| 1. |
- Iglesias, Maria Jesus, et al.
(författare)
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Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Venous thromboembolism (VTE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. Here we show, using proteomics profiling to screen plasma from patients with suspected acute VTE, and several case-control studies for VTE, how Complement Factor H Related 5 protein (CFHR5), a regulator of the alternative pathway of complement activation, is a VTE-associated plasma biomarker. In plasma, higher CFHR5 levels are associated with increased thrombin generation potential and recombinant CFHR5 enhanced platelet activation in vitro. GWAS analysis of ~52,000 participants identifies six loci associated with CFHR5 plasma levels, but Mendelian randomization do not demonstrate causality between CFHR5 and VTE. Our results indicate an important role for the regulation of the alternative pathway of complement activation in VTE and that CFHR5 represents a potential diagnostic and/or risk predictive plasma biomarker.
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| 2. |
- Andersson, Annika, et al.
(författare)
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Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease
- 2019
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Ingår i: Clinica Chimica Acta. - : Elsevier B.V.. - 0009-8981 .- 1873-3492. ; 494, s. 79-93
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Tidskriftsartikel (refereegranskat)abstract
- Detailed knowledge of protein changes in cerebrospinal fluid (CSF) across healthy and diseased individuals would provide a better understanding of the onset and progression of neurodegenerative disorders. In this study, we selected 20 brain-enriched proteins previously identified in CSF by antibody suspension bead arrays (SBA) to be potentially biomarkers for Alzheimer's disease (AD) and verified these using an orthogonal approach. We examined the same set of 94 CSF samples from patients affected by AD (including preclinical and prodromal), mild cognitive impairment (MCI), non-AD dementia and healthy individuals, which had previously been analyzed by SBA. Twenty-eight parallel reaction monitoring (PRM) assays were developed and 13 of them could be validated for protein quantification. Antibody profiles were verified by PRM. For seven proteins, the antibody profiles were highly correlated with the PRM results (r > 0.7) and GAP43, VCAM1 and PSAP were identified as potential markers of preclinical AD. In conclusion, we demonstrate the usefulness of targeted mass spectrometry as a tool for the orthogonal verification of antibody profiling data, suggesting that these complementary methods can be successfully applied for comprehensive exploration of CSF protein levels in neurodegenerative disorders.
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| 3. |
- Edfors, Fredrik, et al.
(författare)
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Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics
- 2019
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 18:7, s. 2706-2718
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Tidskriftsartikel (refereegranskat)abstract
- The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts, and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labeled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labeled recombinant proteins before trypsin digestion enables short digestion protocols (<= 60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINAS, and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.
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| 4. |
- Hober, Andreas, 1992-, et al.
(författare)
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Evaluation of an enhanced antibody-validation strategy for Western blot applications based on migration pattern recognition
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The use of affinity reagents, such as antibodies, for studying specific molecules in complex backgrounds are some of the most powerful tools for researchers in molecular biology. However, all experiments performed using affinity reagents are directly affected by each reagent’s context-dependent ability to bind specifically to a target of interest. A growing issue with non-validated, or poorly validated affinity reagents, has been highlighted by the International Working Group for Antibody Validation (IWGAV). It has been suggested that antibodies should be evaluated in an application-specific manner since they can perform well in one application but fail to deliver reproducible results in another. One of the most commonly used antibody-based applications is the Western blot (WB) technology. When evaluating the result from a WB experiment, the initial measure used for determining whether or not the antibody binds the protein of interest is to determine the molecular weight of the protein detected by the antibody compared to a set of reference proteins. As WB relies on the SDS-PAGE for separating differently sized proteins, the comparison is actually based on protein migration during electrophoresis. It is, however, well known that the migration of a protein can differ significantly from how the reference proteins migrate. Here, we suggest a method for determining the actual migration patterns of proteins instead of relying on the theoretical molecular weight of the protein. Using this approach, called migration capture mass spectrometry (MS), a dataset containing the migration patterns of more than 39,000 protein products from more than 10,500 genes across eleven cell lines and tissues has been created. This migration capture MS approach has been validated using k-fold cross validation against 249 siRNA knockdown WBs showing that the method has a sensitivity of 96.4%, specificity of 87.4% and accuracy of 91.9%, which makes the dataset a useful resource that can facilitate antibody validation strategies in a fit-for-purpose manner. The data set has allowed the automatic evaluation of more than 12,000 antibodies in the Human Protein Atlas using the method.
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| 5. |
- Hober, Andreas, 1992-, et al.
(författare)
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Targeted Proteomics Using Stable Isotope Labeled Protein Fragments Enables Precise and Robust Determination of Total Apolipoprotein(a) in Human Plasma
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Lipoprotein(a), also known as Lp(a), is an LDL-like particle composed of apolipoprotein(a) (apo(a)) bound covalently to apolipoprotein B100. Plasma concentrations of Lp(a) are highly heritable and vary widely between individuals. Elevated plasma concentration of Lp(a) is considered as an independent, causal risk factor of cardiovascular disease (CVD). Targeted mass spectrometry (LC-SRM/MS) combined with stable isotope-labeled recombinant proteins provides robust and precise quantification of proteins in the blood, making LC-SRM/MS assays appealing for monitoring plasma proteins for clinical implications. This study presents a novel quantitative approach, based on proteotypic peptides, to determine the absolute concentration of apo(a) from two microliters of plasma and qualified according to guideline requirements for targeted proteomics assays. After optimization, assay parameters such as linearity, lower limits of quantification (LLOQ), intra-assay variability (CV: 4.7%) and inter-assay repeatability (CV: 7.8%) were determined and the LC-SRM/MS results were benchmarked against a commercially available immunoassay. In summary, the measurements of an apo(a) single copy specific peptide and a kringle 4 specific peptide allows for the determination of molar concentration and relative size of apo(a) in individuals.
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| 6. |
- Hober, Andreas, 1992-, et al.
(författare)
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Targeted proteomics using stable isotope labeled protein fragments enables precise and robust determination of total apolipoprotein(a) in human plasma
- 2023
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 18:2 February
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Tidskriftsartikel (refereegranskat)abstract
- Lipoprotein(a), also known as Lp(a), is an LDL-like particle composed of apolipoprotein(a) (apo(a)) bound covalently to apolipoprotein B100. Plasma concentrations of Lp(a) are highly heritable and vary widely between individuals. Elevated plasma concentration of Lp(a) is considered as an independent, causal risk factor of cardiovascular disease (CVD). Targeted mass spectrometry (LC-SRM/MS) combined with stable isotope-labeled recombinant proteins provides robust and precise quantification of proteins in the blood, making LC-SRM/ MS assays appealing for monitoring plasma proteins for clinical implications. This study presents a novel quantitative approach, based on proteotypic peptides, to determine the absolute concentration of apo(a) from two microliters of plasma and qualified according to guideline requirements for targeted proteomics assays. After optimization, assay parameters such as linearity, lower limits of quantification (LLOQ), intra-assay variability (CV: 4.7%) and inter-assay repeatability (CV: 7.8%) were determined and the LC-SRM/MS results were benchmarked against a commercially available immunoassay. In summary, the measurements of an apo(a) single copy specific peptide and a kringle 4 specific peptide allow for the determination of molar concentration and relative size of apo(a) in individuals.
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| 7. |
- Hong, Mun-Gwan, et al.
(författare)
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Profiles of histidine-rich glycoprotein associate with age and risk of all-cause mortality
- 2020
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Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 3:10, s. e202000817-
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Tidskriftsartikel (refereegranskat)abstract
- Despite recognizing aging as a common risk factor of many human diseases, little is known about its molecular traits. To identify age-associated proteins circulating in human blood, we screened 156 individuals aged 50–92 using exploratory and multiplexed affinity proteomics assays. Profiling eight additional study sets (N = 3,987), performing antibody validation, and conducting a meta-analysis revealed a consistent age association (P = 6.61 × 10−6) for circulating histidine-rich glycoprotein (HRG). Sequence variants of HRG influenced how the protein was recognized in the immunoassays. Indeed, only the HRG profiles affected by rs9898 were associated with age and predicted the risk of mortality (HR = 1.25 per SD; 95% CI = 1.12–1.39; P = 6.45 × 10−5) during a follow-up period of 8.5 yr after blood sampling (IQR = 7.7–9.3 yr). Our affinity proteomics analysis found associations between the particular molecular traits of circulating HRG with age and all-cause mortality. The distinct profiles of this multipurpose protein could serve as an accessible and informative indicator of the physiological processes related to biological aging.
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| 8. |
- Johansson, Camilla, et al.
(författare)
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Monitoring Biomarker Study in Becker Muscular Dystrophy using Data Independent Acquisition LC-MS/MS
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Becker muscular dystrophy (BMD) is a rare and heterogenous form of dystrophinopathy caused by reduced expression of altered dystrophin protein. Gene therapies and exon-skipping therapies for the more severe form of dystrophinopathy, Duchenne muscular dystrophy (DMD), assumes that by promoting partial dystrophin expression in DMD patients, their disease progression could be reduced. Several studies have identified potential progression biomarkers for DMD and hypothesised in their usefulness in monitoring pharmacodynamic response in gene-therapy clinical trials. However, knowledge of progression changes of blood proteome in BMD is lacking. In this study, we aimed at exploring differences in proteomic changes between DMD and BMD in a prospective longitudinal 4-year study as well as explore what proteins relate to functional performance in BMD patients. Serum from 48 BMD patients and 19 DMD patients were analysed using Data Independent Acquisition Tandem Mass Spectrometry (DIA-MS). Linear mixed effects models identified 17 proteins with altered longitudinal signatures between DMD and BMD, among these CKM, PKM and ALDOA. Furthermore, bikunin (product of AMBP gene), C3 and IGHG2 were found related to functional performance in BMD patients.
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| 9. |
- Karlsson, Max J., et al.
(författare)
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Inflammation and Apolipoproteins Are Potential Biomarkers for Stratification of Cutaneous Melanoma Patients for Immunotherapy and Targeted Therapy
- 2021
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Ingår i: Cancer Research. - : American Association for Cancer Research (AACR). - 0008-5472 .- 1538-7445. ; 81:9, s. 2545-2555
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Tidskriftsartikel (refereegranskat)abstract
- Malignant cutaneous melanoma is one of the most common cancers in young adults. During the last decade, targeted and immunotherapies have significantly increased the overall survival of patients with malignant cutaneous melanoma. Nevertheless, disease progression is common, and a lack of predictive biomarkers of patient response to therapy hinders individualized treatment strategies. To address this issue, we performed a longitudinal study using an unbiased proteomics approach to identify and quantify proteins in plasma both before and during treatment from 109 patients treated with either targeted or immunotherapy. Linear modeling and machine learning approaches identified 43 potential prognostic and predictive biomarkers. A reverse correlation between apolipoproteins and proteins related to inflammation was observed. In the immunotherapy group, patients with low pretreatment expression of apolipoproteins and high expression of inflammation markers had shorter progression-free survival. Similarly, increased expression of LDHB during treatment elicited a significant impact on response to immunotherapy. Overall, we identified potential common and treatment-specific biomarkers in malignant cutaneous melanoma, paving the way for clinical use of these biomarkers following validation on a larger cohort. Significance: This study identifies a potential biomarker panel that could improve the selection of therapy for patients with cutaneous melanoma.
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| 10. |
- Kotol, David, et al.
(författare)
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Absolute Quantification of Pan-Cancer Plasma Proteomes Reveals Unique Signature in Multiple Myeloma
- 2023
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 15:19
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry based on data-independent acquisition (DIA) has developed into a powerful quantitative tool with a variety of implications, including precision medicine. Combined with stable isotope recombinant protein standards, this strategy provides confident protein identification and precise quantification on an absolute scale. Here, we describe a comprehensive targeted proteomics approach to profile a pan-cancer cohort consisting of 1800 blood plasma samples representing 15 different cancer types. We successfully performed an absolute quantification of 253 proteins in multiplex. The assay had low intra-assay variability with a coefficient of variation below 20% (CV = 17.2%) for a total of 1013 peptides quantified across almost two thousand injections. This study identified a potential biomarker panel of seven protein targets for the diagnosis of multiple myeloma patients using differential expression analysis and machine learning. The combination of markers, including the complement C1 complex, JCHAIN, and CD5L, resulted in a prediction model with an AUC of 0.96 for the identification of multiple myeloma patients across various cancer patients. All these proteins are known to interact with immunoglobulins.
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| 11. |
- Kotol, David, et al.
(författare)
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Longitudinal Plasma Protein Profiling Using Targeted Proteomics and Recombinant Protein Standards
- 2020
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 19:12, s. 4815-4825
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Tidskriftsartikel (refereegranskat)abstract
- Spike-in of standards of known concentrations used in proteomics-based workflows is an attractive approach for both accurate and precise multiplexed protein quantification. Here, a quantitative method based on targeted proteomics analysis of plasma proteins using isotope-labeled recombinant standards originating from the Human Protein Atlas project has been established. The standards were individually quantified prior to being employed in the final multiplex assay. The assays are mainly directed toward actively secreted proteins produced in the liver, but may also originate from other parts of the human body. This study included 21 proteins classified by the FDA as either drug targets or approved clinical protein biomarkers. We describe the use of this multiplex assay for profiling a well-defined human cohort with sample collection spanning over a one-year period. Samples were collected at four different time points, which allowed for a longitudinal analysis to assess the variable plasma proteome within individuals. Two assays toward APOA1 and APOB had available clinical data, and the two assays were benchmarked against each other. The clinical assay is based on antibodies and shows high correlation between the two orthogonal methods, suggesting that targeted proteomics with highly parallel, multiplex analysis is an attractive alternative to antibody-based protein assays.
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| 12. |
- Kotol, David
(författare)
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Multiplexed absolute quantification of blood plasma proteins using targeted proteomics
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are the molecular building blocks of all living organisms. They are the functioning actors in metabolism and communication, and they give specific architecture and purpose to all tissues. Their abundance is dynamic and can be interpreted and associated to different physiological states or diseases. Blood plasma, the liquid component of blood, is a proximal fluid to all organs in the human body. As a consequence, it contains an immense amount of information about wide variety of biological processes. This makes it a great sample to study but also demanding due to the complexity and dynamic range of protein concentrations. A promising technology capable of extracting the information is targeted proteomics. Especially in combination with heavy labelled standards it combines liquid chromatography and tandem mass spectrometry (LC-MS/MS) into state-of-the-art quantification on an absolute scale. This toolbox is capable of detecting the smallest perturbations thanks to its precision and multiplexing capability. In this thesis, I describe our efforts to develop and use a novel platform for targeted proteomics suitable for clinical applications with high demands on accuracy and reliability.In Paper I, a deep dive into the resource of standards coming from The Human Protein Atlas was performed. It was possible to identify their great analytical performance and generate LC-MS/MS coordinates for over 25,000 proteotypic peptides from over 10,000 proteins. A proof-of-concept experiment was designed based on the screening results where 23 proteins in blood plasma were absolutely quantified. The data allowed us to identify distinct clusters of individuals based on their LPA, APOE, SERPINA5 and TFRC levels. Paper II directly builds on the findings from Paper I and applies them on a well-defined cohort of 94 individuals whose blood plasma samples were collected over one-year period in four different time-points. The longitudinal protein profiling allowed to assess the variability of plasma proteome within individuals with the developed assay. The applied assay included 52 target proteins with mainly actively secreted liver proteins and 21 FDA approved targets. The results were benchmarked with clinical data for APOA1 and APOB and showed high correlation. This suggests that targeted proteomics and multiplex absolute quantification has an appealing potential as an alternative to antibody-based assays.In Paper III, the labelled standards underwent a thorough investigation in regard to their stability and reproducibility of quantification. Also, a novel formulation of standards was devised. Here, the standards were stored in chaotropic agent and subsequently dried down. This allows for addition only protocols, upscaling, increased reproducibility, and storage at ambient temperature for at least one month with retained analytical performance. This novel workflow was used for multiplex quantification of 100 clinically relevant protein targets in blood plasma using almost 300 peptides with median coefficient of variation below 10%.Paper IV expands on Paper III and applies the technology to profile 1,800 individuals in a pan-cancer study. The blood plasma was collected from individuals suffering from one of 15 different cancer types. The applied assay targeted 253 proteins and was able to quantify 1,013 peptides with low variation across the four months of analysis time. Several potential biomarkers for multiple myeloma were identified with the most useful targets consisting of the complement complex C1 and the JCHAIN and CD5L proteins.
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| 13. |
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| 14. |
- Kotol, David, et al.
(författare)
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Targeted proteomics analysis of plasma proteins using recombinant protein standards for addition only workflows
- 2021
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 71:3, s. 473-483
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Tidskriftsartikel (refereegranskat)abstract
- Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation <10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed.
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| 15. |
- Li, Xiangyu, et al.
(författare)
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Discovery of Functional Alternatively Spliced PKM Transcripts in Human Cancers
- 2021
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:2, s. 1-23
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Pyruvate kinase muscle type (PKM) is a key enzyme in glycolysis and is a mediator of the Warburg effect in tumors. The association of PKM with survival of cancer patients is controversial. In this study, we investigated the associations of the alternatively spliced transcripts of PKM with cancer patients' survival outcomes and explained the conflicts in previous studies. We discovered three poorly studied alternatively spliced PKM transcripts that exhibited opposite prognostic indications in different human cancers based on integrative systems analysis. We also detected their protein products and explored their potential biological functions based on in-vitro experiments. Our analysis demonstrated that alternatively spliced transcripts of not only PKM but also other genes should be considered in cancer studies, since it may enable the discovery and targeting of the right protein product for development of the efficient treatment strategies. Pyruvate kinase muscle type (PKM) is a key enzyme in glycolysis and plays an important oncological role in cancer. However, the association of PKM expression and the survival outcome of patients with different cancers is controversial. We employed systems biology methods to reveal prognostic value and potential biological functions of PKM transcripts in different human cancers. Protein products of transcripts were shown and detected by western blot and mass spectrometry analysis. We focused on different transcripts of PKM and investigated the associations between their mRNA expression and the clinical survival of the patients in 25 different cancers. We find that the transcripts encoding PKM2 and three previously unstudied transcripts, namely ENST00000389093, ENST00000568883, and ENST00000561609, exhibited opposite prognostic indications in different cancers. Moreover, we validated the prognostic effect of these transcripts in an independent kidney cancer cohort. Finally, we revealed that ENST00000389093 and ENST00000568883 possess pyruvate kinase enzymatic activity and may have functional roles in metabolism, cell invasion, and hypoxia response in cancer cells. Our study provided a potential explanation to the controversial prognostic indication of PKM, and could invoke future studies focusing on revealing the biological and oncological roles of these alternative spliced variants of PKM.
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| 16. |
- Malm, Magdalena, 1983-, et al.
(författare)
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Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.
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| 17. |
- Malm, Magdalena, 1983-, et al.
(författare)
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Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins
- 2022
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 171-187
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Tidskriftsartikel (refereegranskat)abstract
- Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.
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| 18. |
- Sporre, Emil, et al.
(författare)
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Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Metabolite-level regulation of enzyme activity is important for coping with environmental shifts. Recently developed proteomics methodologies allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feedback and feedforward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify metabolite-protein interactions in four Calvin-cycle harboring bacteria, including two cyanobacteria and two chemolithoautotrophs. We identified widespread protein interactions with the metabolites GAP, ATP, and AcCoA in all strains. Some species-specific interactions were also observed, such as sugar phosphates in Cupravidus necator and glyoxylate in Synechocystis sp. PCC 6803. We screened some metabolites with LiP interactions for their effects on kinetics of the enzymes F/SBPase and transketolase, two enzymatic steps of the Calvin cycle. For both Synechocystis and Cupriavidus F/SBPase, GAP showed an activating effect that may be part of feed-forward regulation in the Calvin cycle. While we verified multiple enzyme inhibitors on transketolase, the effect on kinetics was often small. Incorporation of F/SBPase and transketolase regulations into a kinetic metabolic model of Synechocystis central metabolism resulted in a general decreased stability of the network, and altered flux control coefficients of transketolase as well as other reactions. The LiP-SMap methodology is promising for uncovering new modes of metabolic regulation, but will benefit from improved peptide quantification and higher peptide coverage of enzymes, as known interactions are often not detected for low-coverage proteins. . Furthermore, not all LiP interactions appear to be relevant for catalysis, as 4/8 (transketolase) and 5/6 (F/SBPase) of the tested LiP effectors had an effect in in vitroassays.
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| 19. |
- Sporre, Emil, et al.
(författare)
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Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation
- 2022
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering to improve industrial phenotypes. Recently developed chemoproteomics workflows allow for genome-wide detection of metabolite-protein interactions that may regulate pathway activity. We applied limited proteolysis small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions showed that some metabolites interacted in a species-specific manner, such as interactions of glucose-6-phosphate in Cupriavidus necator and of glyoxylate in Synechocystis sp PCC 6803. These are interpreted in light of the different central carbon conversion pathways present. Metabolites interacting with the Calvin cycle enzymes fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) and transketolase were tested for effects on catalytic activity in vitro. The Calvin cycle intermediate glyceraldehyde-3-phosphate activated both Synechocystis and Cupriavidus F/SBPase, which suggests a feed-forward activation of the cycle in both photoautotrophs and chemolithoautotrophs. In contrast to the stimulating effect in reduced conditions, glyceraldehyde-3-phosphate inactivated the Synechocystis F/SBPase in oxidized conditions by accelerating protein aggregation. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated and may act in addition to redox regulation.
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| 20. |
- Sporre, Emil, et al.
(författare)
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Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation
- 2023
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering for biotechnology. Here we apply limited proteolysis-small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions shows that some metabolites interact in a species-specific manner. We estimate that approximately 35% of interacting metabolites affect enzyme activity in vitro, and the effect is often minor. Using LiP-SMap data as a guide, we find that the Calvin cycle intermediate glyceraldehyde-3-phosphate enhances activity of fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) from Synechocystis sp. PCC 6803 and Cupriavidus necator in reducing conditions, suggesting a convergent feed-forward activation of the cycle. In oxidizing conditions, glyceraldehyde-3-phosphate inhibits Synechocystis F/SBPase by promoting enzyme aggregation. In contrast, the glycolytic intermediate glucose-6-phosphate activates F/SBPase from Cupriavidus necator but not F/SBPase from Synechocystis. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated.
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| 21. |
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| 22. |
- Uhlén, Mathias, et al.
(författare)
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The human secretome
- 2019
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 12:609
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Tidskriftsartikel (refereegranskat)abstract
- The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immuno-assays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.
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| 23. |
- Woessmann, Jakob, et al.
(författare)
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Addressing the Protease Bias in Quantitative Proteomics
- 2022
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 21:10, s. 2526-2534
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Tidskriftsartikel (refereegranskat)abstract
- Protein quantification strategies using multiple proteases have been shown to deliver poor interprotease accuracy in label-free mass spectrometry experiments. By utilizing six different proteases with different cleavage sites, this study explores the protease bias and its effect on accuracy and precision by using recombinant protein standards. We established 557 SRM assays, using a recombinant protein standard resource, toward 10 proteins in human plasma and determined their concentration with multiple proteases. The quantified peptides of these plasma proteins spanned 3 orders of magnitude (0.02-70 μM). In total, 60 peptides were used for absolute quantification and the majority of the peptides showed high robustness. The retained reproducibility was achieved by quantifying plasma proteins using spiked stable isotope standard recombinant proteins in a targeted proteomics workflow.
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| 24. |
- Woessmann, Jakob, et al.
(författare)
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Assessing the Role of Trypsin in Quantitative Plasma and Single-Cell Proteomics toward Clinical Application
- 2023
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 95:36, s. 13649-13658
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry-based bottom-up proteomics is rapidly evolving and routinely applied in large-scale biomedical studies. Proteases are a central component of every bottom-up proteomics experiment, digesting proteins into peptides. Trypsin has been the most widely applied protease in proteomics due to its characteristics. With ever-larger cohort sizes and possible future clinical application of mass spectrometry-based proteomics, the technical impact of trypsin becomes increasingly relevant. To assess possible biases introduced by trypsin digestion, we evaluated the impact of eight commercially available trypsins in a variety of bottom-up proteomics experiments and across a range of protease concentrations and storage times. To investigate the universal impact of these technical attributes, we included bulk HeLa cell lysate, human plasma, and single HEK293 cells, which were analyzed over a range of selected reaction monitoring (SRM), data-independent acquisition (DIA), and data-dependent acquisition (DDA) instrument methods on three LC-MS instruments. The quantification methods employed encompassed both label-free approaches and absolute quantification utilizing spike-in heavy-labeled recombinant protein fragment standards. Based on this extensive data set, we report variations between commercial trypsins, their source, and their concentration. Furthermore, we provide suggestions on the handling of trypsin in large-scale studies.
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| 25. |
- Zheng, Tianyu, et al.
(författare)
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Characterization of reduced astrocyte creatine kinase levels in Alzheimer's disease
- 2024
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Ingår i: Glia. - : WILEY. - 0894-1491 .- 1098-1136.
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Tidskriftsartikel (refereegranskat)abstract
- The creatine-phosphocreatine cycle serves as a crucial temporary energy buffering system in the brain, regulated by brain creatine kinase (CKB), in maintaining Adenosine triphosphate (ATP) levels. Alzheimer's disease (AD) has been linked to increased CKB oxidation and loss of its regulatory function, although specific pathological processes and affected cell types remain unclear. In our study, cerebral cortex samples from individuals with AD, dementia with Lewy bodies (DLB), and age-matched controls were analyzed using antibody-based methods to quantify CKB levels and assess alterations associated with disease processes. Two independently validated antibodies exclusively labeled astrocytes in the human cerebral cortex. Combining immunofluorescence (IF) and mass spectrometry (MS), we explored CKB availability in AD and DLB cases. IF and Western blot analysis demonstrated a loss of CKB immunoreactivity correlated with increased plaque load, severity of tau pathology, and Lewy body pathology. However, transcriptomics data and targeted MS demonstrated unaltered total CKB levels, suggesting posttranslational modifications (PTMs) affecting antibody binding. This aligns with altered efficiency at proteolytic cleavage sites indicated in the targeted MS experiment. These findings highlight that the proper function of astrocytes, understudied in the brain compared with neurons, is highly affected by PTMs. Reduction in ATP levels within astrocytes can disrupt ATP-dependent processes, such as the glutamate-glutamine cycle. As CKB and the creatine-phosphocreatine cycle are important in securing constant ATP availability, PTMs in CKB, and astrocyte dysfunction may disturb homeostasis, driving excitotoxicity in the AD brain. CKB and its activity could be promising biomarkers for monitoring early-stage energy deficits in AD. Brain creatine kinase (CKB) mainly expressed in strocytes in the human brain. A loss of CKB immunoreactivity correlated with increased plaque load. CKB antibody binding might be affected by posttranslational modifications. image
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