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Andersson, A., Remnestål, J., Nellgård, B., Vunk, H., Kotol, D., Edfors, F., . . . Fredolini, C. (2019). Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease. Clinica Chimica Acta, 494, 79-93
Open this publication in new window or tab >>Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease
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2019 (English)In: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 494, p. 79-93Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier B.V., 2019
Keywords
AD, Alzheimer's disease, Biomarkers, Cerebrospinal fluid, Parallel reaction monitoring (PRM), Suspension bead array (SBA), alpha 1 aantitrypsin, alpha 1 antichymotrypsin, apolipoprotein, biological marker, cathepsin D, cholecystokinin, creatine kinase B type, dickkopf related protein 3, fibrinogen alpha, fructose bisphosphate aldolase C, glucose regulated protein 94, inter alpha trypsin inhibitor heavy chain H1, leucine rich alpha 2 glycoprotein, neurobeachin, neurofilament medium polypeptide, neuromodulin, plasminogen, prosaposin, protein S100B, SPARC like protein 1, unclassified drug, vascular cell adhesion protein 1, adult, aged, Alzheimer disease, Article, clinical article, cohort analysis, controlled study, correlational study, disease course, female, human, male, mass spectrometry, middle aged, mild cognitive impairment, multiple reaction monitoring, priority journal, protein blood level, protein cerebrospinal fluid level, protein microarray, suspension bead array, very elderly
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-252444 (URN)10.1016/j.cca.2019.03.243 (DOI)000470950400013 ()2-s2.0-85063002689 (Scopus ID)
Note

QC 20190715

Available from: 2019-07-15 Created: 2019-07-15 Last updated: 2019-07-15Bibliographically approved
Koivula, R. W., Schwenk, J. M. & Franks, P. W. (2019). Discovery of biomarkers for glycaemic deterioration before and after the onset of type 2 diabetes: descriptive characteristics of the epidemiological studies within the IMI DIRECT Consortium. Diabetologia, 62(9), 1601-1615
Open this publication in new window or tab >>Discovery of biomarkers for glycaemic deterioration before and after the onset of type 2 diabetes: descriptive characteristics of the epidemiological studies within the IMI DIRECT Consortium
2019 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 62, no 9, p. 1601-1615Article in journal (Refereed) Published
Abstract [en]

Here, we describe the characteristics of the Innovative Medicines Initiative (IMI) Diabetes Research on Patient Stratification (DIRECT) epidemiological cohorts at baseline and follow-up examinations (18, 36 and 48 months of follow-up). Methods From a sampling frame of 24,682 adults of European ancestry enrolled in population-based cohorts across Europe, participants at varying risk of glycaemic deterioration were identified using a risk prediction algorithm (based on age, BMI, waist circumference, use of antihypertensive medication, smoking status and parental history of type 2 diabetes) and enrolled into a prospective cohort study (n = 2127) (cohort 1, prediabetes risk). We also recruited people from clinical registries with type 2 diabetes diagnosed 6-24 months previously (n = 789) into a second cohort study (cohort 2, diabetes). Follow-up examinations took place at similar to 18 months (both cohorts) and at similar to 48 months (cohort 1) or similar to 36 months (cohort 2) after baseline examinations. The cohorts were studied in parallel using matched protocols across seven clinical centres in northern Europe. Results Using ADA 2011 glycaemic categories, 33% (n = 693) of cohort 1 (prediabetes risk) had normal glucose regulation and 67% (n = 1419) had impaired glucose regulation. Seventy-six per cent of participants in cohort 1 was male. Cohort 1 participants had the following characteristics (mean +/- SD) at baseline: age 62 (6.2) years; BMI 27.9 (4.0) kg/m(2); fasting glucose 5.7 (0.6) mmol/l; 2 h glucose 5.9 (1.6) mmol/l. At the final follow-up examination the participants' clinical characteristics were as follows: fasting glucose 6.0 (0.6) mmol/l; 2 h OGTT glucose 6.5 (2.0) mmol/l. In cohort 2 (diabetes), 66% (n = 517) were treated by lifestyle modification and 34% (n = 272) were treated with metformin plus lifestyle modification at enrolment. Fifty-eight per cent of participants in cohort 2 was male. Cohort 2 participants had the following characteristics at baseline: age 62 (8.1) years; BMI 30.5 (5.0) kg/m(2); fasting glucose 7.2 (1.4) mmol/l; 2 h glucose 8.6 (2.8) mmol/l. At the final follow-up examination, the participants' clinical characteristics were as follows: fasting glucose 7.9 (2.0) mmol/l; 2 h mixed-meal tolerance test glucose 9.9 (3.4) mmol/l. Conclusions/interpretation The IMI DIRECT cohorts are intensely characterised, with a wide-variety of metabolically relevant measures assessed prospectively. We anticipate that the cohorts, made available through managed access, will provide a powerful resource for biomarker discovery, multivariate aetiological analyses and reclassification of patients for the prevention and treatment of type 2 diabetes.

Place, publisher, year, edition, pages
SPRINGER, 2019
Keywords
Diet, Ectopic fat, Genome, Glycaemic control, Insulin secretion, Insulin sensitivity, Personalised medicine, Physical activity, Prediabetes, Type 2 diabetes
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:kth:diva-257545 (URN)10.1007/s00125-019-4906-1 (DOI)000478770300009 ()31203377 (PubMedID)2-s2.0-85067006221 (Scopus ID)
Note

QC 20190925

Available from: 2019-09-25 Created: 2019-09-25 Last updated: 2019-09-25Bibliographically approved
Wilman, H. R., Parisinos, C. A., Atabaki-Pasdar, N., Kelly, M., Thomas, E. L., Neubauer, S., . . . Yaghootkar, H. (2019). Genetic studies of abdominal MRI data identify genes regulating hepcidin as major determinants of liver iron concentration. Journal of Hepatology, 71(3), 594-602
Open this publication in new window or tab >>Genetic studies of abdominal MRI data identify genes regulating hepcidin as major determinants of liver iron concentration
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2019 (English)In: Journal of Hepatology, ISSN 0168-8278, E-ISSN 1600-0641, Vol. 71, no 3, p. 594-602Article in journal (Refereed) Published
Abstract [en]

Background & Aims: Excess liver iron content is common and is linked to the risk of hepatic and extrahepatic diseases. We aimed to identify genetic variants influencing liver iron content and use genetics to understand its link to other traits and diseases. Methods: First, we performed a genome-wide association study (GWAS) in 8,289 individuals from UK Biobank, whose liver iron level had been quantified by magnetic resonance imaging, before validating our findings in an independent cohort (n = 1,513 from IMI DIRECT). Second, we used Mendelian randomisation to test the causal effects of 25 predominantly metabolic traits on liver iron content. Third, we tested phenome-wide associations between liver iron variants and 770 traits and disease outcomes. Results: We identified 3 independent genetic variants (rs1800562 [C282Y] and rs1799945 [H63D] in HFE and rs855791 [V736A] in TMPRSS6) associated with liver iron content that reached the GWAS significance threshold (p <5 × 10−8). The 2 HFE variants account for ∼85% of all cases of hereditary haemochromatosis. Mendelian randomisation analysis provided evidence that higher central obesity plays a causal role in increased liver iron content. Phenome-wide association analysis demonstrated shared aetiopathogenic mechanisms for elevated liver iron, high blood pressure, cirrhosis, malignancies, neuropsychiatric and rheumatological conditions, while also highlighting inverse associations with anaemias, lipidaemias and ischaemic heart disease. Conclusion: Our study provides genetic evidence that mechanisms underlying higher liver iron content are likely systemic rather than organ specific, that higher central obesity is causally associated with higher liver iron, and that liver iron shares common aetiology with multiple metabolic and non-metabolic diseases. Lay summary: Excess liver iron content is common and is associated with liver diseases and metabolic diseases including diabetes, high blood pressure, and heart disease. We identified 3 genetic variants that are linked to an increased risk of developing higher liver iron content. We show that the same genetic variants are linked to higher risk of many diseases, but they may also be associated with some health advantages. Finally, we use genetic variants associated with waist-to-hip ratio as a tool to show that central obesity is causally associated with increased liver iron content.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Genetics, Genome-wide association study, Iron, Magnetic resonance imaging, Metabolic syndrome, Metabolism
National Category
Gastroenterology and Hepatology
Identifiers
urn:nbn:se:kth:diva-262512 (URN)10.1016/j.jhep.2019.05.032 (DOI)000481571400017 ()31226389 (PubMedID)2-s2.0-85068854139 (Scopus ID)
Note

QC 20191128

Available from: 2019-10-29 Created: 2019-10-29 Last updated: 2019-10-29Bibliographically approved
Pernemalm, M., Sandberg, A., Zhu, Y., Boekel, J., Tamburro, D., Schwenk, J. M., . . . Lehtio, J. (2019). In-depth human plasma proteome analysis captures tissue proteins and transfer of protein variants across the placenta. eLIFE, 8, Article ID e41608.
Open this publication in new window or tab >>In-depth human plasma proteome analysis captures tissue proteins and transfer of protein variants across the placenta
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2019 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e41608Article in journal (Refereed) Published
Abstract [en]

Here, we present a method for in-depth human plasma proteome analysis based on high-resolution isoelectric focusing HiRIEF LC-MS/MS, demonstrating high proteome coverage, reproducibility and the potential for liquid biopsy protein profiling. By integrating genomic sequence information to the MS-based plasma proteome analysis, we enable detection of single amino acid variants and for the first time demonstrate transfer of multiple protein variants between mother and fetus across the placenta. We further show that our method has the ability to detect both low abundance tissue-annotated proteins and phosphorylated proteins in plasma, as well as quantitate differences in plasma proteomes between the mother and the newborn as well as changes related to pregnancy.

Place, publisher, year, edition, pages
ELIFE SCIENCES PUBLICATIONS LTD, 2019
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-252639 (URN)10.7554/eLife.41608 (DOI)000468060900001 ()30958262 (PubMedID)2-s2.0-85066163570 (Scopus ID)
Note

QC 20190610

Available from: 2019-06-10 Created: 2019-06-10 Last updated: 2019-06-10Bibliographically approved
Thomas, C. E., Häussler, R. S., Hong, M.-G., Raverdy, V., Dale, M., Vinuela, A., . . . Schwenk, J. M. (2019). Individual effects of gastric bypass surgery on longitudinal blood protein profiles: an IMI DIRECT study. Paper presented at 55th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), SEP 16-20, 2019, Barcelona, SPAIN. Diabetologia, 62, S271-S271
Open this publication in new window or tab >>Individual effects of gastric bypass surgery on longitudinal blood protein profiles: an IMI DIRECT study
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2019 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 62, p. S271-S271Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2019
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-261975 (URN)000485303802156 ()
Conference
55th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), SEP 16-20, 2019, Barcelona, SPAIN
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2019-10-14 Created: 2019-10-14 Last updated: 2019-10-14Bibliographically approved
Lorenzen, E., Dodig-Crnkovic, T., Kotliar, I. B., Pin, E., Ceraudo, E., Vaughan, R. D., . . . Sakmar, T. P. (2019). Multiplexed analysis of the secretin-like GPCR-RAMP interactome. Science Advances, 5(9), Article ID eaaw2778.
Open this publication in new window or tab >>Multiplexed analysis of the secretin-like GPCR-RAMP interactome
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2019 (English)In: Science Advances, E-ISSN 2375-2548, Vol. 5, no 9, article id eaaw2778Article in journal (Refereed) Published
Abstract [en]

Receptor activity-modifying proteins (RAMPs) have been shown to modulate the functions of several G protein-coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

Place, publisher, year, edition, pages
AMER ASSOC ADVANCEMENT SCIENCE, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-263373 (URN)10.1126/sciadv.aaw2778 (DOI)000491128800060 ()31555726 (PubMedID)2-s2.0-85072340840 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20191115

Available from: 2019-11-15 Created: 2019-11-15 Last updated: 2019-11-15Bibliographically approved
Lorenzen, E., Dodig-Crnkovic, T., Kotliar, I. B., Pin, E., Ceraudo, E., Vaughan, R. D., . . . Sakmar, T. P. (2019). Multiplexed analysis of the secretin-like GPCR-RAMP interactome. Science Advances, 5(9), Article ID eaaw2778.
Open this publication in new window or tab >>Multiplexed analysis of the secretin-like GPCR-RAMP interactome
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2019 (English)In: Science Advances, E-ISSN 2375-2548, Vol. 5, no 9, article id eaaw2778Article in journal (Refereed) Published
Abstract [en]

Receptor activity–modifying proteins (RAMPs) have been shown to modulate the functions of several G protein–coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues. Copyright

Place, publisher, year, edition, pages
American Association for the Advancement of Science, 2019
Keywords
Antibodies, Cell culture, Chemokine receptors, Coupled receptors, Direct interactions, Membrane proteins, Multiplexed analysis, Native proteins, Receptor activity, Suspension bead arrays, Epitopes
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-263565 (URN)10.1126/sciadv.aaw2778 (DOI)000491128800060 ()31555726 (PubMedID)2-s2.0-85072340840 (Scopus ID)
Note

QC20191120

Available from: 2019-11-20 Created: 2019-11-20 Last updated: 2019-11-20Bibliographically approved
Edfors, F., Forsström, B., Vunk, H., Kotol, D., Fredolini, C., Maddalo, G., . . . Uhlén, M. (2019). Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics. Journal of Proteome Research, 18(7), 2706-2718
Open this publication in new window or tab >>Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics
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2019 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 7, p. 2706-2718Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2019
Keywords
targeted proteomics, stable isotope standards, mass spectrometry, protein quantification, recombinant proteins, protein fragment, trypsin digestion, spectral library, assay generation, peptide formation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-255390 (URN)10.1021/acs.jproteome.8b00924 (DOI)000474795500003 ()31094526 (PubMedID)2-s2.0-85067403932 (Scopus ID)
Note

QC 20190730

Available from: 2019-07-30 Created: 2019-07-30 Last updated: 2019-07-30Bibliographically approved
Fredolini, C., Byström, S., Sanchez-Rivera, L., Ioannou, M., Tamburro, D., Pontén, F., . . . Schwenk, J. M. (2019). Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles. Scientific Reports, 9, Article ID 8324.
Open this publication in new window or tab >>Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 8324Article in journal (Refereed) Published
Abstract [en]

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here, we applied a magnetic bead assisted workflow and immunoprecipitation mass spectrometry (IP-MS/MS) to assess antibody selectivity for the detection of proteins in human plasma. A resource was built on 414 IP experiments using 157 antibodies (targeting 120 unique proteins) in assays with heat-treated or untreated EDTA plasma. For each protein we determined their antibody related degrees of enrichment using z-scores and their frequencies of identification across all IP assays. Out of 1,313 unique endogenous proteins, 426 proteins (33%) were detected in >20% of IPs, and these background components were mainly comprised of proteins from the complement system. For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score >= 3). Among these 70 antibodies, 59 (84%) co-enriched other proteins beside the intended target and mainly due to sequence homology or protein abundance. We also detected protein interactions in plasma, and for IGFBP2 confirmed these using several antibodies and sandwich immunoassays. The protein enrichment data with plasma provide a very useful and yet lacking resource for the assessment of antibody selectivity. Our insights will contribute to a more informed use of affinity reagents for plasma proteomics assays.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:kth:diva-254077 (URN)10.1038/s41598-019-43552-5 (DOI)000470243800005 ()31171813 (PubMedID)2-s2.0-85067067842 (Scopus ID)
Note

QC 20190624

Available from: 2019-06-24 Created: 2019-06-24 Last updated: 2019-06-24Bibliographically approved
Häussler, R. S., Bendes, A., Iglesias, M. J., Sanchez-Rivera, L., Dodig-Crnkovic, T., Byström, S., . . . Schwenk, J. M. (2019). Systematic Development of Sandwich Immunoassays for the Plasma Secretome. Proteomics, Article ID 1900008.
Open this publication in new window or tab >>Systematic Development of Sandwich Immunoassays for the Plasma Secretome
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2019 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, article id 1900008Article in journal (Refereed) Published
Abstract [en]

The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity-free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

Place, publisher, year, edition, pages
Wiley, 2019
Keywords
antibodies, plasma, sandwich assays, screening, secreted proteins
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-255741 (URN)10.1002/pmic.201900008 (DOI)000477448900001 ()31278833 (PubMedID)2-s2.0-85069914143 (Scopus ID)
Note

QC 20190812

Available from: 2019-08-12 Created: 2019-08-12 Last updated: 2019-10-04Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-8141-8449

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