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Development of novel affinity enrichment strategies for clinical applications using selected reaction monitoring
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8947-2562
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are key components of any living organism and an essential part of life itself. They can provide cells with structure and perform life-sustaining intracellular reactions. As organisms grow more complex, this task expands even further. The proteins’ areas of responsibility suddenly also include communication and coordination between cells and throughout entire organisms, such as the human body. Everything that can be touched and felt on a living organism is composed of millions and millions of proteins tightly packed together. They are even the molecules responsible for propagating the signals that make up the sense of feeling. Understanding the role of proteins in the complex system of life is essential for understanding what makes up a healthy human and what causes disease. This knowledge makes up the foundation of modern medicine, and to further this knowledge, allowing for new treatments and preventative interventions, the study of proteins is crucial. The large-scale study of proteins, proteomics, is an extensive field of research where a vast toolbox of technologies has been implemented. The foundation for this toolbox is made up of mass spectrometry- and affinity-based technologies.

In this thesis, both mass spectrometry-based proteomics and affinity-based proteomics will be explored. The first part, Paper I and Paper II, describe the use of selected reaction monitoring for measuring proteins of clinical relevance in human blood plasma. The second part, Paper III and Paper IV, highlight the importance of validating reagents used for affinity-based proteomics and how this can be achieved in a high throughput manner. Lastly, Paper V showcases how a combined strategy, relying on both affinity-based proteomics and mass spectrometry-based proteomics, can capitalize on the best properties of each technology and how this combined strategy can even be utilized for diagnostic purposes.

Abstract [sv]

Proteiner är livsviktiga molekyler som både förser celler med struktur och utför diverse reaktioner och uppgifter som håller cellerna vid liv. Ju mer komplex en organism är, desto svårare blir proteinernas uppgifter. I en organism som består av flera celler, såsom människor och djur, räcker det inte längre att varje cell sköter sina reaktioner och undertaganden separat, utan alla sådana processer måste koordineras. Denna koordinering utförs också av proteinerna. Proteinerna utgör en så stor del av livet att om du rör vid något levande så är det i regel proteiner som bygger upp den yta du känner. De är till och med så tätt ordnade att varje liten cell består av miljoner och åter miljoner proteiner. Inte nog med detta. Att du ens kan känna att du tar på en annan varelse eller föremål är också något som proteiner ser till. De ansvarar för att föra vidare signalerna från handen till hjärnan och att du sedan uppfattar detta som ett föremål. Det är därför inte förvånande att man måste förstå proteinernas uppgifter i alla möjliga situationer för att kunna veta hur en frisk människa fungerar och därmed avgöra när någon är sjuk. Detta ligger till grund för hela medicinfältet. För att kunna komma på nya behandlingar och för att rentav kunna förebygga sjukdomar är det nödvändigt att ha så mycket kunskap som möjligt om hur proteiner fungerar. Att studera proteiner i stor skala brukar kallas för proteomik och detta område har utvecklats något oerhört de senaste årtionden och det finns en mängd olika tekniker för att undersöka proteiner på. De flesta av dessa tekniker bygger dock på två huvudområden: masspektrometri och affinitetsreagens.

I den här avhandlingen har båda dessa områden utforskats. Den första delen av avhandlingen, som utgörs av Artikel I och Artikel II, bygger på masspektrometri. Här används så kallad riktad proteomik för att mäta proteinnivåerna av kliniska markörer i blodplasma. I del två, som utgörs av Artikel III och Artikel IV, undersöks istället affinitetsreagens och hur man kan försäkra sig om att de binder till de protein som man tror att de binder till i en stor skala. Slutligen kombineras båda dessa två områden i Artikel V och används för att undersöka förekomsten av SARS-CoV-2 i en asymtomatisk grupp människor.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2022. , p. 91
Series
TRITA-CBH-FOU ; 2022:47
Keywords [en]
proteomics, mass spectrometry, selected reaction monitoring, absolute quantification, antibody validation, precision medicine
National Category
Biochemistry and Molecular Biology Medical and Health Sciences Medical Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-318308ISBN: 978-91-8040-357-3 (print)OAI: oai:DiVA.org:kth-318308DiVA, id: diva2:1697291
Public defence
2022-10-14, Gradängsalen, Teknikringen 1, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Knut and Alice Wallenberg Foundation
Note

QC 2022-09-20

Available from: 2022-09-20 Created: 2022-09-20 Last updated: 2023-12-07Bibliographically approved
List of papers
1. Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay
Open this publication in new window or tab >>Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay
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2019 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, no 12, p. 2433-2446Article in journal (Refereed) Published
Abstract [en]

Stable isotope-labeled standard (SIS) peptides are used as internal standards in targeted proteomics to provide robust protein quantification, which is required in clinical settings. However, SIS peptides are typically added post trypsin digestion and, as the digestion efficiency can vary significantly between peptides within a protein, the accuracy and precision of the assay may be compromised. These drawbacks can be remedied by a new class of internal standards introduced by the Human Protein Atlas project, which are based on SIS recombinant protein fragments called SIS PrESTs. SIS PrESTs are added initially to the sample and SIS peptides are released on trypsin digestion. The SIS PrEST technology is promising for absolute quantification of protein biomarkers but has not previously been evaluated in a clinical setting. An automated and scalable solid phase extraction workflow for desalting and enrichment of plasma digests was established enabling simultaneous preparation of up to 96 samples. Robust high-precision quantification of 13 apolipoproteins was achieved using a novel multiplex SIS PrEST-based LC-SRM/MS Tier 2 assay in non-depleted human plasma. The assay exhibited inter-day coefficients of variation between 1.5% and 14.5% (median = 3.5%) and was subsequently used to investigate the effects of omega-3 carboxylic acids (OM3-CA) and fenofibrate on these 13 apolipoproteins in human plasma samples from a randomized placebo-controlled trial, EFFECT I (NCT02354976). No significant changes were observed in the OM3-CA arm, whereas treatment with fenofibrate significantly increased apoAII and reduced apoB, apoCI, apoE and apoCIV levels. The reduction in apoCIV following fenofibrate treatment is a novel finding. The study demonstrates that SIS PrESTs can facilitate the generation of robust multiplexed biomarker Tier 2 assays for absolute quantification of proteins in clinical studies. Applications of LC-SRM in clinical research are still limited. SIS PrEST are a novel class of standards added prior to trypsinization. We have developed a semi-automated sample preparation workflow and a SIS PrEST LC-SRM/MS Tier 2 assay for absolute quantification of 13 apolipoproteins in human plasma and applied it on clinical samples from the EFFECT I study. We demonstrate, for the first time, that SIS PrEST can be applied for exploratory biomarker research in clinical settings and capture drug effects.

Place, publisher, year, edition, pages
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2019
Keywords
Clinical trials, assay development, targeted mass spectrometry, selected reaction monitoring, absolute quantification, apolipoproteins, fenofibrate and omega-3 carboxylic acids, NAFLD, SIS PrEST
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-266286 (URN)10.1074/mcp.RA119.001765 (DOI)000501288700007 ()31591263 (PubMedID)2-s2.0-85075958078 (Scopus ID)
Note

QC 20200108

Available from: 2020-01-08 Created: 2020-01-08 Last updated: 2022-10-24Bibliographically approved
2. Targeted Proteomics Using Stable Isotope Labeled Protein Fragments Enables Precise and Robust Determination of Total Apolipoprotein(a) in Human Plasma
Open this publication in new window or tab >>Targeted Proteomics Using Stable Isotope Labeled Protein Fragments Enables Precise and Robust Determination of Total Apolipoprotein(a) in Human Plasma
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

Keywords
apolipoprotein(a) (apo(a)), apolipoproteins, quantification, stable isotope labeled recombinant protein fragment standard, liquid chromatography, tandem mass spectrometry
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-318202 (URN)
Note

QC 20220927

Available from: 2022-09-19 Created: 2022-09-19 Last updated: 2023-12-07Bibliographically approved
3. Enhanced validation of antibodies for research applications
Open this publication in new window or tab >>Enhanced validation of antibodies for research applications
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2018 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 9, article id 4130Article in journal (Refereed) Published
Abstract [en]

There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-237096 (URN)10.1038/s41467-018-06642-y (DOI)000446566000016 ()30297845 (PubMedID)2-s2.0-85054574300 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Note

QC 20181030

Available from: 2018-10-30 Created: 2018-10-30 Last updated: 2024-03-15Bibliographically approved
4. Evaluation of an enhanced antibody-validation strategy for Western blot applications based on migration pattern recognition
Open this publication in new window or tab >>Evaluation of an enhanced antibody-validation strategy for Western blot applications based on migration pattern recognition
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

Keywords
antibody validation, Western blot, SDS-PAGE, mass spectrometry, gel electrophoresis, proteomics
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-318304 (URN)
Note

QC 20220927

Available from: 2022-09-19 Created: 2022-09-19 Last updated: 2023-12-07Bibliographically approved
5. Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis
Open this publication in new window or tab >>Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis
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2021 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 10, article id e70843Article in journal (Refereed) Published
Abstract [en]

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct <= 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

Place, publisher, year, edition, pages
eLIFE SCIENCES PUBL LTD, 2021
Keywords
SARS CoV-2, COVID-19, SISCAPA, proteomics, diagnostics, mass spectrometry, Human
National Category
Infectious Medicine
Identifiers
urn:nbn:se:kth:diva-306455 (URN)10.7554/eLife.70843 (DOI)000723865100001 ()34747696 (PubMedID)2-s2.0-85120041769 (Scopus ID)
Note

See also peer review documents at DOI 10.7554/eLife.70843.sa0  10.7554/eLife.70843.sa1 10.7554/eLife.70843.sa2

QC 20211217

Available from: 2021-12-17 Created: 2021-12-17 Last updated: 2023-12-07Bibliographically approved

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