| 1. |
- Agaton, C., et al.
(författare)
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Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues
- 2003
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 2, s. 405-
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Tidskriftsartikel (refereegranskat)abstract
- Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.
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| 2. |
- Ahmad, Yasmeen, et al.
(författare)
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Systematic Analysis of Protein Pools, Isoforms, and Modifications Affecting Turnover and Subcellular Localization
- 2012
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 11:3
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Tidskriftsartikel (refereegranskat)abstract
- In higher eukaryotes many genes encode protein isoforms whose properties and biological roles are often poorly characterized. Here we describe systematic approaches for detection of either distinct isoforms, or separate pools of the same isoform, with differential biological properties. Using information from ion intensities we have estimated protein abundance levels and using rates of change in stable isotope labeling with amino acids in cell culture isotope ratios we measured turnover rates and subcellular distribution for the HeLa cell proteome. Protein isoforms were detected using three data analysis strategies that evaluate differences between stable isotope labeling with amino acids in cell culture isotope ratios for specific groups of peptides within the total set of peptides assigned to a protein. The candidate approach compares stable isotope labeling with amino acids in cell culture isotope ratios for predicted isoform- specific peptides, with ratio values for peptides shared by all the isoforms. The rule of thirds approach compares the mean isotope ratio values for all peptides in each of three equal segments along the linear length of the protein, assessing differences between segment values. The three in a row approach compares mean isotope ratio values for each sequential group of three adjacent peptides, assessing differences with the mean value for all peptides assigned to the protein. Protein isoforms were also detected and their properties evaluated by fractionating cell extracts on one- dimensional SDS- PAGE prior to trypsin digestion and MS analysis and independently evaluating isotope ratio values for the same peptides isolated from different gel slices. The effect of protein phosphorylation on turnover rates was analyzed by comparing mean turnover values calculated for all peptides assigned to a protein, either including, or excluding, values for cognate phosphopeptides. Collectively, these experimental and analytical approaches provide a framework for expanding the func- tional annotation of the genome.
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| 3. |
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| 4. |
- 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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| 5. |
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| 6. |
- Asplund, C., et al.
(författare)
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Real-time RT-PCR of protein epitope signature tags
- 2005
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Ingår i: Molecular & Cellular Proteomics. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 1535-9476 .- 1535-9484. ; 4:8, s. S60-S60
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 7. |
- Ayoglu, Burcu, et al.
(författare)
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Autoantibody profiling in multiple sclerosis using arrays of human protein fragments
- 2013
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 12:9, s. 2657-2672
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Tidskriftsartikel (refereegranskat)abstract
- Profiling the autoantibody repertoire with large antigen collections is emerging as a powerful tool for the identification of biomarkers for autoimmune diseases. Here, a systematic and undirected approach was taken to screen for profiles of IgG in human plasma from 90 individuals with multiple sclerosis related diagnoses. Reactivity pattern of 11,520 protein fragments (representing ̃38% of all human protein encoding genes) were generated on planar protein microarrays built within the Human Protein Atlas. For more than 2,000 antigens IgG reactivity was observed, among which 64% were found only in single individuals. We used reactivity distributions among multiple sclerosis subgroups to select 384 antigens, which were then reevaluated on planar microarrays, corroborated with suspension bead arrays in a larger cohort (n = 376) and confirmed for specificity in inhibition assays. Among the heterogeneous pattern within and across multiple sclerosis subtypes, differences in recognition frequencies were found for 51 antigens, which were enriched for proteins of transcriptional regulation. In conclusion, using protein fragments and complementary high-throughput protein array platforms facilitated an alternative route to discovery and verification of potentially disease-associated autoimmunity signatures, that are now proposed as additional antigens for large-scale validation studies across multiple sclerosis biobanks.
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| 8. |
- Baan, Bart, et al.
(författare)
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In situ proximity ligation detection of c-Jun/AP-1 dimers reveals increased levels of c-Jun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo
- 2010
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 9:9, s. 1982-1990
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Tidskriftsartikel (refereegranskat)abstract
- Genetic and biochemical studies have shown that selective interactions between the Jun, Fos, and activating transcription factor (ATF) components of transcription factor activating protein 1 (AP-1) exhibit specific and critical functions in the regulation of cell proliferation, differentiation, and survival. For instance, the ratio between c-Jun/c-Fos and c-Jun/ATF2 dimers in the cell can be a determining factor in the cellular response to oncogenic or apoptotic stimuli. Until recently, no methods were available to detect endogenous AP-1 complexes in cells and tissues in situ. Here, we validated the proximity ligation assay (PLA) for its ability to specifically visualize and quantify changes in endogenous c-Jun/c-Fos, c-Jun/ATF2, and c-Jun/Fra1 complexes by using, among others, partner-selective c-Jun mutants. Furthermore, we examined the levels of c-Jun/AP-1 dimers in cell lines representing different types of human breast cancer and found that aggressive basal-like breast cancer cells can be discriminated from much less invasive luminal-like cells by PLA detection of c-Jun/Fra1 rather than of c-Jun/ATF2 and c-Jun/c-Fos. Also in tumor tissue derived from highly metastatic basal-like MDA-MB231 cells, high levels of c-Jun/Fra1 complexes were detected. Together, these results demonstrate that in situ PLA is a powerful diagnostic tool to analyze and quantify the amounts of biologically critical AP-1 dimers in fixed cells and tissue material.
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| 9. |
- Balonova, Lucie, et al.
(författare)
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Characterization of protein glycosylation in Francisella tularensis subsp holarctica
- 2012
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 11:7
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Tidskriftsartikel (refereegranskat)abstract
- FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.015016, 1-12, 2012.
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| 10. |
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| 11. |
- Barbe, Laurent, et al.
(författare)
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Toward a confocal subcellular atlas of the human proteome
- 2008
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Ingår i: Molecular and cellular proteomics. - 1535-9476 .- 1535-9484. ; 7:3, s. 499-508
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Tidskriftsartikel (refereegranskat)abstract
- Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.
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| 12. |
- Barranco, Isabel, et al.
(författare)
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The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions
- 2023
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Ingår i: Molecular & Cellular Proteomics. - : ELSEVIER. - 1535-9476 .- 1535-9484. ; 22:4
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Tidskriftsartikel (refereegranskat)abstract
- Seminal plasma contains many morphologically hetero-geneous extracellular vesicles (sEVs). These are sequentially released by cells of the testis, epididymis, and accessory sex glands and involved in male and fe-male reproductive processes. This study aimed to define in depth sEV subsets isolated by ultrafiltration and size exclusion chromatography, decode their proteomic profiles using liquid chromatography-tandem mass spectrometry, and quantify identified proteins using sequential window acquisition of all theoretical mass spectra. The sEV subsets were defined as large (L-EVs) or small (S-EVs) by their protein concentration, morphology, size distribution, and EV-specific protein markers and purity. Liquid chromatography-tandem mass spectrometry identified a total of 1034 proteins, 737 of them quantified by SWATH in S-EVs, L-EVs, and non-EVs-enriched samples (18-20 size exclusion chromatography-eluted fractions). The differential expression analysis revealed 197 differentially abundant proteins between both EV subsets, S-EVs and L-EVs, and 37 and 199 between S-EVs and L-EVs versus non-EVs-enriched samples, respectively. The gene ontology enrichment analysis of differentially abundant proteins suggested, based on the type of protein detected, that S-EVs could be mainly released through an apocrine blebbing pathway and be involved in modulating the im-mune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, L-EVs could be released by fusion of multivesicular bodies with the plasma membrane becoming involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV subsets, indicating different sources and biological functions for the sEVs.
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| 13. |
- Berggrund, Malin, et al.
(författare)
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Identification of candidate plasma protein biomarkers for cervical cancer using the multiplex proximity extension assay
- 2019
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 18:4, s. 735-743
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Tidskriftsartikel (refereegranskat)abstract
- Human papillomavirus (HPV) is recommended as the primary test in cervical cancer screening, with co-testing by cytology for HPV-positive women to identify cervical lesions. Cytology has low sensitivity and there is a need to identify biomarkers that could identify dysplasia that are likely to progress to cancer. We searched for plasma proteins that could identify women with cervical cancer using the multiplex proximity extension assay (PEA). The abundance of 100 proteins were measured in plasma collected at the time of diagnosis of patients with invasive cervical cancer and in population controls using the Olink Multiplex panels CVD II, INF I, and ONC II. Eighty proteins showed increased levels in cases compared to controls. We identified a signature of 11 proteins (PTX3, ITGB1BP2, AXIN1, STAMPB, SRC, SIRT2, 4E-BP1, PAPPA, HB-EGF, NEMO and IL27) that distinguished cases and controls with a sensitivity of 0.96 at a specificity of 1.0. This signature was evaluated in a prospective replication cohort with samples collected before, at or after diagnosis and achieved a sensitivity of 0.78 and a specificity 0.56 separating samples collected at the time of diagnosis of invasive cancer from samples collected prior to diagnosis. No difference in abundance was seen between samples collected prior to diagnosis or after treatment as compared to population controls, indicating that this protein signature is mainly informative close to time of diagnosis. Further studies are needed to determine the optimal window in time prior to diagnosis for these biomarker candidates.
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| 14. |
- Berglund, Lisa, et al.
(författare)
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A genecentric Human Protein Atlas for expression profiles based on antibodies
- 2008
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:10, s. 2019-2027
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Forskningsöversikt (refereegranskat)abstract
- An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.
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| 15. |
- Bjorling, E., et al.
(författare)
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Human protein atlas, version 2
- 2006
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Ingår i: Molecular & Cellular Proteomics. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 1535-9476 .- 1535-9484. ; 5:10, s. S328-S328
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 16. |
- Bjorling, E., et al.
(författare)
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The creation and usage of a human protein atlas database
- 2005
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Ingår i: Molecular & Cellular Proteomics. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 1535-9476 .- 1535-9484. ; 4:8, s. S18-S18
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 17. |
- Björkesten, Johan, et al.
(författare)
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Stability of Proteins in Dried Blood Spot Biobanks.
- 2017
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 16:7, s. 1286-1296
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Tidskriftsartikel (refereegranskat)abstract
- An important motivation for the construction of biobanks is to discover biomarkers that identify diseases at early, potentially curable stages. This will require biobanks from large numbers of individuals, preferably sampled repeatedly, where the samples are collected and stored under conditions that preserve potential biomarkers. Dried blood samples are attractive for biobanking because of the ease and low cost of collection and storage. Here we have investigated their suitability for protein measurements. 92 proteins with relevance for oncology were analyzed using multiplex proximity extension assays (PEA) in dried blood spots collected on paper and stored for up to 30 years at either +4°C or -24°C. Our main findings were that 1) the act of drying only slightly influenced detection of blood proteins (average correlation of 0.970), and in a reproducible manner (correlation of 0.999), 2) detection of some proteins was not significantly affected by storage over the full range of three decades (34% and 76% of the analyzed proteins at +4°C and -24°C, respectively), while levels of others decreased slowly during storage with half-lives in the range of 10 to 50 years, and 3) detectability of proteins was less affected in dried samples stored at -24°C compared to at +4°C, as the median protein abundance had decreased to 80% and 93% of starting levels after 10 years of storage at +4°C or -24°C, respectively. The results of our study are encouraging as they suggest an inexpensive means to collect large numbers of blood samples, even by the donors themselves, and to transport, and store biobanked samples as spots of whole blood dried on paper. Combined with emerging means to measure hundreds or thousands of protein, such biobanks could prove of great medical value by greatly enhancing discovery as well as routine analysis of blood biomarkers.
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| 18. |
- Björling, Erik, et al.
(författare)
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A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues
- 2008
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:5, s. 825-844
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Tidskriftsartikel (refereegranskat)abstract
- Here we report the development of a publicly available Web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed toward human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas to discover potential protein biomarkers. Such biomarkers include tissue-specific markers, cell type-specific markers, tumor type-specific markers, markers of malignancy, and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies utilizing more classical proteomics tools.
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| 19. |
- Blokzijl, Andries, et al.
(författare)
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Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens
- 2016
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 15:6, s. 1848-1856
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Tidskriftsartikel (refereegranskat)abstract
- The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF- signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF- signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF- signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA. Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.
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| 20. |
- Boulund, Fredrik, 1985, et al.
(författare)
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Typing and Characterization of Bacteria Using Bottom-up Tandem Mass Spectrometry Proteomics
- 2017
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 16:6, s. 1052-1063
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Tidskriftsartikel (refereegranskat)abstract
- Methods for rapid and reliable microbial identification are essential in modern healthcare. The ability to detect and correctly identify pathogenic species and their resistance phenotype is necessary for accurate diagnosis and efficient treatment of infectious diseases. Bottom-up tandem mass spectrometry (MS) proteomics enables rapid characterization of large parts of the expressed genes of microorganisms. However, the generated data are highly fragmented, making downstream analyses complex. Here we present TCUP, a new computational method for typing and characterizing bacteria using proteomics data from bottom-up tandem MS. TCUP compares the generated protein sequence data to reference databases and automatically finds peptides suitable for characterization of taxonomic composition and identification of expressed antimicrobial resistance genes. TCUP was evaluated using several clinically relevant bacterial species (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae), using both simulated data generated by in silico peptide digestion and experimental proteomics data generated by liquid chromatography-tandem mass spectrometry (MS/MS). The results showed that TCUP performs correct peptide classifications at rates between 90.3 and 98.5% at the species level. The method was also able to estimate the relative abundances of individual species in mixed cultures. Furthermore, TCUP could identify expressed beta-lactamases in an extended spectrum beta-lactamase-producing (ESBL) E.coli strain, even when the strain was cultivated in the absence of antibiotics. Finally, TCUP is computationally efficient, easy to integrate in existing bioinformatics workflows, and freely available under an open source license for both Windows and Linux environments.
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| 21. |
- Burnum-Johnson, Kristin E., et al.
(författare)
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New Views of Old Proteins : Clarifying the Enigmatic Proteome
- 2022
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Ingår i: Molecular & Cellular Proteomics. - : Elsevier BV. - 1535-9476 .- 1535-9484. ; 21:7
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Tidskriftsartikel (refereegranskat)abstract
- All human diseases involve proteins, yet our current tools to characterize and quantify them are limited. To better elucidate proteins across space, time, and molecular composition, we provide a >10 years of projection for technologies to meet the challenges that protein biology presents. With a broad perspective, we discuss grand opportunities to transition the science of proteomics into a more propulsive enterprise. Extrapolating recent trends, we describe a next generation of approaches to define, quantify, and visualize the multiple dimensions of the proteome, thereby transforming our understanding and interactions with human disease in the coming decade.
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| 22. |
- Buus, S., et al.
(författare)
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High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays
- 2012
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 11:12, s. 1790-1800
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level.
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| 23. |
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| 24. |
- Chahal, Gurdeep, et al.
(författare)
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A Complex Connection Between the Diversity of Human Gastric Mucin O-Glycans, Helicobacter pylori Binding, Helicobacter Infection and Fucosylation
- 2022
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Ingår i: Molecular & Cellular Proteomics. - : Elsevier BV. - 1535-9476 .- 1535-9484. ; 21:11
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Tidskriftsartikel (refereegranskat)abstract
- Helicobacter pylori colonizes the stomach of half of the human population. Most H. pylori are located in the mucus layer, which is mainly comprised by glycosylated mucins. Using mass spectrometry, we identified 631 glycans (whereof 145 were fully characterized and the remainder assigned as compositions) on mucins isolated from 14 Helicobacter spp.-infected and 14 Helicobacter spp.-noninfected stomachs. Only six identified glycans were common to all individuals, from a total of 60 to 189 glycans in each individual. An increased number of unique glycan structures together with an increased intra-individual diversity and larger interindividual variation were identified among O-glycans from Helicobacter spp.-infected stomachs compared with noninfected stom-achs. H. pylori strain J99, which carries the blood group antigen-binding adhesin (BabA), the sialic acid-binding adhesin (SabA), and the LacdiNAc-binding adhesin, bound both to Lewis b (Leb)-positive and Leb-negative mucins. Among Leb-positive mucins, H. pylori J99 bind-ing was higher to mucins from Helicobacter spp.-infected individuals than noninfected individuals. Statistical corre-lation analysis, binding experiments with J99 wt, and J99 Delta babA Delta sabA and inhibition experiments using syn-thetic glycoconjugates demonstrated that the differences in H. pylori-binding ability among these four groups were governed by BabA-dependent binding to fucosylated structures. LacdiNAc levels were lower in mucins that bound to J99 lacking BabA and SabA than in mucins that did not, suggesting that LacdiNAc did not significantly contribute to the binding. We identified 24 O-glycans from Leb-negative mucins that correlated well with H. pylori binding whereof 23 contained alpha 1,2-linked fucosylation. The large and diverse gastric glycan library identified, including structures that correlated with H. pylori binding, could be used to select glycodeterminants to experimen-tally investigate further for their importance in host- pathogen interactions and as candidates to develop glycan-based therapies.
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| 25. |
- Chornoguz, Olesya, et al.
(författare)
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Proteomic Pathway Analysis Reveals Inflammation Increases Myeloid-Derived Suppressor Cell Resistance to Apoptosis
- 2011
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory settings. Proteomic pathway analysis identified the Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by increasing MDSC resistance to Fas-mediated apoptosis. The MS findings were validated and extended by biological studies. Using activated caspase 3 and caspase 8 as indicators of apoptosis, flow cytometry, confocal microscopy, and Western blot analyses demonstrated that inflammation-induced MDSC treated with a Fas agonist contain lower levels of activated caspases, suggesting that inflammation enhances resistance to Fas-mediated apoptosis. Resistance to Fas-mediated apoptosis was confirmed by viability studies of MDSC treated with a Fas agonist. These results suggest that an inflammatory environment, which is frequently present in tumor-bearing individuals, protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life, and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings.
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