrates the potential of the CD9-HPLC-IAC method for the efficient isolation and proteomic characterization of EVs from a microscale volume of serum.

PRM-MS Quantitative Analysis of Isomeric N-Glycopeptides Derived from Human Serum Haptoglobin of Patients with Cirrhosis and Hepatocellular Carcinoma.

Currently, surveillance strategies have inadequate performance for cirrhosis and early detection of hepatocellular carcinoma (HCC). The glycosylation of serum haptoglobin has shown to have significant differences between cirrhosis and HCC, thus can be used for diagnosis. We performed a comprehensive liquid chromatography-parallel reaction monitoring-mass spectrometry (LC-PRM-MS) approach, where a targeted parallel reaction monitoring (PRM) strategy was coupled to a powerful LC system, to study the site-specific isomerism of haptoglobin (Hp) extracted from cirrhosis and HCC patients. We found that our strategy was able to identify a large number of isomeric N-glycopeptides, mainly located in the Hp glycosylation site Asn207. Four N-glycopeptides were found to have significant changes in abundance between cirrhosis and HCC samples (p < 0.05). Strategic combinations of the significant N-glycopeptides, either with alpha-fetoprotein (AFP) or themselves, better estimate the areas under the curve (AUC) of their respective receiver operating characteristic (ROC) curves with respect to AFP. The combination of AFP with the isomeric sialylated fucosylated N-glycopeptides Asn207 + 5-6-1-2 and Asn207 + 5-6-1-3, resulted with an AUC value of 0.98, while the AUC value for AFP alone was 0.85. When comparing cirrhosis vs. early HCC, the isomeric N-glycopeptide Asn207 + 5-6-0-1 better estimated AUC with respect to AFP (AUCAFP = 0.81, and AUCAsn207 + 5-6-0-1 = 0.88, respectively).

Label-free quantitative proteomic analysis of serum extracellular vesicles differentiating patients of alcoholic and nonalcoholic fatty liver diseases.

Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are typically asymptomatic and slow-progressing but potentially fatal diseases that are common causes of liver cirrhosis and related complications. Exosomes are nano-sized extracellular vesicles that have been linked to various intercellular communication processes and can carry biological materials reflecting the state and severity of disease. In this study, shotgun proteomic analysis of the protein expression profiles of extracellular vesicles, including exosomes and microvesicles, enriched from human serum samples of 24 patients diagnosed with various fatty liver diseases was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS) followed by protein identification and label-free quantification using the MaxQuant platform. A total of 329 proteins, including 190 previously reported exosome-specific proteins, were identified from four types of liver disease, where significant differences in protein expression were found in apolipoproteins, immunoglobulins, and other previously reported markers of liver disease. Principal component analysis of 61 proteins identified from MaxQuant analysis of the LC-MS/MS data provided a confident differentiation between ALD and NAFLD. SIGNIFICANCE: The current investigation revealed the difference among various types of liver disease using LC-MS/MS of exosomes enriched from human serum samples of 24 patients where the most significantly up-regulation proteins were alpha-2-macroglobulin for alcoholic hepatitis and apolipoprotein C3 for nonalcoholic fatty liver disease.