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Sundberg, Mårten
Alternative names
Publications (10 of 14) Show all publications
Strage, E. M., Sundberg, M., Holst, B. S., Andersson Franko, M., Ramström, M., Fall, T. & Lewitt, M. (2018). Effect of insulin treatment on circulating insulin-like growth factor I and IGF-binding proteins in cats with diabetes mellitus.. Journal of Veterinary Internal Medicine, 32(5), 1579-1590
Open this publication in new window or tab >>Effect of insulin treatment on circulating insulin-like growth factor I and IGF-binding proteins in cats with diabetes mellitus.
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2018 (English)In: Journal of Veterinary Internal Medicine, ISSN 0891-6640, E-ISSN 1939-1676, Vol. 32, no 5, p. 1579-1590Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Insulin-like growth factor-I (IGF-I) is used to screen for acromegaly in diabetic cats. In humans, most circulating IGF-I forms ternary complexes (TC) with IGF-binding protein (IGFBP-3) and an acid-labile subunit. Compared to humans, the amount of TC in cats is more variable. Insulin-like growth factor-I concentrations are reported to increase during insulin treatment, more rapidly in cats achieving remission.

OBJECTIVES: To investigate (i) factors associated with circulating IGF-I concentrations, including IGFBP-profiles (ii) effect of insulin treatment on IGF-I concentrations and (iii) IGF-I as prognostic marker of diabetes mellitus remission.

ANIMALS: Thirty-one privately owned diabetic cats of which 24 were followed 1 year, and 13 healthy cats.

METHODS: Prospective study. Serum insulin, IGF-I, glucose, and fructosamine concentrations were measured. IGF-binding forms were determined by chromatography in 14 diabetic and 13 healthy cats; and IGF-I, IGF-II, IGFBP-3, and IGFBP-5 by mass spectrometry in 3 cats achieving remission.

RESULTS: Insulin-like growth factor-I median (interquartile range) before start of insulin treatment was 300 (160-556) ng/mL. Insulin-like growth factor-I was positively associated with TC (P < .0001) and endogenous insulin (P = .005) and negatively associated with fructosamine (P < .0001). Median IGF-I was higher 2-4 weeks after start of insulin treatment compared with baseline (300 versus 670 ng/mL, P = .0001) and predicted future remission (P = .046). In cats that went into remission, the amount of TC and IGFBP-3 increased, suggesting increase in IGF-I is dependent on TC formation.

CONCLUSIONS: Insulin treatment should be accounted for when interpreting IGF-I in diabetic cats. Insulin-like growth factor-I 2-4 weeks after initiation of insulin treatment shows promise as prognostic marker for remission in diabetic cats.

Keywords
IGF-II, IGFBP-3, insulin, ternary complex
National Category
Clinical Science
Identifiers
urn:nbn:se:uu:diva-366083 (URN)10.1111/jvim.15243 (DOI)000447414300011 ()30112786 (PubMedID)
Note

Fall och Lewitt delar sistaförfattarskapet.

Available from: 2018-11-16 Created: 2018-11-16 Last updated: 2018-12-11Bibliographically approved
Sundberg, M., Bergquist, J. & Ramström, M. (2015). High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry. Biochemistry and Biophysics Reports, 3, 68-75
Open this publication in new window or tab >>High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry
2015 (English)In: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 3, p. 68-75Article in journal (Refereed) Published
Abstract [en]

As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals. In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study.

Keywords
Cerebrospinal fluid, Dog, High-abundant protein depletion, Shotgun proteomics, Orbitrap, Mass spectrometry
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-259611 (URN)10.1016/j.bbrep.2015.07.013 (DOI)
Available from: 2015-08-10 Created: 2015-08-10 Last updated: 2015-10-01Bibliographically approved
Sundberg, M. (2015). Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody.

Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity.

Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. p. 82
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1272
Keywords
Mass spectrometry, proteomics, microarray, protein, antibody, antigen, affinity, validation
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-259623 (URN)978-91-554-9300-4 (ISBN)
Public defence
2015-09-25, C4:305, BMC, Husargatan 3, Uppsala, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2015-09-03 Created: 2015-08-10 Last updated: 2015-10-01
Colwill, K., Nilsson, P., Sundberg, M., Sjöberg, R., Sivertsson, Å., Schwenk, J. M., . . . Gräslund, S. (2011). A roadmap to generate renewable protein binders to the human proteome. Nature Methods, 8(7), 551-8
Open this publication in new window or tab >>A roadmap to generate renewable protein binders to the human proteome
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2011 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, no 7, p. 551-8Article in journal (Refereed) Published
Abstract [en]

Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders.

Keywords
Immunology, High-Throughput Generation, Phage Display, Recombinant Antibodies, Affinity Reagents
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-208948 (URN)10.1038/nmeth.1607 (DOI)000292194500016 ()21572409 (PubMedID)
Note

Author count: 53. QC 20120214

Available from: 2012-02-14 Created: 2013-10-11 Last updated: 2018-06-05
Sundberg, M. (2011). Protein microarrays for validation of affinity binders. (Licentiate dissertation). Stockholm: KTH Royal Institute of Technology
Open this publication in new window or tab >>Protein microarrays for validation of affinity binders
2011 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. p. 31
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2011:23
Keywords
Microarray, protein, antibody, antigen, affinity, validation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-208950 (URN)978-91-7501-149-3 (ISBN)
Presentation
2011-12-09, FB42, AlbaNova Universitetscentrum, Roslagstullsbacken 21, 10:00 (Swedish)
Opponent
Supervisors
Projects
Development and applications of protein microarraysThe Swedish Human Proteome Resource (HPR) program
Note

QC 20111117

Available from: 2013-10-14 Created: 2013-10-11 Last updated: 2018-06-05Bibliographically approved
Sjöberg, R., Sundberg, M., Gundberg, A., Sivertsson, Å., Schwenk, J. M., Uhlén, M. & Nilsson, P. (2011). Validation of affinity reagents using antigen microarrays. New Biotechnology, 29(5), 555-563
Open this publication in new window or tab >>Validation of affinity reagents using antigen microarrays
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2011 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, no 5, p. 555-563Article in journal (Other academic) Published
Abstract [en]

There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

Keywords
protein microarray, antibody validation, affinity reagent, antigen, specificity, SH2
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-208949 (URN)10.1016/j.nbt.2011.11.009 (DOI)000305606500007 ()
Available from: 2011-11-17 Created: 2013-10-11 Last updated: 2018-06-05
Rimini, R., Schwenk, J. M., Sundberg, M., Sjöberg, R., Klevebring, D., Gry, M., . . . Nilsson, P. (2009). Validation of serum protein profiles by a dual antibody array approach. Journal of Proteomics, 73(2), 252-266
Open this publication in new window or tab >>Validation of serum protein profiles by a dual antibody array approach
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2009 (English)In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 73, no 2, p. 252-266Article in journal (Refereed) Published
Abstract [en]

In recent years, affinity-based technologies have become important tools for serum profiling to uncover protein expression patterns linked to disease state or therapeutic effects. In this study, we describe a path towards the production of an antibody microarray to allow protein profiling of biotinylated human serum samples with reproducible sensitivity in the picomolar range. With the availability of growing numbers of affinity reagents, protein profiles are to be validated in efficient manners and we describe a cross-platform strategy based on data concordance with a suspension bead array to interrogate the identical set of antibodies with the same cohort of serum samples. Comparative analysis enabled to screen for high-performing antibodies, which were displaying consistent results across the two platforms and targeting known serum components. Moreover, data processing methods such as sample referencing and normalization were evaluated for their effects on inter-platform agreement. Our work suggests that mutual validation of protein expression profiles using alternative microarray platforms holds great potential in becoming an important and valuable component in affinity-based high-throughput proteomic screenings as it allows to narrow down the number of discovered targets prior to orthogonal, uniplexed validation approaches.

Keywords
Antibody microarray, Planar microarray, Suspension bead array, Serum, profiling, Antibody proteomics, systematic variation, microarray analysis, pancreatic-cancer, plasma-proteome, normalization, identification, immunoassays, generation, specimens, support
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-208951 (URN)10.1016/j.jprot.2009.09.009 (DOI)000272564000007 ()
Note

QC 20100525

Available from: 2010-08-05 Created: 2013-10-11 Last updated: 2018-06-05
Lundberg, E., Sundberg, M., Gräslund, T., Uhlén, M. & Andersson-Svahn, H. (2007). A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase. JIM - Journal of Immunological Methods, 322(1-2), 40-49
Open this publication in new window or tab >>A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase
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2007 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 322, no 1-2, p. 40-49Article in journal (Refereed) Published
Abstract [en]

Fluorescently labeled antibodies are very important tools in cell biology, providing for specific and quantitative detection of antigens. To date, fluorophore labeling of antibodies has been performed in solution and has been limited by low-throughput methods requiring a substantial amount of pure antibody sample at a high concentration. We have developed a novel solid-phase labeling protocol for small amounts (i.e. micrograms) of antibodies with fluorescent dyes. Protein A affinity medium was used as solid support in a micropipette tip format. This solid-phase approach, including the advantage of the strong and specific interaction between Protein A and antibodies, allows for simultaneous purification, labeling and concentration of the antibody sample, making it possible to start with unpure antibody samples at low concentrations. We have optimized the protocol with regard to reaction pH, time, temperature and amount of amine reactive dye. In addition, we have evaluated the stability and activity of the labeled antibodies. To evaluate the reproducibility and robustness of this method we labeled eight antibodies with amine reactive fluorescent dyes followed by evaluation of antibody specificity on protein arrays. Interestingly, this gave an extremely high conformity in the degree of labeling, showing the robustness of the method. The solid-phase method also gave predictable and reproducible results and by varying the amount of reactive dye, the desired degree of labeling can easily be achieved. Antibodies labeled using this solid-phase method were similar in stability and activity to antibodies labeled in solution. This novel solid-phase antibody labeling method may also be applicable for other conjugation chemistries and labels, and has potential for throughput applications.

Keywords
labeling; solid phase; protein A; antibody; Alexa fluor 555; Alexa fluor 647
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-208955 (URN)10.1016/j.jim.2007.01.023 (DOI)000246422100005 ()
Note

QC 20100824

Available from: 2008-09-16 Created: 2013-10-11 Last updated: 2017-12-06
Schwenk, J. M., Lindberg, J., Sundberg, M., Uhlén, M. & Nilsson, P. (2007). Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics. Molecular & Cellular Proteomics, 6(1), 125-132
Open this publication in new window or tab >>Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics
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2007 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 1, p. 125-132Article in journal (Refereed) Published
Abstract [en]

One of the major challenges of antibody-based proteomics is the quality assurance of the generated antibodies to ensure specificity to the target protein. Here we describe a single tube multiplex approach to simultaneously analyze the binding of antibodies to a large number of different antigens. This bead-based assay utilizes the full multiplexing capacity theoretically offered by the Luminex suspension array technology. A protocol for an increased coupling throughput for the immobilization of antigens was developed and used to set up complex and stabile 100-plex bead mixtures. The possibility of using a two-dimensional multiplexing, in terms of high numbers of both analytes and samples or as in this case antigens and antibodies, enables the specificity of 96 antibodies versus 100 different antigens to be determined in 2 h. This high throughput analysis will potentially have great impact on the possibility for the utilization of different antibody proteomics approaches where the quality assessment of antibodies is of the utmost importance.

Keywords
protein microarrays, atlas
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-208952 (URN)10.1074/mcp.T60035-MCP200 (DOI)000243312000011 ()
Note

QC 20100525

Available from: 2010-08-05 Created: 2013-10-11 Last updated: 2018-06-05
Eriksson, C., Agaton, C., Kånge, R., Sundberg, M., Nilsson, P., Ek, B., . . . Hober, S. (2006). Microfluidic analysis of antibody specificity in a compact disk format. Journal of Proteome Research, 5(7), 1568-1574
Open this publication in new window or tab >>Microfluidic analysis of antibody specificity in a compact disk format
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2006 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 7, p. 1568-1574Article in journal (Refereed) Published
Abstract [en]

A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His(6)-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three- dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.

Keywords
microfluidics, miniaturization, compact disk, biochip, gyrolab, antibody specificity, IMMUNOSORBENT-ASSAY ELISA, PROTEOMICS, MICROARRAYS, GENOME, CHIP
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-208954 (URN)10.1021/pr050447c (DOI)000238838500007 ()
Note

QC 20100712

Available from: 2010-07-12 Created: 2013-10-11 Last updated: 2018-06-05
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