| 41. |
- Fisher, Jane, et al.
(författare)
-
Proteome profiling of recombinant DNase therapy in reducing NETs and aiding recovery in COVID-19 patients
- 2021
-
Ingår i: Molecular and Cellular Proteomics. - : Elsevier BV. - 1535-9484 .- 1535-9476. ; 20
-
Tidskriftsartikel (refereegranskat)abstract
- Severe COVID-19 can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hall mark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe COVID-19 patients reveal high levels of neutrophil extracellular trap(s) (NETs) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and can lead to acute respiratory distress syndrome (ARDS). Recombinant human DNase (rhDNase/Pulmozyme) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated 5 COVID-19 patients presenting acute symptoms with clinically approved aerosolized Pulmozyme. No adverse reactions to the drug were seen, and improved oxygen saturation and recovery in all severely ill COVID-19 patients was observed after therapy. Immunofluorescence and proteome analysis of sputum and blood plasma samples after treatment revealed a marked reduction of NETs and a set of statistically significant proteome changes that indicate reduction of haemorrhage, plasma leakage and inflammation in the airways, and reduced systemic inflammatory state in the blood plasma of patients. Taken together, the results indicate that NETs contribute to acute respiratory failure in COVID-19 and that degrading NETs may reduce dependency on external high flow oxygen therapy in patients. Targeting NETs using rhDNase may have significant therapeutic implications in COVID-19 disease and warrants further studies.
|
|
| 42. |
- Forsström, Bjorn, et al.
(författare)
-
Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays
- 2014
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 13:6, s. 1585-1597
-
Tidskriftsartikel (refereegranskat)abstract
- Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on-and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.
|
|
| 43. |
- Fälth, Maria, et al.
(författare)
-
Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides
- 2007
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 6:7, s. 1188-1197
-
Tidskriftsartikel (refereegranskat)abstract
- A new approach using targeted sequence collections has been developed for identifying endogenous peptides. This approach enables a fast, specific, and sensitive identification of endogenous peptides. Three different sequence collections were constituted in this study to mimic the peptidomic samples: SwePep precursors, SwePep peptides, and SwePep predicted. The searches for neuropeptides performed against these three sequence collections were compared with searches performed against the entire mouse proteome, which is commonly used to identify neuropeptides. These four sequence collections were searched with both Mascot and X! Tandem. Evaluation of the sequence collections was achieved using a set of manually identified and previously verified peptides. By using the three new sequence collections, which more accurately mimic the sample, 3 times as many peptides were significantly identified, with a false-positive rate below 1%, in comparison with the mouse proteome. The new sequence collections were also used to identify previously uncharacterized peptides from brain tissue; 27 previously uncharacterized peptides and potentially bioactive neuropeptides were identified. These novel peptides are cleaved from the peptide precursors at sites that are characteristic for prohormone convertases, and some of them have post-translational modifications that are characteristic for neuropeptides. The targeted protein sequence collections for different species are publicly available for download from SwePep.
|
|
| 44. |
- Fälth, Maria, et al.
(författare)
-
SwePep – A database designed for endogenous peptides and mass spectrometry
- 2006
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 5:6, s. 998-1005
-
Tidskriftsartikel (refereegranskat)abstract
- A new database, SwePep, specifically designed for endogenous peptides, has been constructed to significantly speed up the identification process from complex tissue samples utilizing mass spectrometry. In the identification process the experimental peptide masses are compared with the peptide masses stored in the database both with and without possible post-translational modifications. This intermediate identification step is fast and singles out peptides that are potential endogenous peptides and can later be confirmed with tandem mass spectrometry data. Successful applications of this methodology are presented. The SwePep database is a relational database developed using MySql and Java. The database contains 4180 annotated endogenous peptides from different tissues originating from 394 different species as well as 50 novel peptides from brain tissue identified in our laboratory. Information about the peptides, including mass, isoelectric point, sequence, and precursor protein, is also stored in the database. This new approach holds great potential for removing the bottleneck that occurs during the identification process in the field of peptidomics. The SwePep database is available to the public.
|
|
| 45. |
- Geiger, T., et al.
(författare)
-
Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse
- 2013
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 12:6, s. 1709-1722
-
Tidskriftsartikel (refereegranskat)abstract
- Identifying the building blocks of mammalian tissues is a precondition for understanding their function. In particular, global and quantitative analysis of the proteome of mammalian tissues would point to tissue-specific mechanisms and place the function of each protein in a whole-organism perspective. We performed proteomic analyses of 28 mouse tissues using high-resolution mass spectrometry and used a mix of mouse tissues labeled via stable isotope labeling with amino acids in cell culture as a "spike-in" internal standard for accurate protein quantification across these tissues. We identified a total of 7,349 proteins and quantified 6,974 of them. Bioinformatic data analysis showed that physiologically related tissues clustered together and that highly expressed proteins represented the characteristic tissue functions. Tissue specialization was reflected prominently in the proteomic profiles and is apparent already in their hundred most abundant proteins. The proportion of strictly tissue-specific proteins appeared to be small. However, even proteins with household functions, such as those in ribosomes and spliceosomes, can have dramatic expression differences among tissues. We describe a computational framework with which to correlate proteome profiles with physiological functions of the tissue. Our data will be useful to the broad scientific community as an initial atlas of protein expression of a mammalian species.
|
|
| 46. |
|
|
| 47. |
- Ghoshal, Biraja, et al.
(författare)
-
DeepHistoClass : A Novel Strategy for Confident Classification of Immunohistochemistry Images Using Deep Learning
- 2021
-
Ingår i: Molecular & Cellular Proteomics. - : Elsevier. - 1535-9476 .- 1535-9484. ; 20
-
Tidskriftsartikel (refereegranskat)abstract
- A multitude of efforts worldwide aim to create a single-cell reference map of the human body, for fundamental understanding of human health, molecular medicine, and targeted treatment. Antibody-based proteomics using immunohistochemistry (IHC) has proven to be an excellent technology for integration with large-scale single-cell transcriptomics datasets. The golden standard for evaluation of IHC staining patterns is manual annotation, which is expensive and may lead to subjective errors. Artificial intelligence holds much promise for efficient and accurate pattern recognition, but confidence in prediction needs to be addressed. Here, the aim was to present a reliable and comprehensive framework for automated annotation of IHC images. We developed a multilabel classification of 7848 complex IHC images of human testis corresponding to 2794 unique proteins, generated as part of the Human Protein Atlas (HPA) project. Manual annotation data for eight different cell types was generated as a basis for training and testing a proposed Hybrid Bayesian Neural Network. By combining the deep learning model with a novel uncertainty metric, DeepHistoClass (DHC) Confidence Score, the average diagnostic performance improved from 86.9% to 96.3%. This metric not only reveals which images are reliably classified by the model, but can also be utilized for identification of manual annotation errors. The proposed streamlined workflow can be developed further for other tissue types in health and disease and has important implications for digital pathology initiatives or large-scale protein mapping efforts such as the HPA project.
|
|
| 48. |
- Gloriam, David E., et al.
(författare)
-
A Community Standard Format for the Representation of Protein Affinity Reagents
- 2010
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 9:1, s. 1-10
-
Tidskriftsartikel (refereegranskat)abstract
- Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one online warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site. Molecular & Cellular Proteomics 9: 1-10, 2010.
|
|
| 49. |
- Götzke, Hansjörg, et al.
(författare)
-
Identification of Putative Substrates for the Periplasmic Chaperone YfgM in Escherichia coli Using Quantitative Proteomics
- 2015
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 14:1, s. 216-226
-
Tidskriftsartikel (refereegranskat)abstract
- How proteins are trafficked, folded, and assembled into functional units in the cell envelope of Gram-negative bacteria is of significant interest. A number of chaperones have been identified, however, the molecular roles of these chaperones are often enigmatic because it has been challenging to assign substrates. Recently we discovered a novel periplasmic chaperone, called YfgM, which associates with PpiD and the SecYEG translocon and operates in a network that contains Skp and SurA. The aim of the study presented here was to identify putative substrates of YfgM. We reasoned that substrates would be incorrectly folded or trafficked when YfgM was absent from the cell, and thus more prone to proteolysis (the loss-of-function rationale). We therefore used a comparative proteomic approach to identify cell envelope proteins that were lower in abundance in a strain lacking yfgM, and strains lacking yfgM together with either skp or surA. Sixteen putative substrates were identified. The list contained nine inner membrane proteins (CusS, EvgS, MalF, OsmC, TdcB, TdcC, WrbA, YfhB, and YtfH) and seven periplasmic proteins (HdeA, HdeB, AnsB, Ggt, MalE, YcgK, and YnjE), but it did not include any lipoproteins or outer membrane proteins. Significantly, AnsB (an asparaginase) and HdeB (a protein involved in the acid stress response), were lower in abundance in all three strains lacking yfgM. For both genes, we ruled out the possibility that they were transcriptionally down-regulated, so it is highly likely that the corresponding proteins are misfolded/mistargeted and turned-over in the absence of YfgM. For HdeB we validated this conclusion in a pulse-chase experiment. The identification of HdeB and other cell envelope proteins as potential substrates will be a valuable resource for follow-up experiments that aim to delineate molecular the function of YfgM.
|
|
| 50. |
- Hamsten, Carl, 1981-, et al.
(författare)
-
Recombinant surface proteomics as a tool to analyze humoral immune responses in bovines infected by Mycoplasma mycoides subsp. mycoides SC
- 2009
-
Ingår i: Molecular & Cellular Proteomics. - Stanford : HighWire Press. - 1535-9476 .- 1535-9484. ; 8:11, s. 2544-2554
-
Tidskriftsartikel (refereegranskat)abstract
- A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causing agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP affected cattle and controls were monitored against one third of the surface proteins of M. mycoides SC in a high-throughput magnetic bead based assay. First, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in E. coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP positive and negative sera. Signals were proven to be protein-specific by inhibition experiments and results agreed with western blot experiments. The assay's potential to monitor IgG, IgM and IgA responses over time was shown in a proof-of-concept study with 116 sera from 8 animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M mycoides SC, has been created.
|
|
