| 1. |
- Noborn, Fredrik, et al.
(författare)
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Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans.
- 2015
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Ingår i: Molecular & cellular proteomics : MCP. - 1535-9484 .- 1535-9476. ; 14:1, s. 41-9
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Tidskriftsartikel (refereegranskat)abstract
- Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.
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| 2. |
- Häggmark, Anna, 1985-, et al.
(författare)
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A High-throughput Bead-based Affinity Assay Enables Analysis of Genital Protein Signatures in Women At Risk of HIV Infection
- 2019
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Ingår i: Molecular & Cellular Proteomics. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 1535-9476 .- 1535-9484. ; 18:3, s. 461-476
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Tidskriftsartikel (refereegranskat)abstract
- Women at high risk of HIV infection, including sex workers and those with active genital inflammation, have molecular signatures of immune activation and epithelial barrier remodeling in samples of their genital mucosa. These alterations in the local immunological milieu are likely to impact HIV susceptibility. We here analyze host genital protein signatures in HIV uninfected women, with high frequency of condom use, living in HIV-serodiscordant relationships. Cervicovaginal secretions from women living in HIV-serodiscordant relationships (n = 62) were collected at three time points over 12 months. Women living in HIV-negative seroconcordant relationships (controls, n = 25) were sampled at one time point. All study subjects were examined for demographic parameters associated with susceptibility to HIV infection. The cervicovaginal samples were analyzed using a high-throughput bead-based affinity assay. Proteins involved in epithelial barrier function and inflammation were increased in HIV-serodiscordant women. By combining several methods of analysis, a total of five proteins (CAPG, KLK10, SPRR3, elafin/PI3, CSTB) were consistently associated with this study group. Proteins analyzed using the affinity set-up were further validated by label-free tandem mass spectrometry in a partially overlapping cohort with concordant results. Women living in HIV-serodiscordant relationships thus had elevated levels of proteins involved in epithelial barrier function and inflammation despite low prevalence of sexually transmitted infections and a high frequency of safe sex practices. The identified proteins are important markers to follow during assessment of mucosal HIV susceptibility factors and a high-throughput bead-based affinity set-up could be a suitable method for such evaluation.
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| 3. |
- Karlsson, Roger, 1975, et al.
(författare)
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Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping
- 2020
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 19:3, s. 518-528
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae. The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.
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| 4. |
- Larssen, Pia, et al.
(författare)
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Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assay
- 2017
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 16:3, s. 502-511
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) are membrane-coated objects such as exosomes and microvesicles, released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive potential biomarkers. The ability to determine their cellular origin could greatly move the field forward. We used multiplex proximity extension assays (PEA) to identify with high specificity and sensitivity the protein profiles of exosomes of different origins, including seven cell lines and two different body fluids. By comparing cells and exosomes, we successfully identified the cells originating the exosomes. Furthermore, by principal component analysis of protein patterns human milk EVs and prostasomes released from prostate acinar cells clustered with cell lines from breast and prostate tissues, respectively. Milk exosomes uniquely expressed CXCL5, MIA and KLK6, while prostasomes carried NKX31, GSTP1 and SRC, highlighting that EVs originating from different origins express distinct proteins. In conclusion, PEA provides a powerful protein screening tool in exosome research, for purposes of identifying the cell source of exosomes, or new biomarkers in diseases such as cancer and inflammation.
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| 5. |
- Noborn, Fredrik, et al.
(författare)
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Mapping the Human Chondroitin Sulfate Glycoproteome Reveals an Unexpected Correlation Between Glycan Sulfation and Attachment Site Characteristics. : Chondroitin Sulfation and Attachment Site Characteristics
- 2023
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Ingår i: Molecular & cellular proteomics : MCP. - : Elsevier. - 1535-9484 .- 1535-9476. ; 22:8
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Tidskriftsartikel (refereegranskat)abstract
- Chondroitin sulfate proteoglycans (CSPGs) control key events in human health and disease and are composed of chondroitin sulfate (CS) polysaccharide(s) attached to different core proteins. Detailed information on the biological effects of site-specific CS structures is scarce as the polysaccharides are typically released from their core proteins prior to analysis. Here we present a novel glycoproteomic approach for site-specific sequencing of CS modifications from human urine. Software-assisted and manual analysis revealed that certain core proteins carried CS with abundant sulfate modifications, while others carried CS with lower levels of sulfation. Inspection of the amino acid sequences surrounding the attachment sites indicated that the acidity of the attachment site motifs increased the levels of CS sulfation, and statistical analysis confirmed this relationship. However, not only the acidity but also the sequence and characteristics of specific amino acids in the proximity of the serine glycosylation site correlated with the degree of sulfation. These results demonstrate attachment site-specific characteristics of CS polysaccharides of CSPGs in human urine and indicate that this novel method may assist in elucidating the biosynthesis and functional roles of CSPGs in cellular physiology.
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| 6. |
- Schwenk, Jochen M., et al.
(författare)
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Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays
- 2010
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 9:11, s. 2497-2507
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Tidskriftsartikel (refereegranskat)abstract
- There is a need for high throughput methods for screening patient samples in the quest for potential biomarkers for diagnostics and patient care. Here, we used a combination of undirected target selection, antibody suspension bead arrays, and heat-induced epitope retrieval to allow for protein profiling of human plasma in a novel and systematic manner. Several antibodies were found to reveal altered protein profiles upon epitope retrieval at elevated temperatures with limits of detection improving into lower ng/ml ranges. In a study based on prostate cancer patients, several proteins with differential profiles were discovered and subsequently validated in an independent cohort. For one of the potential biomarkers, the human carnosine dipeptidase 1 protein (CNDP1), the differences were determined to be related to the glycosylation status of the targeted protein. The study shows a path of pursuit for large scale screening of biobank repositories in a flexible and proteome-wide fashion by utilizing heat-induced epitope retrieval and using an antibody suspension bead array format. Molecular & Cellular Proteomics 9:2497-2507, 2010.
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| 7. |
- Toledo, Alejandro Gomez, et al.
(författare)
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Positive mode LC-MS/MS analysis of chondroitin sulfate modified glycopeptides derived from light and heavy chains of the human inter-α-trypsin inhibitor complex : LC-MS/MS of IaI Proteoglycopeptides
- 2015
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Ingår i: Molecular and Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 14:12, s. 3118-3131
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Tidskriftsartikel (refereegranskat)abstract
- © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α- trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcβ4GlcAβ3)n, to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified crosslinked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.
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| 8. |
- Uhlén, Mathias, et al.
(författare)
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Antibody-based Protein Profiling of the Human Chromosome 21
- 2012
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 11:3
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Tidskriftsartikel (refereegranskat)abstract
- The Human Proteome Project has been proposed to create a knowledge-based resource based on a systematical mapping of all human proteins, chromosome by chromosome, in a gene-centric manner. With this background, we here describe the systematic analysis of chromosome 21 using an antibody-based approach for protein profiling using both confocal microscopy and immunohistochemistry, complemented with transcript profiling using next generation sequencing data. We also describe a new approach for protein isoform analysis using a combination of antibody-based probing and isoelectric focusing. The analysis has identified several genes on chromosome 21 with no previous evidence on the protein level, and the isoform analysis indicates that a large fraction of human proteins have multiple isoforms. A chromosome-wide matrix is presented with status for all chromosome 21 genes regarding subcellular localization, tissue distribution, and molecular characterization of the corresponding proteins. The path to generate a chromosome-specific resource, including integrated data from complementary assay platforms, such as mass spectrometry and gene tagging analysis, is discussed.
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| 9. |
- Ali, Neserin, et al.
(författare)
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Proteomics profiling of human synovial fluid suggests increased protein interplay in early-osteoarthritis (OA) that is lost in late-stage OA
- 2022
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Ingår i: Molecular & Cellular Proteomics. - : Elsevier BV. - 1535-9484 .- 1535-9476.
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Tidskriftsartikel (refereegranskat)abstract
- The underlying molecular mechanisms in osteoarthritis (OA) development are largely unknown. This study explores the proteome and the pairwise interplay of proteins in synovial fluid from patients with late-stage knee OA (arthroplasty), early knee OA (arthroscopy due to degenerative meniscal tear) and from deceased controls without knee OA.Synovial fluid samples were analyzed using state-of-the-art mass spectrometry with data-independent acquisition. The differential expression of the proteins detected was clustered and evaluated with data mining strategies and a multilevel model. Group-specific slopes of associations were estimated between expressions of each pair of identified proteins to assess the co-expression (i.e. interplay) between the proteins in each group.More proteins were increased in early-OA vs controls than late-stage OA vs controls. For most of these proteins, the fold changes between late-stage OA vs controls and early stage OA vs controls were remarkably similar suggesting potential involvement in the OA process. Further, for the first time this study illustrated distinct patterns in protein co-expression suggesting that the interplay between the protein machinery is increased in early-OA and lost in late-stage OA. Further efforts should focus on earlier stages of the disease than previously considered.
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| 10. |
- El-Rami, Fadi E., et al.
(författare)
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Quantitative proteomics of the 2016 WHO Neisseria gonorrhoeae reference strains surveys vaccine candidates and antimicrobial resistance determinants
- 2019
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Ingår i: Molecular & Cellular Proteomics. - : American Society for Biochemistry and Molecular Biology. - 1535-9476 .- 1535-9484. ; 18:1, s. 127-150
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Tidskriftsartikel (refereegranskat)abstract
- The sexually transmitted disease gonorrhea (causative agent: Neisseria gonorrhoeae) remains an urgent public health threat globally due to its reproductive health repercussions, high incidence, widespread antimicrobial resistance (AMR), and absence of a vaccine. To mine gonorrhea antigens and enhance our understanding of gonococcal AMR at the proteome level, we performed the first large-scale proteomic profiling of a diverse panel (n=15) of gonococcal strains, including the 2016 World Health Organization (WHO) reference strains. These strains show all existing AMR profiles - established through phenotypic characterization and reference genome publication - and are intended for quality assurance in laboratory investigations. Herein, these isolates were subjected to subcellular fractionation and labeling with tandem mass tags coupled to mass spectrometry and multi-combinatorial bioinformatics. Our analyses detected 904 and 723 common proteins in cell envelope and cytoplasmic subproteomes, respectively. We identified nine novel gonorrhea vaccine candidates. Expression and conservation of new and previously selected antigens were investigated. In addition, established gonococcal AMR determinants were evaluated for the first time using quantitative proteomics. Six new proteins, WHO_F_00238, WHO_F_00635c, WHO_F_00745, WHO_F_01139, WHO_F_01144c, and WHO_F_01126, were differentially expressed in all strains, suggesting that they represent global proteomic AMR markers, indicate a predisposition toward developing or compensating gonococcal AMR, and/or act as new antimicrobial targets. Finally, phenotypic clustering based on the isolates' defined antibiograms and common differentially expressed proteins yielded seven matching clusters between established and proteome-derived AMR signatures. Together, our investigations provide a reference proteomics databank for gonococcal vaccine and AMR research endeavors, which enables microbiological, clinical, or epidemiological projects and enhances the utility of the WHO reference strains.
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