RESUMO
The reinvigoration of anti-tumor T cells in response to immune checkpoint blockade (ICB) therapy is well established. Whether and how ICB therapy manipulates antibody-mediated immune response in cancer environments, however, remains elusive. Using tandem mass spectrometric analysis of modification of immunoglobulin G (IgG) from hepatoma tissues, we identified a role of ICB therapy in catalyzing IgG sialylation in the Fc region. Effector T cells triggered sialylation of IgG via an interferon (IFN)-γ-ST6Gal-I-dependent pathway. DC-SIGN+ macrophages represented the main target cells of sialylated IgG. Upon interacting with sialylated IgG, DC-SIGN stimulated Raf-1-elicited elevation of ATF3, which inactivated cGAS-STING pathway and eliminated subsequent type-I-IFN-triggered antitumorigenic immunity. Although enhanced IgG sialylation in tumors predicted improved therapeutic outcomes for patients receiving ICB therapy, impeding IgG sialylation augmented antitumorigenic T cell immunity after ICB therapy. Thus, targeting antibody-based negative feedback action of ICB therapy has potential for improving efficacy of cancer immunotherapies.
Assuntos
Carcinoma Hepatocelular , Interferon Tipo I , Neoplasias Hepáticas , Humanos , Imunoglobulina G , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Imunoterapia/métodosRESUMO
As an essential enzyme in the uridine diphosphate (UDP)-GlcNAc biosynthesis pathway, the significant role of N-acetylglucosamine phosphate mutase (AGM) remains unknown in plants. In the present study, a functional plant AGM (AtAGM) was identified from Arabidopsis thaliana. AtAGM catalyzes the isomerization of GlcNAc-1-P and GlcNAc-6-P, and has broad catalytic activity on different phosphohexoses. UDP-GlcNAc contents were significantly decreased in AtAGM T-DNA insertional mutants, which caused temperature-dependent growth defects in seedlings and vigorous growth in adult plants. Further analysis revealed that protein O-GlcNAcylation but not N-glycosylation was dramatically impaired in Atagm mutants due to UDP-GlcNAc shortage. Combined with the results from O-GlcNAcylation or N-glycosylation deficient mutants, and O-GlcNAcase inhibitor all suggested that protein O-GlcNAcylation impairment mainly leads to the phenotypic variations of Atagm plants. In conclusion, based on the essential role in UDP-GlcNAc biosynthesis, AtAGM is important for plant growth mainly via protein O-GlcNAcylation-level regulation.
Assuntos
Arabidopsis , Arabidopsis/metabolismo , Acetilglucosamina/metabolismo , Proteínas/metabolismo , Glicosilação , Fosfatos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Dysregulation of protein core-fucosylation plays a pivotal role in the onset, progression, and immunosuppression of cancer. However, analyzing core-fucosylation, especially the accurate determination of the core-fucosylation (CF) site occupancy ratio, remains challenging. To address these problems, we developed a truncation strategy that efficiently converts intact glycopeptides with hundreds of different glycans into two truncated forms, i.e., a monosaccharide HexNAc and a disaccharide HexNAc+core-fucose. Further combination with data-independent analysis to form an integrated platform allowed the measurement of site-specific core-fucosylation abundances and the determination of the CF occupancy ratio with high reproducibility. Notably, three times CF sites were identified using this strategy compared to conventional methods based on intact glycopeptides. Application of this platform to characterize protein core-fucosylation in two breast cancer cell lines, i.e., MDA-MB-231 and MCF7, yields a total of 1615 unique glycosites and about 900 CF sites from one single LC-MS/MS analysis. Differential analysis unraveled the distinct glycosylation pattern for over 201 cell surface drug targets between breast cancer subtypes and provides insights into developing new therapeutic strategies to aid precision medicine. Given the robust performance of this platform, it would have broad application in discovering novel biomarkers based on the CF glycosylation pattern, investigating cancer mechanisms, as well as detecting new intervention targets.
Assuntos
Fucose , Polissacarídeos , Humanos , Polissacarídeos/química , Polissacarídeos/metabolismo , Polissacarídeos/análise , Fucose/química , Fucose/metabolismo , Glicosilação , Espectrometria de Massas em Tandem , Linhagem Celular Tumoral , Glicopeptídeos/química , Glicopeptídeos/análise , Glicopeptídeos/metabolismoRESUMO
The quantitative profiling of residue reactivity in proteins promotes the discovery of covalent druggable targets for precise therapy. Histidine (His) residues, accounting for more than 20% of the active sites in enzymes, have not been systematically characterized for their reactivity, due to lack of labeling probes. Herein, we report a chemical proteomics platform for the site-specific quantitative analysis of His reactivity by combination of acrolein (ACR) labeling and reversible hydrazine chemistry enrichment. Based on this platform, in-depth characterization of His residues was conducted for the human proteome, in which the rich content of His residues (>8200) was quantified, including 317 His hyper-reactive residues. Intriguingly, it was observed that the hyper-reactive residues were less likely to be the sites for phosphorylation, and the possible mechanism of this antagonistic effect still needs to be evaluated in further research. Based on the first comprehensive map of His residue reactivity, many more residues could be adopted as the bindable sites to disrupt the activities of a diverse number of proteins; meanwhile, ACR derivatives could also be used as a novel reactive warhead in the development of covalent inhibitors.
Assuntos
Acroleína , Proteoma , Humanos , Histidina , Fosforilação , ProteômicaRESUMO
Different from N-linked glycosylation, the core structures of mucin type O-glycans are much more diverse, and the sensitive interpretation of O-glycopeptide spectra remains a challenge. The Y-ion pattern, a series of Y-ions with known mass gaps derived from the penta-saccharide core structure of N-linked glycosylation, is exploited to facilitate N-glycopeptide identification from their spectra. However, the pattern of Y ions in O-glycopeptides has not been well studied. In this study, we found that the Y-ion patterns were also frequently observed in the spectra of O-glycopeptides, and a special search approach is presented to identify O-glycopeptides by utilizing the Y-ion patterns. In this strategy, theoretical O-glycan Y-ion patterns are constructed to match the experimental Y-ions in O-glycopeptide spectra, which enables the determination of the mass of some glycans and results in the reduction of searching space. In addition, a Y-ion pattern-based deisotope process is also developed to correct the precursor m/z. The new search strategy was applied to search a human serum data set, and 15.4%-199.0% more O-glycopeptide-spectrum matches (OGPSMs) and 19.6%-107.1% more glycopeptide sequence identifications than other state-of-the-art software tools were observed. This search mode, the O-Search-Pattern, has been implemented into our database search software, MS-Decipher, and is recommended for searching the O-glycopeptide spectra acquired by sceHCD (stepped collision energy higher-energy collisional dissociation).
Assuntos
Glicopeptídeos , Software , Humanos , Sequência de Aminoácidos , Glicopeptídeos/análise , Polissacarídeos/química , ÍonsRESUMO
Mucin-type O-glycosylation (or O-GalNAcylation) takes place on most membrane and secretory proteins and is vital in regulating protein functions and many biological processes. O-GalNAcylation generally exhibits highly diverse and dense O-glycans linked to carrier proteins, which challenges the analysis of O-GalNAc glycoproteome using conventional methodologies. Here, we report an O-glycopeptide truncation strategy for the characterization of protein O-GalNAcylation in biological samples. The O-glycopeptide truncation strategy utilizes proteases or O-glycopeptidases for targeted cleavage of the enriched tryptic O-glycopeptides. It simplifies the O-glycopeptide backbones, O-glycans, or both, and has been shown to aid the improvement of the analytical coverage of O-GalNAc glycopeptides and glycoproteins. Tryptic O-glycopeptides covered with O-glycan clusters and terminal sialic acids could be well isolated by the hydrophilic-based enrichment approaches. The enriched O-glycopeptides are then enzymatically truncated into shorter or less multiply O-glycosylated peptides, which are more favorable for mass spectrometry detection and database search in general bottom-up glycoproteomics. We also investigate different proteolysis which could be well integrated into the O-glycopeptide truncation strategy. For large-scale analysis, we exploit different truncation schemes and identify nearly 2000 O-glycopeptides corresponding to 391 glycoproteins from 75 µL human serum, achieving the deepest-scale coverage of O-glycoproteins compared to other plasma/serum O-glycoproteomic studies. Together, the O-glycopeptide truncation strategy has great potential to facilitate the in-depth study of O-GalNAc glycoproteomics in biological samples.
Assuntos
Glicopeptídeos , Proteômica , Humanos , Glicopeptídeos/análise , Proteômica/métodos , Glicoproteínas/química , Glicosilação , Polissacarídeos/análiseRESUMO
Both N-linked glycosylation and O-linked glycosylation play essential roles in the onset and progression of various diseases including cancer, and N-/O-linked site-specific glycans have been proven to be promising biomarkers for the discrimination of cancer. However, the micro-heterogeneity and low abundance nature of N-/O-linked glycosylation, as well as the time-consuming and tedious procedures for the enrichment of O-linked intact glycopeptides, pose great challenges for their efficient and accurate characterization. In this study, we developed an integrated platform for the simultaneous enrichment and characterization of N- and O-linked intact glycopeptides from the same serum sample. By fine-tuning the experimental conditions, we demonstrated that this platform allowed the selective separation of N- and O-linked intact glycopeptides into two fractions, with 85.1% O-linked intact glycopeptides presented in the first fraction and 93.4% N-linked intact glycopeptides presented in the second fraction. Determined with high reproducibility, this platform was further applied to the differential analysis of serum samples of gastric cancer and health control, which revealed 17 and 181 significantly changed O-linked and N-linked intact glycopeptides. Interestingly, five glycoproteins containing both significant regulation of N- and O-glycosylation were observed, hinting potential co-regulation of different types of glycosylation during tumor progress. In summary, this integrated platform opened a potentially useful avenue for the global analysis of protein glycosylation and can serve as a useful tool for the characterization of N-/O-linked intact glycopeptides at the proteomics scale.
Assuntos
Glicopeptídeos , Glicoproteínas , Glicopeptídeos/análise , Reprodutibilidade dos Testes , Glicoproteínas/química , Glicosilação , Proteômica/métodosRESUMO
The ligand-receptor signaling occurring on the cell surface governs cell growth, proliferation, and survival via rapidly triggering a cascade of events. Here, we for the first time report an in situ perturbation-free and rapid surface proteomic profiling at a temporal resolution of ten seconds. By this innovation, about 1022 cell surface-associated proteins were reproducibly identified and quantified. It is noteworthy that, upon a model ligand insulin stimulus, a few rapid-responding proteins at 10 s to 2 min were identified, e.g., CNNM3. Moreover, temporal response patterns were established for the members of GLUT4 storage vesicles (GSVs; responsible for glucose transportation) and confirmed with five known GSV proteins. This pattern was then exploited to uncover seven new regulatory proteins (LDLR, HFE, ECE1, MRC2, CORO1C, CPD, and BST2). Collectively, we showed a powerful surface proteomic tool to decipher rapid signaling of cell-surface proteins and to uncover new subunits involved in rapidly trafficking vesicles.
Assuntos
Insulina , Proteômica , Ligantes , Membrana Celular/metabolismo , Insulina/metabolismo , Transdução de Sinais , Transporte ProteicoRESUMO
MOTIVATION: The interpretation of mass spectrometry (MS) data is a crucial step in proteomics analysis, and the identification of post-translational modifications (PTMs) is vital for the understanding of the regulation mechanism of the living system. Among various PTMs, glycosylation is one of the most diverse ones. Though many search engines have been developed to decipher proteomic data, some of them are difficult to operate and have poor performance on glycoproteomic datasets compared to advanced glycoproteomic software. RESULTS: To simplify the analysis of proteomic datasets, especially O-glycoproteomic datasets, here, we present a user-friendly proteomic database search platform, MS-Decipher, for the identification of peptides from MS data. Two scoring schemes can be chosen for peptide-spectra matching. It was found that MS-Decipher had the same sensitivity and confidence in peptide identification compared to traditional database searching software. In addition, a special search mode, O-Search, is integrated into MS-Decipher to identify O-glycopeptides for O-glycoproteomic analysis. Compared with Mascot, MetaMorpheus and MSFragger, MS-Decipher can obtain about 139.9%, 48.8% and 6.9% more O-glycopeptide-spectrum matches. A useful tool is provided in MS-Decipher for the visualization of O-glycopeptide-spectra matches. MS-Decipher has a user-friendly graphical user interface, making it easier to operate. Several file formats are available in the searching and validation steps. MS-Decipher is implemented with Java, and can be used cross-platform. AVAILABILITY AND IMPLEMENTATION: MS-Decipher is freely available at https://github.com/DICP-1809/MS-Decipher for academic use. For detailed implementation steps, please see the user guide. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Glicopeptídeos , Proteoma , Glicopeptídeos/análise , Glicopeptídeos/química , Proteômica/métodos , Software , Espectrometria de Massas , Peptídeos/químicaRESUMO
BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a process linked to metastasis and drug resistance with non-coding RNAs (ncRNAs) playing pivotal roles. We previously showed that miR-100 and miR-125b, embedded within the third intron of the ncRNA host gene MIR100HG, confer resistance to cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, in colorectal cancer (CRC). However, whether the MIR100HG transcript itself has a role in cetuximab resistance or EMT is unknown. METHODS: The correlation between MIR100HG and EMT was analyzed by curating public CRC data repositories. The biological roles of MIR100HG in EMT, metastasis and cetuximab resistance in CRC were determined both in vitro and in vivo. The expression patterns of MIR100HG, hnRNPA2B1 and TCF7L2 in CRC specimens from patients who progressed on cetuximab and patients with metastatic disease were analyzed by RNAscope and immunohistochemical staining. RESULTS: The expression of MIR100HG was strongly correlated with EMT markers and acted as a positive regulator of EMT. MIR100HG sustained cetuximab resistance and facilitated invasion and metastasis in CRC cells both in vitro and in vivo. hnRNPA2B1 was identified as a binding partner of MIR100HG. Mechanistically, MIR100HG maintained mRNA stability of TCF7L2, a major transcriptional coactivator of the Wnt/ß-catenin signaling, by interacting with hnRNPA2B1. hnRNPA2B1 recognized the N6-methyladenosine (m6A) site of TCF7L2 mRNA in the presence of MIR100HG. TCF7L2, in turn, activated MIR100HG transcription, forming a feed forward regulatory loop. The MIR100HG/hnRNPA2B1/TCF7L2 axis was augmented in specimens from CRC patients who either developed local or distant metastasis or had disease progression that was associated with cetuximab resistance. CONCLUSIONS: MIR100HG and hnRNPA2B1 interact to control the transcriptional activity of Wnt signaling in CRC via regulation of TCF7L2 mRNA stability. Our findings identified MIR100HG as a potent EMT inducer in CRC that may contribute to cetuximab resistance and metastasis by activation of a MIR100HG/hnRNPA2B1/TCF7L2 feedback loop.
Assuntos
Neoplasias Colorretais , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , MicroRNAs , RNA Longo não Codificante , Linhagem Celular Tumoral , Movimento Celular/genética , Cetuximab/genética , Cetuximab/metabolismo , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
Lysine acetylation is one of the most basic molecular mechanisms to mediate protein functions in living organisms, and its abnormal regulation has been linked to many diseases. The drug development associated to this process is of great significance but severely hindered by the complex interplay of lysine acetylation and deacetylation in thousands of proteins, and we reasoned that targeting a specific protein acetylation or deacetylation event instead of the related enzymes should be a feasible solution to this issue. Toward this goal, we devised an orthogonal lysine acylation and deacylation (OKAD) system, which potentially could precisely dissect the biological consequence of an individual acetylation or deacetylation event in living cells. The system includes a genetically encoded acylated lysine (PhOAcK) that is not a substrate of endogenous deacetylases, and an evolved sirtuin (CobB2/CobB3) that displays PhOAcK deacylase activities as well as reduced deacetylase activities. We believe the strategy introduced here holds potential for future in-depth biological applications.
Assuntos
Histona Desacetilases/metabolismo , Lisina/metabolismo , Acilação , Lisina/química , Estrutura MolecularRESUMO
Post-translational modifications (PTMs) occurring on lysine residues, especially diverse forms of acylations, have seen rapid growth over the past two decades. Among them, lactylation and ß-hydroxybutyrylation of lysine side-chains are newly identified histone marks and their implications in physiology and diseases have aroused broad research interest. Meanwhile, lysine lipoylation is highly conserved in diverse organisms and well known for its pivotal role in central metabolic pathways. Recent findings in the proteomic profiling of protein lipoylation have nonetheless suggested a pressing need for an extensive investigation. For both basic and applied research, it is necessary to prepare PTM-bearing proteins particularly in a site-specific manner. Herein, we use genetic code expansion to site-specifically generate these lysine PTMs, including lactylation, ß-hydroxybutyrylation and lipoylation in proteins in E.â coli and mammalian cells. Notably, using strategies including activity-based selection, screening and rational design, unique pyrrolysyl-tRNA synthetase variants were successfully evolved for each of the three non-canonical amino acids, which enabled efficient production of recombinant proteins. Through encoding these ncAAs, we examined the deacylase activities of mammalian sirtuins to these modifications, and importantly we unfold the lipoamidase activity of several sirtuins.
Assuntos
Aminoacil-tRNA Sintetases , Sirtuínas , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Lipoilação , Lisina/metabolismo , Mamíferos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Proteínas Recombinantes/genética , Sirtuínas/metabolismoRESUMO
Lysine crotonylation (Kcr) is increasingly recognized as a key protein post-translational modification. However, selective detection and enrichment of crotonylated proteins remains a challenging task. Herein we present a covalent binder for the selective recognition of protein crotonylation. Based on proximity-induced crosslinking, a bacterial sirtuin (CobB) was remodeled with genetically installed thiol-bearing noncanonical amino acids at the Kcr-interacting site, which subsequently could react with Kcr sites in a unique NAD+ -dependent manner. The covalent binder has been used to selectively recognize crotonylated proteins in extracted histone samples and in fixed cells.
Assuntos
Sirtuínas , Histonas/química , Lisina/química , Processamento de Proteína Pós-Traducional , Sirtuínas/metabolismoRESUMO
To selectively enrich O-linked ß-N-acetylglucosamine (O-GlcNAc) peptides in their original form from complex samples, we report the first reversible chemoenzymatic labeling approach for proteomic analysis. In this strategy, the O-GlcNAc moieties are ligated with long N-glycans using an Endo-M mutant, which enables the enrichment of the labeled glycopeptides by hydrophilic interaction liquid chromatography (HILIC). The attached glycans on the enriched glycopeptides are removed by wild-type Endo-M/S to restore the O-GlcNAc moiety. Compared with classic chemoenzymatic labeling, this approach enables the tag-free identification, and eliminates the interference of bulky tags in glycopeptide detection. This approach presents a unique avenue for the proteome-wide analysis of protein O-GlcNAcylation to promote its mechanism research.
Assuntos
Glicopeptídeos , Proteômica , Acetilglucosamina/metabolismo , Cromatografia Líquida/métodos , Glicopeptídeos/química , Polissacarídeos/química , Proteoma/análiseRESUMO
N-linked glycosylation plays important roles in multiple physiological and pathological processes, while the analysis coverage is still limited due to the insufficient digestion of glycoproteins, as well as incomplete ion fragments for intact glycopeptide determination. Herein, a mirror-cutting-based digestion strategy was proposed by combining two orthogonal proteases of LysargiNase and trypsin to characterize the macro- and micro-heterogeneity of protein glycosylation. Using the above two proteases, the b- or y-ion series of peptide sequences were, respectively, enhanced in MS/MS, generating the complementary spectra for peptide sequence identification. More than 27% (489/1778) of the site-specific glycoforms identified by LysargiNase digestion were not covered by trypsin digestion, suggesting the elevated coverage of protein sequences and site-specific glycoforms by the mirror-cutting method. Totally, 10,935 site-specific glycoforms were identified from mouse brain tissues in the 18 h MS analysis, which significantly enhanced the coverage of protein glycosylation. Intriguingly, 27 mannose-6-phosphate (M6P) glycoforms were determined with core fucosylation, and 23 of them were found with the "Y-HexNAc-Fuc" ions after manual checking. This is hitherto the first report of M6P and fucosylation co-modifications of glycopeptides, in which the mechanism and function still needs further exploration. The mirror-cutting digestion strategy also has great application potential in the exploration of missing glycoproteins from other complex samples to provide rich resources for glycobiology research.
Assuntos
Glicopeptídeos , Espectrometria de Massas em Tandem , Animais , Digestão , Glicopeptídeos/análise , Glicoproteínas/química , Glicosilação , Camundongos , Espectrometria de Massas em Tandem/métodosRESUMO
Protein methylation, especially that occurs on arginine and lysine residues, is one of the most important post-translational modifications involved in various cellular processes including RNA splicing, DNA repair, and so forth. Systematic analysis of protein methylation would facilitate the understanding of its regulatory mechanisms. Strong cation chromatography has been used to globally analyze arginine/lysine methylation at the proteome scale with good performance. However, the co-enriched histidine-containing peptides severely interfere with the detection of low-abundance methylpeptides. Here, we developed a novel chemical strategy which enabled almost complete depletion of histidine-containing peptides in the protein digest, thereby resulting in the identification of more low-abundance arginine/lysine methylpeptides. Totally, 333 arginine and lysine methylation forms from 207 proteins were identified in this study. Overall, the number of methylation identifications increased about 50% by using our new method. Data are available via ProteomeXchange with the identifier PXD023845.
Assuntos
Histidina , Proteoma , Arginina/metabolismo , Lisina/metabolismo , Metilação , Peptídeos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Protein O-GlcNAcylation has been implicated in a broad range of cellular processes, while the functional research is still lagging behind other post-translational modification (PTMs), as a result of the low stoichiometry and limited enrichment efficiency. Herein, a strategy, named CHO-GlcNAc, was developed for O-GlcNAc glycopeptide enrichment. In this strategy, the O-GlcNAc glycopeptides were first enzymatically labeled with a Gal moiety, followed by chemical oxidation to efficiently introduce the aldehyde groups. The labeled O-GlcNAc glycopeptides could be efficiently enriched based on the equilibrium between the hydrazine and oxime bonds. Good specificity of the glycopeptide enrichment was observed from the mixtures of glycopeptide and non-glycopeptides using the CHO-GlcNAc method. Then, it was applied to analyze O-GlcNAcylation in the nucleus of HeLa cells, and 829 potential O-GlcNAcylation sites on 274 glycoproteins were identified, including the two readers of m6A (YTHDF1 and YTHDF3), which could provide clues for the mechanism of crosstalk between O-GlcNAcylation and other PTMs of proteins and RNA. Thus, this method could be a versatile tool for the proteomic analysis of O-GlcNAcylation.
Assuntos
Glicopeptídeos , Proteômica , Células HeLa , Humanos , HidrazinasRESUMO
Proteomics analysis of O-GalNAc glycosylation is important for the screening of biomarkers and the assessment of therapeutic responses. However, its analysis still faces challenges due to the poor performance of currently available enrichment methods. In this study, an enrichment method was established on the basis of Ti-IMAC(IV) materials, which could enrich the intact O-GalNAc glycopeptides via both the hydrophilic interaction and affinity interaction. This method enabled nearly 200 intact O-GalNAc glycopeptides identified from only 0.1 µL of human serum. This was nearly 2-fold different from that of the HILIC method. An in-depth analysis of the O-GalNAc glycosylation was performed, and 2093 intact glycopeptides were identified from 7.2 µL of human serum samples. This is the largest O-GalNAc glycosylation database of human serum from a trace amount of sample. Furthermore, 52 significantly changed intact O-GalNAc glycopeptides were determined by the quantitative analysis of hepatocellular carcinoma (HCC) and control serum samples, indicating the potential applications of this enrichment method in biomarker discovery.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Cromatografia de Afinidade , Glicopeptídeos , Humanos , Interações Hidrofóbicas e Hidrofílicas , ProteômicaRESUMO
Bottom-up proteomics has been increasingly applied in clinical research to study the disease pathophysiology and to discover disease biomarkers. However, glycoproteomic analysis always requires tedious experimental steps for intact glycopeptide enrichment, which has been the technique bottleneck for large-scale analysis of clinical samples. Herein, we developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min. It showed higher enrichment specificity, more intact glycopeptide identifications, and better quantitative reproducibility than the traditional manual method using microtip enrichment devices. We further applied this method to investigate the serum site-specific N-glycosylation changes between four patients with pancreatic cancer and seven healthy controls. The principal component analysis of intact N-glycopeptides showed good clustering across cancer and normal groups. Furthermore, we found that the site-specific glycoforms, monofucosylated and nonsialylated oligosaccharides, on IgG1 site 180 expressed a significant decrease in pancreatic cancer patients compared to healthy controls. Together, the automated method is a powerful tool for site-specific N-glycoproteomic analysis of complex biological samples, and it has great potential for clinical utilities.
Assuntos
Glicopeptídeos , Proteoma , Glicosilação , Humanos , Proteômica , Reprodutibilidade dos TestesRESUMO
D-dimer is an important marker of different coagulation diseases, such as venous thromboembolism (including deep vein thrombosis and pulmonary embolism) and disseminated intravascular coagulation. Though it is frequently used in many clinical diagnostic situations, the D-dimer assays currently lack standardization due to its inherent heterogeneity which makes the antibody-based methods have different quantitative results and cutoffs to define an abnormal value. In this study, we report the first antibody-free D-dimer quantification method. In the method, a cross-linked peptide of fibrin D domain carboxyl terminal cross-linked by the factor XIIIa was used to represent the D-dimer. By using a filter-aided sample preparation and a nickel immobilized metal affinity chromatography enrichment strategy, the complexity of the plasma sample was significantly reduced, and the cross-linked peptide was enriched effectively for analysis with parallel reaction monitoring in mass spectrometry. The linear range of this method was 3.125-400 nmol/L which spans over two magnitudes. Recovery and reproducibility of the method were found to be good. To further demonstrate the performance of our method, D-dimer concentrations of 25 human plasma samples were analyzed, and the results had a good correlation between with the commercial D-dimer assay kit used in hospitals. This method was completely antibody-free and has the potential to promote the standardization of D-dimer analysis.