ABSTRACT
Lung cancer is a severe disease for which better diagnostic and therapeutic approaches are urgently needed. Increasing evidence implies that aberrant protein glycosylation plays a crucial role in the pathogenesis and progression of lung cancer. Differences in glycosylation patterns have been previously observed between healthy and cancerous samples as well as between different lung cancer subtypes, which suggests untapped diagnostic potential. In addition, understanding the changes mediated by glycosylation may shed light on possible novel therapeutic targets and personalized treatment strategies for lung cancer patients. Mass spectrometry based glycomics and glycoproteomics have emerged as powerful tools for in-depth characterization of changes in protein glycosylation, providing valuable insights into the molecular basis of lung cancer. This paper reviews the literature on the analysis of protein glycosylation in lung cancer using mass spectrometry, which is dominated by manuscripts published over the past 5 years. Studies analyzing N-glycosylation, O-glycosylation, and glycosaminoglycan patterns in tissue, serum, plasma, and rare biological samples of lung cancer patients are highlighted. The current knowledge on the potential utility of glycan and glycoprotein biomarkers is also discussed.
ABSTRACT
The technical difficulty of separating extracellular vesicles (EVs) from plasma proteins in human blood presents a significant hurdle in EV research, particularly during nano ultra-high-performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) analysis, where detecting "vesicular" proteins among abundant plasma proteins is challenging. Standardisation is a pressing issue in EV research, prompting collaborative global efforts to address it. While the MISEV guidelines offer valuable recommendations, unanswered questions remain, particularly regarding sample storage. We compared size exclusion chromatography (SEC) columns with pore sizes of 35 nm and 70 nm to identify fractions with minimal contaminating proteins and the highest concentration of small EVs (sEVs). Following column selection, we explored potential differences in the quality and quantity of sEVs isolated from platelet-free plasma (PFP) after long-term storage at -80 °C (>2.5 years) compared to freshly drawn blood. Our methodologically rigorous study indicates that prolonged storage, under correct storage and processing conditions, does not compromise sEV quality. Both columns effectively isolated vesicles, with the 70 nm column exhibiting a higher abundance of "vesicular" proteins. We propose a relatively rapid and moderately efficient protocol for obtaining a comparatively pure sEV fraction from plasma, facilitating sEV processing in clinical trials.
ABSTRACT
Heparan sulphates (HSs), a specific class of glycosaminoglycans (GAGs), are important participants of cellular signalling. Analytical characterization of GAGs requires a complex sample preparation workflow. Although a detailed stability and recovery study is available for the chondroitin sulphate GAG class, the literature concerning HS is incomplete in this regard. Therefore, our aim was to systematically investigate various parameters that could potentially influence the stability and recovery of HS samples when performing disaccharide analysis using high-performance liquid chromatography-mass spectrometry. First, effects concerning vacuum evaporation and freezing were investigated. Next, the storage stability of the HS disaccharides was analysed under several conditions such as temperature, pH, digestion buffers, injection solvents and storage vessels. We have identified several critical parameters influencing the stability and recovery of HS disaccharides. We concluded that major sample loss is expected when Tris-HCl is used as digestion buffer, followed by vacuum evaporation at elevated temperatures, or samples are stored under alkaline conditions. Following the practical considerations of this paper can contribute to increasing the reliability of future analytical measurements.
ABSTRACT
The release of extracellular vesicles (EVs) is increased under cellular stress and cardiomyocyte damaging conditions. However, whether the cardiomyocyte-derived EVs eventually reach the systemic circulation and whether their number in the bloodstream reflects cardiac injury, remains unknown. Wild type C57B/6 and conditional transgenic mice expressing green fluorescent protein (GFP) by cardiomyocytes were studied in lipopolysaccharide (LPS)-induced systemic inflammatory response syndrome (SIRS). EVs were separated both from platelet-free plasma and from the conditioned medium of isolated cardiomyocytes of the left ventricular wall. Size distribution and concentration of the released particles were determined by Nanoparticle Tracking Analysis. The presence of GFP + cardiomyocyte-derived circulating EVs was monitored by flow cytometry and cardiac function was assessed by echocardiography. In LPS-treated mice, systemic inflammation and the consequent cardiomyopathy were verified by elevated plasma levels of TNFα, GDF-15, and cardiac troponin I, and by a decrease in the ejection fraction. Furthermore, we demonstrated elevated levels of circulating small- and medium-sized EVs in the LPS-injected mice. Importantly, we detected GFP+ cardiomyocyte-derived EVs in the circulation of control mice, and the number of these circulating GFP+ vesicles increased significantly upon intraperitoneal LPS administration (P = 0.029). The cardiomyocyte-derived GFP+ EVs were also positive for intravesicular troponin I (cTnI) and muscle-associated glycogen phosphorylase (PYGM). This is the first direct demonstration that cardiomyocyte-derived EVs are present in the circulation and that the increased number of cardiac-derived EVs in the blood reflects cardiac injury in LPS-induced systemic inflammation (SIRS).
Subject(s)
Cell Movement , Extracellular Vesicles/metabolism , Myocardium/pathology , Myocytes, Cardiac/pathology , Systemic Inflammatory Response Syndrome/pathology , Animals , Cell Movement/drug effects , Clusterin/metabolism , Extracellular Vesicles/drug effects , Glycogen Phosphorylase/metabolism , Green Fluorescent Proteins/metabolism , Integrases/metabolism , Lipopolysaccharides , Male , Mice, Inbred C57BL , Mice, Transgenic , Myocardium/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Organ Specificity/drug effects , Phenotype , Systemic Inflammatory Response Syndrome/blood , Systemic Inflammatory Response Syndrome/physiopathology , Tamoxifen/pharmacology , Troponin I/metabolismABSTRACT
Lung cancer is one of the most commonly diagnosed cancer types. Studying the molecular changes that occur in lung cancer is important to understand tumor formation and identify new therapeutic targets and early markers of the disease to decrease mortality. Glycosaminoglycan chains play important roles in various signaling events in the tumor microenvironment. Therefore, we have determined the quantity and sulfation characteristics of chondroitin sulfate and heparan sulfate in formalin-fixed paraffin-embedded human lung tissue samples belonging to different lung cancer types as well as tumor adjacent normal areas. Glycosaminoglycan disaccharide analysis was performed using HPLC-MS following on-surface lyase digestion. Significant changes were identified predominantly in the case of chondroitin sulfate; for example, the total amount was higher in tumor tissue compared to the adjacent normal tissue. We also observed differences in the degree of sulfation and relative proportions of individual chondroitin sulfate disaccharides between lung cancer types and adjacent normal tissue. Furthermore, the differences in the 6-O-/4-O-sulfation ratio of chondroitin sulfate were different between the lung cancer types. Our pilot study revealed that further investigation of the role of chondroitin sulfate chains and enzymes involved in their biosynthesis is an important aspect of lung cancer research.
Subject(s)
Glycosaminoglycans , Lung Neoplasms , Humans , Chondroitin Sulfates , Pilot Projects , Heparitin Sulfate , Disaccharides , Tumor MicroenvironmentABSTRACT
Tumors are intricate ecosystems where cancer cells and non-malignant stromal cells, including cancer-associated fibroblasts (CAFs), engage in complex communication. In this study, we investigated the interaction between poorly (HLE) and well-differentiated (HuH7) hepatoma cells and LX2 fibroblasts. We explored various communication channels, including soluble factors, metabolites, extracellular vesicles (EVs), and miRNAs. Co-culture with HLE cells induced LX2 to produce higher levels of laminin ß1, type IV collagen, and CD44, with pronounced syndecan-1 shedding. Conversely, in HuH7/LX2 co-culture, fibronectin, thrombospondin-1, type IV collagen, and cell surface syndecan-1 were dominant matrix components. Integrins α6ß4 and α6ß1 were upregulated in HLE, while α5ß1 and αVß1 were increased in HuH7. HLE-stimulated LX2 produced excess MMP-2 and 9, whereas HuH7-stimulated LX2 produced excess MMP-1. LX2 activated MAPK and Wnt signaling in hepatoma cells, and conversely, hepatoma-derived EVs upregulated MAPK and Wnt in LX2 cells. LX2-derived EVs induced over tenfold upregulation of SPOCK1/testican-1 in hepatoma EV cargo. We also identified liver cancer-specific miRNAs in hepatoma EVs, with potential implications for early diagnosis. In summary, our study reveals tumor type-dependent communication between hepatoma cells and fibroblasts, shedding light on potential implications for tumor progression. However, the clinical relevance of liver cancer-specific miRNAs requires further investigation.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/genetics , Syndecan-1 , Collagen Type IV , Ecosystem , Liver Neoplasms/genetics , Fibroblasts , Communication , ProteoglycansABSTRACT
Pulmonary adenocarcinomas (pADCs) with an ALK rearrangement are a rare cancer subtype, necessitating comprehensive molecular investigations to unravel their heterogeneity and improve therapeutic strategies. In this pilot study, we employed spatial transcriptomic (NanoString GeoMx) and proteomic profiling to investigate seven treatment-naïve pADCs with an ALK rearrangement. On each FFPE tumor slide, 12 smaller and 2-6 larger histopathologically annotated regions were selected for transcriptomic and proteomic analysis, respectively. The correlation between proteomics and transcriptomics was modest (average Pearson's r = 0.43 at the gene level). Intertumoral heterogeneity was more pronounced than intratumoral heterogeneity, and normal adjacent tissue exhibited distinct molecular characteristics. We identified potential markers and dysregulated pathways associated with tumors, with a varying extent of immune infiltration, as well as with mucin and stroma content. Notably, some markers appeared to be specific to the ALK-driven subset of pADCs. Our data showed that within tumors, elements of the extracellular matrix, including FN1, exhibited substantial variability. Additionally, we mapped the co-localization patterns of tumor microenvironment elements. This study represents the first spatially resolved profiling of ALK-driven pADCs at both the gene and protein expression levels. Our findings may contribute to a better understanding of this cancer type prior to treatment with ALK inhibitors.
Subject(s)
Adenocarcinoma of Lung , Adenocarcinoma , Lung Neoplasms , Humans , Anaplastic Lymphoma Kinase/genetics , Anaplastic Lymphoma Kinase/metabolism , Lung Neoplasms/pathology , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Adenocarcinoma/pathology , Transcriptome , Pilot Projects , Proteomics , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/pathology , Gene Rearrangement , Tumor Microenvironment/geneticsABSTRACT
Chronic liver diseases have both high incidence and mortality rates; therefore, a deeper understanding of the underlying molecular mechanisms is essential. We have determined the content and sulfation pattern of chondroitin sulfate (CS) and heparan sulfate (HS) in human hepatocellular carcinoma and cirrhotic liver tissues, considering the etiology of the diseases. A variety of pathological conditions such as alcoholic liver disease, hepatitis B and C virus infections, and primary sclerosing cholangitis were studied. Major differences were observed in the total abundance and sulfation pattern of CS and HS chains. For example, the 6-O-sulfation of CS is fundamentally different regarding etiologies of cirrhosis, and a 2-threefold increase in HS N-sulfation/O-sulfation ratio was observed in hepatocellular carcinoma compared to cirrhotic tissues.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Chondroitin Sulfates , Glycosaminoglycans/metabolism , Heparitin Sulfate , Humans , Liver Cirrhosis , Pilot ProjectsABSTRACT
Cell surface proteins, including transmembrane and other surface-anchored proteins, play a key role in several critical cellular processes and have a strong diagnostic value. The development of quick and robust experimental methods remains vital for the accurate and comprehensive characterization of the cell surface subproteome of individual cells. Here we present a high-throughput technique which relies on the biotinylation of the accessible primary amino groups in the extracellular segments of the proteins, using HL60 as a model cell line. Several steps of the method have been thoroughly optimized to capture labeled surface proteins selectively and in larger quantities. These include the following: improving the efficiency of the cell surface biotinylation; reducing the endogen protease activity; applying an optimal amount of affinity column and elution steps for labeled peptide enrichment; and examining the effect of various solid-phase extraction methods, different HPLC gradients, and various tandem mass spectrometry settings. Using the optimized workflow, we identified at least 1700 surface-associated individual labeled peptides (~6000-7000 redundant peptides) from the model cell surface in a single nanoHPLC-MS/MS run. The presented method can provide a comprehensive and specific list of the cell surface available protein segments that could be potential targets in various bioinformatics and molecular biology research.
Subject(s)
Membrane Proteins , Tandem Mass Spectrometry , Biotinylation , Membrane Proteins/metabolism , Tandem Mass Spectrometry/methods , Peptides/chemistry , Cell Membrane/metabolismABSTRACT
The optimization of solid-phase extraction (SPE) purification and chromatographic separation is usually neglected during proteomics studies. However, the effects on detection performance are not negligible, especially when working with highly glycosylated samples. We performed a comparative study of different SPE setups, including an in-house optimized method and reversed-phase chromatographic gradients for the analysis of highly glycosylated plasma fractions as a model sample for glycopeptide analysis. The in-house-developed SPE method outperformed the graphite-based and hydrophilic interaction liquid chromatography (HILIC) purification methods in detection performance, recovery, and repeatability. During optimization of the chromatography, peak distribution was maximized to increase the peptide detection rate. As a result, we present sample purification and chromatographic separation methods optimized for the analysis of hydrophilic samples, the most important of which is heavily N-glycosylated protein mixtures.
Subject(s)
Graphite , Chromatography, Liquid/methods , Glycopeptides/chemistry , Hydrophobic and Hydrophilic Interactions , Peptides , Solid Phase Extraction/methodsABSTRACT
Chondroitin sulfate (CS) is a widely studied class of glycosaminoglycans, responsible for diverse biological functions. Structural analysis of CS is generally based on disaccharide analysis. Sample preparation is a key analytical issue in this case. However, a detailed study on the stability and recovery of CS-derived species has been lacking so far. We have found that for solvent exchange, in general, vacuum evaporation (SpeedVac) is much preferable than lyophilization. Moreover, in the case of aqueous solutions, higher recovery was experienced than in solutions with high organic solvent content. Storage of the resulting disaccharide mixture in typical HPLC injection solvents is also critical; decomposition starts after 12 h at 4 °C; therefore, the mixtures should not be kept in the sample tray of an automatic injector for a long time. The study, therefore, lays down suggestions on proper sample preparation and measurement conditions for biologically derived chondroitin sulfate species.
Subject(s)
Chemistry Techniques, Analytical , Chondroitin Sulfates/chemistry , Disaccharides/chemistry , Glycosaminoglycans/chemistry , Acetonitriles/chemistry , Chromatography, High Pressure Liquid , Chromatography, Liquid , Freeze Drying , Mass Spectrometry , Methanol/chemistry , Organic Chemicals , Solvents/chemistryABSTRACT
Rigorous validation of amino acid sequence is fundamental in the characterization of original and biosimilar protein biopharmaceuticals. Widely accepted workflows are based on bottom-up mass spectrometry, and they often require multiple techniques and significant manual work. Here, we demonstrate that optimization of a set of tandem mass spectroscopy (MS/MS) collision energies and automated combination of all available information in the measurements can increase the sequence validated by one technique close to the inherent limits. We created a software (called "Serac") that consumes results of the Mascot database search engine and identifies the amino acids validated by bottom-up MS/MS experiments using the most rigorous, industrially acceptable definition of sequence coverage (we term this "confirmed sequence coverage"). The software can combine spectra at the level of amino acids or fragment ions to exploit complementarity, provides full transparency to justify validation, and reduces manual effort. With its help, we investigated collision energy dependence of confirmed sequence coverage of individual peptides and full proteins on trypsin-digested monoclonal antibody samples (rituximab and trastuzumab). We found the energy dependence to be modest, but we demonstrated the benefit of using spectra taken at multiple energies. We describe a workflow based on 2-3 LC-MS/MS runs, carefully selected collision energies, and a fragment ion level combination, which yields â¼85% confirmed sequence coverage, 25%-30% above that from a basic proteomics protocol. Further increase can mainly be expected from alternative digestion enzymes or fragmentation techniques, which can be seamlessly integrated to the processing, thereby allowing effortless validation of full sequences.
Subject(s)
Rituximab/analysis , Rituximab/chemistry , Sequence Analysis, Protein/methods , Trastuzumab/analysis , Trastuzumab/chemistry , Amino Acid Sequence , Biosimilar Pharmaceuticals/analysis , Biosimilar Pharmaceuticals/chemistry , Chromatography, Liquid , Computational Biology , Peptides/analysis , Peptides/chemistry , Proteolysis , Software , Tandem Mass Spectrometry/methods , Trypsin/chemistryABSTRACT
The cellular vesicle is a fluid-filled structure separated from the surrounding environment by a biological membrane. Here, we isolated nanovesicles (NVs) from the juice of clementines using a discontinuous density gradient ultracentrifugation method. To gain information about the protein content of vesicles, mass spectrometry-based organelle proteomics and bioinformatics were applied to the exosome-like vesicle fraction isolated in the 1 mol/L sucrose/D2O cushion. Analysis of 1018 identified proteins revealed a highly complex mixture of different intra, extracellular and artificially-formed vesicle populations. In particular, clathrin-coated vesicles were significantly expressed in this sample. Membrane transporters are significantly represented in clementines nanovesicles. We have found 162 proteins associated with the transport Gene Ontology term (GO: 0006810) which includes; 71 transmembrane transport related, 53 vesicle mediated and 50 intracellular transporters. Platellin-3 like carrier protein containing a Sec14 domain is known to have a role in plant-virus interaction and that is one of the most abundant proteins in our dataset. The presence of transmembrane transporters like ATPases, aquaporins, ATP Binding Cassette (ABC) transporters and tetraspanins, regulators of protein trafficking suggests that nanovesicles of clementines can actively interact with their environment in a controlled way.
Subject(s)
Citrus , Extracellular Vesicles , Fruit and Vegetable Juices , Membrane Transport Proteins , Nanoparticles/chemistry , Plant Proteins , Citrus/genetics , Citrus/metabolism , Extracellular Vesicles/chemistry , Extracellular Vesicles/genetics , Extracellular Vesicles/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
Regulatory T cells (Treg) are mandatory elements in the maintenance of human pregnancy, but their de novo differentiation has not been completely exposed. HSPE1 chaperone expressing trophoblast cells may have a role in it. Trophoblast-derived extracellular vesicles (EVs), either at the feto-maternal interface or in circulation, target CD4+ T cells. We hypothesized that HSPE1-associated trophoblastic cell line (BeWo)-derived EVs are active mediators of Treg cell differentiation. We proved at first that recombinant HSPE1 promote human Treg cell differentiation in vitro. Developing a CRISPR-Cas9 based HSPE1 knockout BeWo cell line we could also demonstrate, that EV-associated HSPE1 induces Treg development. Next-generation sequencing of miRNA cargo of BeWo-EVs characterized the regulatory processes of Treg polarization. By the use of single-cell transcriptomics analysis, seven Treg cell subtypes were distinguished and we demonstrated for the first time that the expression level of HSPE1 was Treg subtype dependent, and CAPG expression is characteristic to memory phenotype of T cells. Our data indicate that HSPE1 and CAPG may be used as markers for identification of Treg subtypes. Our results suggest, that trophoblastic-derived iEVs-associated HSPE1 and miRNA cargo have an important role in Treg cell expansion in vitro and HSPE1 is a useful marker of Treg subtype characterization.
Subject(s)
Cell Communication , Cell Differentiation , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism , Trophoblasts/metabolism , Cell Proliferation , Extracellular Vesicles/metabolism , Gene Expression Profiling , Gene Expression Regulation , Humans , Proteome , Proteomics/methods , T-Lymphocytes, Regulatory/immunology , TranscriptomeABSTRACT
Collision energy is a key parameter determining the information content of beam-type collision induced dissociation tandem mass spectrometry (MS/MS) spectra, and its optimal choice largely affects successful peptide and protein identification in MS-based proteomics. For an MS/MS spectrum, quality of peptide match based on sequence database search, often characterized in terms of a single score, is a complex function of spectrum characteristics, and its collision energy dependence has remained largely unexplored. We carried out electrospray ionization-quadrupole-time of flight (ESI-Q-TOF)-MS/MS measurements on 2807 peptides from tryptic digests of HeLa and E. coli at 21 different collision energies. Agglomerative clustering of the resulting Mascot score versus energy curves revealed that only few of them display a single, well-defined maximum; rather, they feature either a broad plateau or two clear peaks. Nonlinear least-squares fitting of one or two Gaussian functions allowed the characteristic energies to be determined. We found that the double peaks and the plateaus in Mascot score can be associated with the different energy dependence of b- and y-type fragment ion intensities. We determined that the energies for optimum Mascot scores follow separate linear trends for the unimodal and bimodal cases with rather large residual variance even after differences in proton mobility are taken into account. This leaves room for experiment optimization and points to the possible influence of further factors beyond m/ z.
Subject(s)
Peptide Fragments/analysis , Proteomics/methods , Tandem Mass Spectrometry/methods , Amino Acid Sequence , Escherichia coli Proteins , HeLa Cells , Humans , Normal Distribution , Spectrometry, Mass, Electrospray Ionization , Trypsin/metabolismABSTRACT
A straightforward approach has been developed to distinguish core and antenna fucosylation in glycopeptides. The method does not require derivatization and can be easily adapted into a proteomics workflow. The key aspect is to use low collision energy collision-induced dissociation (CID) (on a quadrupole time-of-flight type instrument) when only single-step fragmentation processes occur. Low collision energy should show the precursor ion as the largest peak in the spectrum; the survival yield should be ideally over 50%, and this is obtained at a collision energy ca. 30% of that typically used for proteomics. In such a case, interfering processes like fucose migration or consecutive reactions are minimized. Core and antenna fucosylation can be discriminated using various ion abundance ratios. Low-energy CID spectra are very "clean" (no chemical noise), and the ions used for locating the fucose are among the major peaks, making the method well-suited for analytical work. Monitoring the change in the proportion of core and antenna fucosylation at the same glycosylation site is also feasible.
Subject(s)
Fucose/analysis , Glycopeptides/analysis , Orosomucoid/analysis , Prostate-Specific Antigen/analysis , Fucose/chemistry , Glycopeptides/chemistry , Glycosylation , Humans , Molecular Structure , Orosomucoid/chemistry , Prostate-Specific Antigen/chemistry , Proteomics , Tandem Mass Spectrometry/methodsABSTRACT
AIMS: The prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths. In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer. In addition, we also studied blood plasma and pancreatic cancer cell line-derived EVs. METHODS: Comparative proteomic analysis was performed of 102â¯EV preparations from human pancreatic juices, blood, and pancreatic cancer cell lines Capan-1 and MIA PaCa-2. EV preparations were also characterized by electron microscopy, tunable resistive pulse sensing, and flow cytometry. RESULTS: Here we describe the presence of EVs in human pancreatic juice samples. Pancreatic juice EV-associated proteins that we identified as possible candidate markers for pancreatic cancer included mucins, such as MUC1, MUC4, MUC5AC, MUC6 and MUC16, CFTR, and MDR1 proteins. These candidate biomarkers could also be detected by flow cytometry in EVs found in pancreatic juice and those secreted by pancreatic cancer cell lines. CONCLUSIONS: Together our data show that detection and characterization of EVs directly in pancreatic juice is feasible and may prove to be a valuable source of potential biomarkers of pancreatic cancer.
Subject(s)
Adenocarcinoma/diagnosis , Biomarkers, Tumor/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Extracellular Vesicles/chemistry , Mucins/genetics , Pancreatic Neoplasms/diagnosis , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Diagnosis, Differential , Extracellular Vesicles/metabolism , Gene Expression , Humans , Mucins/metabolism , Pancreas , Pancreatic Juice/chemistry , Pancreatic Juice/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pancreatitis, Chronic/diagnosis , Pancreatitis, Chronic/genetics , Pancreatitis, Chronic/metabolism , Pancreatitis, Chronic/pathology , Prognosis , Proteome/genetics , Proteome/metabolism , ProteomicsABSTRACT
RATIONALE: Protein citrullination (deimination) is a post-translational modification of proteins converting arginine(s) into citrulline(s). "Overcitrullination" could be associated with severe pathological conditions. Mass spectrometric analysis of modified proteins is hindered by several problems. A comprehensive study of the fragmentation of deiminated peptides is not yet available. In this paper we have made an attempt to describe the characteristics of these processes, based on the studies of epitope model oligopeptides derived from clinically relevant proteins. METHODS: Solutions of purified model peptides containing either one or two citrulline residues as well as their native variants were injected directly into the electrospray source of a high accuracy and resolution quadrupole-time-of-flight instrument and were analysed by tandem mass spectrometry using low-energy collision-induced dissociation. RESULTS: Loss of isocyanic acid from citrulline residues is a preferred fragmentation route for deiminated peptides, which yields ornithine residues in the sequence. However, simultaneous detection of both the isocyanic acid loss and sequence fragments is often compromised. A preferential cleavage site was observed between citrulline and any other following amino acids yielding intensive complementary b- and y-type ions. Also, citrulline positioned at the C-termini displays a preferential cleavage N-terminal to this residue yielding characteristic y1 ions. These phenomena are described here for the first time and are referred to as the "citrulline effect". CONCLUSIONS: We found that the citrulline effect is very pronounced and could be used as a complementary tool for the confirmation of modification sites in addition to losses of isocyanic acids from the protonated molecules or from fragment ions. Low collision energy applied to peptide ions having partially mobile protons reveals the site of modification by generating specific and intensive fragments of the sequence. On the other hand, fragmenting precursor ions with mobile protons usually allows full sequence coverage, although citrulline-specific fragments may exhibit lower intensities compared to other fragments.
Subject(s)
Citrulline/chemistry , Peptides/chemistry , Tandem Mass Spectrometry/methods , Arthritis, Rheumatoid/immunology , Epitopes/chemistry , Humans , Peptide Fragments/analysis , Peptide Fragments/chemistry , Peptide Mapping/methods , Peptides/analysis , Peptides/metabolism , Protein Processing, Post-Translational , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
INTRODUCTION: Extracellular vesicles are emerging sources of biomarkers for modern preventive and precision medicine. Extracellular vesicles in body fluids offer a unique opportunity for integrative biomarker approaches due to their complex biocargo that includes proteins, lipids, nucleic acids and metabolites. Mass spectrometry-based proteomics data suggest that a significant portion of human proteins are sorted into extracellular vesicles and amenable for biomarker discovery schemes. Areas covered: this review focuses on key aspects of isolation, quality control and subsequent analysis of blood plasma- and conditioned medium-derived extracellular vesicle proteins, and summarizes the current state-of-the-art in the field. Furthermore, it provides introduction and guidelines for mass spectrometry-based proteomic analysis of extracellular vesicles. Expert commentary: Comparison of newly developed isolation and purification techniques with classical ultracentrifugation-based approaches are highly recommended. It is also essential to use multiple analytical approaches to characterize the isolated extracellular vesicles prior to characterization of their biocargo. Rigor in data reproducibility, critical data analysis, awareness of potential pitfalls, standardization and benchmarking are required for extracellular vesicle research to fulfil the current expectation that these subcellular structures can become a valid source of next generation biomarkers.