Publications by authors named "Rainer Bischoff"

190 Publications

Meta-Inflammation and Metabolic Reprogramming of Macrophages in Diabetes and Obesity: The Importance of Metabolites.

Front Immunol 2021 5;12:746151. Epub 2021 Nov 5.

Department of Molecular Pharmacology, University of Groningen, Groningen, Netherlands.

Diabetes mellitus type II and obesity are two important causes of death in modern society. They are characterized by low-grade chronic inflammation and metabolic dysfunction (meta-inflammation), which is observed in all tissues involved in energy homeostasis. A substantial body of evidence has established an important role for macrophages in these tissues during the development of diabetes mellitus type II and obesity. Macrophages can activate into specialized subsets by cues from their microenvironment to handle a variety of tasks. Many different subsets have been described and in diabetes/obesity literature two main classifications are widely used that are also defined by differential metabolic reprogramming taking place to fuel their main functions. Classically activated, pro-inflammatory macrophages (often referred to as M1) favor glycolysis, produce lactate instead of metabolizing pyruvate to acetyl-CoA, and have a tricarboxylic acid cycle that is interrupted at two points. Alternatively activated macrophages (often referred to as M2) mainly use beta-oxidation of fatty acids and oxidative phosphorylation to create energy-rich molecules such as ATP and are involved in tissue repair and downregulation of inflammation. Since diabetes type II and obesity are characterized by metabolic alterations at the organism level, these alterations may also induce changes in macrophage metabolism resulting in unique macrophage activation patterns in diabetes and obesity. This review describes the interactions between metabolic reprogramming of macrophages and conditions of metabolic dysfunction like diabetes and obesity. We also focus on different possibilities of measuring a range of metabolites intra-and extracellularly in a precise and comprehensive manner to better identify the subsets of polarized macrophages that are unique to diabetes and obesity. Advantages and disadvantages of the currently most widely used metabolite analysis approaches are highlighted. We further describe how their combined use may serve to provide a comprehensive overview of the metabolic changes that take place intracellularly during macrophage activation in conditions like diabetes and obesity.
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http://dx.doi.org/10.3389/fimmu.2021.746151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8602812PMC
November 2021

Adsorptive Microtiter Plates As Solid Supports in Affinity Purification Workflows.

J Proteome Res 2021 Nov 20;20(11):5218-5221. Epub 2021 Oct 20.

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, 9700 AB Groningen, The Netherlands.

Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biological samples. These ligands are generally immobilized onto solid supports which facilitate the separation of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochemical detection (e.g., immunoassays) but hardly ever prior to liquid chromatography-mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., soluble receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-κB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purification workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research.
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http://dx.doi.org/10.1021/acs.jproteome.1c00623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8576839PMC
November 2021

Enrichment and Liquid Chromatography-Mass Spectrometry Analysis of Trastuzumab and Pertuzumab Using Affimer Reagents.

Anal Chem 2021 10 28;93(40):13597-13605. Epub 2021 Sep 28.

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Trastuzumab and pertuzumab are monoclonal antibodies used in the treatment of human epidermal growth factor receptor-2 (HER2)-positive breast cancer. Therapeutic proteins may undergo chemical modifications that may affect the results of bioanalytical assays, as well as their therapeutic efficacy. Modifications may arise during production and storage, as well as after administration to patients. Studying in vivo biotransformation of monoclonal, therapeutic antibodies requires their enrichment from plasma to discriminate them from endogenous antibodies, as well as from other plasma proteins. To this end, we screened Affimer reagents for selectivity toward trastuzumab or pertuzumab. Affimer reagents are alternative binding proteins possessing two variable binding loops that are based on the human protease inhibitor stefin A or phytocystatin protein scaffolds. Affimer reagents were selected from an extensive library by phage display. The four best-performing binders for each therapeutic antibody were prioritized using a microtiter plate-based approach combined with liquid chromatography-mass spectrometry (LC-MS) in the selected reaction monitoring (SRM) mode. These Affimer reagents were immobilized via engineered 6-His or Cys tags to Ni- or maleimide beads, respectively. Recovery values of 70% and higher were obtained for both trastuzumab and pertuzumab when spiked at 100, 150, and 200 μg/mL concentrations in human plasma followed by trypsin digestion in the presence of 0.5% sodium deoxycholate and 10 mM dithiothreitol (DTT). Notably, the maleimide beads showed undetectable unspecific binding to endogenous immunoglobulin G (IgGs) or other plasma proteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enrichment method was applied to samples from stress tests of the antibodies at 37 °C to mimic in vivo conditions.
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http://dx.doi.org/10.1021/acs.analchem.1c02807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8515351PMC
October 2021

Molecular markers for cervical cancer screening.

Expert Rev Proteomics 2021 Aug 29;18(8):675-691. Epub 2021 Sep 29.

Department of Analytical Biochemistry, University of Groningen, Groningen, The Netherlands.

Introduction: Cervical cancer remains a significant healthcare problem, notably in low- to middle-income countries. While a negative test for hrHPV has a predictive value of more than 99.5%, its positive predictive value is less than 10% for CIN2+ stages. This makes the use of a so-called triage test indispensable for population-based screening to avoid referring women, that are ultimately at low risk of developing cervical cancer, to a gynecologist. This review will give an overview of tests that are based on epigenetic marker panels and protein markers.

Areas Covered: There is a medical need for molecular markers with a better predictive value to discriminate hrHPV-positive women that are at risk of developing cervical cancer from those that are not. Areas covered are epigenetic and protein markers as well as health economic considerations in view of the fact that most cases of cervical cancer arise in low-to-middle-income countries.

Expert Opinion: While there are biomarker assays based on changes at the nucleic acid (DNA methylation patterns, miRNAs) and at the protein level, they are not widely used in population screening. Combining nucleic acid-based and protein-based tests could improve the overall specificity for discriminating CIN2+ lesions that carry a low risk of progressing to cervical cancer within the screening interval from those that carry an elevated risk. The challenge is to reduce unnecessary referrals without an undesired increase in false-negative diagnoses resulting in cases of cervical cancer that could have been prevented. A further challenge is to develop tests for low-and middle-income countries, which is critical to reduce the worldwide burden of cervical cancer.
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http://dx.doi.org/10.1080/14789450.2021.1980387DOI Listing
August 2021

Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes-a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC-MS.

Anal Chem 2021 09 14;93(38):12872-12880. Epub 2021 Sep 14.

Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck 6020, Austria.

Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope-labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated, enabling accurate relative quantification of site-specific lysine acetylation dynamics in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics.
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http://dx.doi.org/10.1021/acs.analchem.1c01359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8482368PMC
September 2021

The 'un-shrunk' partial correlation in Gaussian graphical models.

BMC Bioinformatics 2021 Sep 7;22(1):424. Epub 2021 Sep 7.

Bernoulli Institute, University of Groningen, Groningen, 9747 AG, The Netherlands.

Background: In systems biology, it is important to reconstruct regulatory networks from quantitative molecular profiles. Gaussian graphical models (GGMs) are one of the most popular methods to this end. A GGM consists of nodes (representing the transcripts, metabolites or proteins) inter-connected by edges (reflecting their partial correlations). Learning the edges from quantitative molecular profiles is statistically challenging, as there are usually fewer samples than nodes ('high dimensional problem'). Shrinkage methods address this issue by learning a regularized GGM. However, it remains open to study how the shrinkage affects the final result and its interpretation.

Results: We show that the shrinkage biases the partial correlation in a non-linear way. This bias does not only change the magnitudes of the partial correlations but also affects their order. Furthermore, it makes networks obtained from different experiments incomparable and hinders their biological interpretation. We propose a method, referred to as 'un-shrinking' the partial correlation, which corrects for this non-linear bias. Unlike traditional methods, which use a fixed shrinkage value, the new approach provides partial correlations that are closer to the actual (population) values and that are easier to interpret. This is demonstrated on two gene expression datasets from Escherichia coli and Mus musculus.

Conclusions: GGMs are popular undirected graphical models based on partial correlations. The application of GGMs to reconstruct regulatory networks is commonly performed using shrinkage to overcome the 'high-dimensional problem'. Besides it advantages, we have identified that the shrinkage introduces a non-linear bias in the partial correlations. Ignoring this type of effects caused by the shrinkage can obscure the interpretation of the network, and impede the validation of earlier reported results.
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http://dx.doi.org/10.1186/s12859-021-04313-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8424921PMC
September 2021

Change of charge variant composition of trastuzumab upon stressing at physiological conditions.

J Chromatogr A 2021 Oct 28;1655:462506. Epub 2021 Aug 28.

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, A Deusinglaan 1, 9713 AV Groningen, the Netherlands. Electronic address:

Cation-exchange chromatography is a widely used approach to study charge heterogeneity of monoclonal antibodies. Heterogeneity may arise both in vitro and in vivo because of the susceptibility of monoclonal antibodies to undergo chemical modifications. Modifications may adversely affect the potency of the drug, induce immunogenicity or affect pharmacokinetics. In this study, we evaluated the application of optimized pH gradient systems for the separation of charge variants of trastuzumab after forced degradation study. pH gradient-based elution resulted in high-resolution separation of some 20 charge variants after 3 weeks at 37°C under physiological conditions. The charge variants were further characterized by LC-MS-based peptide mapping. There was no significant difference in the binding properties to HER2 or a range of Fcγ receptors between non-stressed and stressed trastuzumab.
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http://dx.doi.org/10.1016/j.chroma.2021.462506DOI Listing
October 2021

Pipelines and Systems for Threshold-Avoiding Quantification of LC-MS/MS Data.

Anal Chem 2021 08 6;93(32):11215-11224. Epub 2021 Aug 6.

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands.

The accurate processing of complex liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) data from biological samples is a major challenge for metabolomics, proteomics, and related approaches. Here, we present the pipelines and systems for threshold-avoiding quantification (PASTAQ) LC-MS/MS preprocessing toolset, which allows highly accurate quantification of data-dependent acquisition LC-MS/MS datasets. PASTAQ performs compound quantification using single-stage (MS1) data and implements novel algorithms for high-performance and accurate quantification, retention time alignment, feature detection, and linking annotations from multiple identification engines. PASTAQ offers straightforward parameterization and automatic generation of quality control plots for data and preprocessing assessment. This design results in smaller variance when analyzing replicates of proteomes mixed with known ratios and allows the detection of peptides over a larger dynamic concentration range compared to widely used proteomics preprocessing tools. The performance of the pipeline is also demonstrated in a biological human serum dataset for the identification of gender-related proteins.
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http://dx.doi.org/10.1021/acs.analchem.1c01892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8374884PMC
August 2021

Correction to: An antibody-free LC-MS/MS method for the quantification of intact insulin-like growth factors 1 and 2 in human plasma.

Anal Bioanal Chem 2021 Sep 16;413(21):5529. Epub 2021 Jul 16.

Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713, GZ, Groningen, The Netherlands.

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http://dx.doi.org/10.1007/s00216-021-03507-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8587368PMC
September 2021

Online-2D NanoLC-MS for Crude Serum Proteome Profiling: Assessing Sample Preparation Impact on Proteome Composition.

Anal Chem 2021 07 8;93(28):9663-9668. Epub 2021 Jul 8.

Thermo Fisher Scientific, Dornierstrasse 4, 82110 Germering, Germany.

Although current LC-MS technology permits scientists to efficiently screen clinical samples in translational research, e.g., steroids, biogenic amines, and even plasma or serum proteomes, in a daily routine, maintaining the balance between throughput and analytical depth is still a limiting factor. A typical approach to enhance the proteome depth is employing offline two-dimensional (2D) fractionation techniques before reversed-phase nanoLC-MS/MS analysis (1D-nanoLC-MS). These additional sample preparation steps usually require extensive sample manipulation, which could result in sample alteration and sample loss. Here, we present and compare 1D-nanoLC-MS with an automated online-2D high-pH RP × low pH RP separation method for deep proteome profiling using a nanoLC system coupled to a high-resolution accurate-mass mass spectrometer. The proof-of-principle study permitted the identification of ca. 500 proteins with ∼10,000 peptides in 15 enzymatically digested crude serum samples collected from healthy donors in 3 laboratories across Europe. The developed method identified 60% more peptides in comparison with conventional 1D nanoLC-MS/MS analysis with ca. 4 times lower throughput while retaining the quantitative information. Serum sample preparation related changes were revealed by applying unsupervised classification techniques and, therefore, must be taken into account while planning multicentric biomarker discovery and validation studies. Overall, this novel method reduces sample complexity and boosts the number of peptide and protein identifications without the need for extra sample handling procedures for samples equivalent to less than 1 μL of blood, which expands the space for potential biomarker discovery by looking deeper into the composition of biofluids.
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http://dx.doi.org/10.1021/acs.analchem.1c01291DOI Listing
July 2021

Chemical isotope labeling for quantitative proteomics.

Mass Spectrom Rev 2021 Jun 6. Epub 2021 Jun 6.

Department of Analytical Biochemistry and Interfaculty Mass Spectrometry Center, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.

Advancements in liquid chromatography and mass spectrometry over the last decades have led to a significant development in mass spectrometry-based proteome quantification approaches. An increasingly attractive strategy is multiplex isotope labeling, which significantly improves the accuracy, precision and throughput of quantitative proteomics in the data-dependent acquisition mode. Isotope labeling-based approaches can be classified into MS1-based and MS2-based quantification. In this review, we give an overview of approaches based on chemical isotope labeling and discuss their principles, benefits, and limitations with the goal to give insights into fundamental questions and provide a useful reference for choosing a method for quantitative proteomics. As a perspective, we discuss the current possibilities and limitations of multiplex, isotope labeling approaches for the data-independent acquisition mode, which is increasing in popularity.
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http://dx.doi.org/10.1002/mas.21709DOI Listing
June 2021

The Isotopic Ac-IP Tag Enables Multiplexed Proteome Quantification in Data-Independent Acquisition Mode.

Anal Chem 2021 06 31;93(23):8196-8202. Epub 2021 May 31.

Department of Analytical Biochemistry and Interfaculty Mass Spectrometry Center, Groningen Research Institute of Pharmacy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Data-independent acquisition (DIA) is an increasingly used approach for quantitative proteomics. However, most current isotope labeling strategies are not suitable for DIA as they lead to more complex MS2 spectra or severe ratio distortion. As a result, DIA suffers from a lower throughput than data-dependent acquisition (DDA) due to a lower level of multiplexing. Herein, we synthesized an isotopically labeled acetyl-isoleucine-proline (Ac-IP) tag for multiplexed quantification in DIA. Differentially labeled peptides have distinct precursor ions carrying the quantitative information but identical MS2 spectra since the isotopically labeled Ac-Ile part leaves as a neutral loss upon collision-induced dissociation, while fragmentation of the peptide backbone generates regular fragment ions for identification. The Ac-IP-labeled samples can be analyzed using general DIA liquid chromatography-mass spectrometry settings, and the data obtained can be processed with established approaches. Relative quantification requires deconvolution of the isotope envelope of the respective precursor ions. Suitability of the Ac-IP tag is demonstrated with a triplex-labeled yeast proteome spiked with bovine serum albumin that was mixed at 10:5:1 ratios, resulting in measured ratios of 9.7:5.3:1.1.
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http://dx.doi.org/10.1021/acs.analchem.1c00453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209779PMC
June 2021

Soluble receptor for advanced glycation end products (sRAGE) as a biomarker of COPD.

Respir Res 2021 Apr 27;22(1):127. Epub 2021 Apr 27.

Research and Development, GlaxoSmithKline, Collegeville, PA, USA.

Background: Soluble receptor for advanced glycation end products (sRAGE) is a proposed emphysema and airflow obstruction biomarker; however, previous publications have shown inconsistent associations and only one study has investigate the association between sRAGE and emphysema. No cohorts have examined the association between sRAGE and progressive decline of lung function. There have also been no evaluation of assay compatibility, receiver operating characteristics, and little examination of the effect of genetic variability in non-white population. This manuscript addresses these deficiencies and introduces novel data from Pittsburgh COPD SCCOR and as well as novel work on airflow obstruction. A meta-analysis is used to quantify sRAGE associations with clinical phenotypes.

Methods: sRAGE was measured in four independent longitudinal cohorts on different analytic assays: COPDGene (n = 1443); SPIROMICS (n = 1623); ECLIPSE (n = 2349); Pittsburgh COPD SCCOR (n = 399). We constructed adjusted linear mixed models to determine associations of sRAGE with baseline and follow up forced expiratory volume at one second (FEV) and emphysema by quantitative high-resolution CT lung density at the 15th percentile (adjusted for total lung capacity).

Results: Lower plasma or serum sRAGE values were associated with a COPD diagnosis (P < 0.001), reduced FEV (P < 0.001), and emphysema severity (P < 0.001). In an inverse-variance weighted meta-analysis, one SD lower log-transformed sRAGE was associated with 105 ± 22 mL lower FEV and 4.14 ± 0.55 g/L lower adjusted lung density. After adjusting for covariates, lower sRAGE at baseline was associated with greater FEV decline and emphysema progression only in the ECLIPSE cohort. Non-Hispanic white subjects carrying the rs2070600 minor allele (A) and non-Hispanic African Americans carrying the rs2071288 minor allele (A) had lower sRAGE measurements compare to those with the major allele, but their emphysema-sRAGE regression slopes were similar.

Conclusions: Lower blood sRAGE is associated with more severe airflow obstruction and emphysema, but associations with progression are inconsistent in the cohorts analyzed. In these cohorts, genotype influenced sRAGE measurements and strengthened variance modelling. Thus, genotype should be included in sRAGE evaluations.
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http://dx.doi.org/10.1186/s12931-021-01686-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076883PMC
April 2021

Isolation of extracellular vesicles with combined enrichment methods.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Apr 27;1169:122604. Epub 2021 Feb 27.

Department of Pediatrics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands. Electronic address:

Extracellular vesicles (EVs) are currently of tremendous interest in many research disciplines and EVs have potential for development of EV diagnostics or therapeutics. Most well-known single EV isolation methods have their particular advantages and disadvantages in terms of EV purity and EV yield. Combining EV isolation methods provides additional potential to improve the efficacy of both purity and yield. This review assesses the contribution and efficacy of using combined EV isolation methods by performing a two-step systematic literature analysis from all papers applying EV isolation in the year 2019. This resulted in an overview of the various methods being applied for EV isolations. A second database was generated for all studies within the first database that fairly compared multiple EV isolation methods by determining both EV purity and EV yield after isolation. From these databases it is shown that the most used EV isolation methods are not per definition the best methods based on EV purity or EV yield, indicating that more factors play a role in the choice which EV isolation method to choose than only the efficacy of the method. From the included studies it is shown that ~60% of all the included EV isolations were performed with combined EV isolation methods. The majority of EV isolations were performed with differential ultracentrifugation alone or in combination with differential ultrafiltration. When efficacy of EV isolation methods was determined in terms of EV purity and EV yield, combined EV isolation methods clearly outperformed single EV isolation methods, regardless of the type of starting material used. A recommended starting point would be the use of size-exclusion chromatography since this method, especially when combined with low-speed centrifugation, resulted in the highest EV purity, while still providing a reasonable EV yield.
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http://dx.doi.org/10.1016/j.jchromb.2021.122604DOI Listing
April 2021

Data Sharing Under the General Data Protection Regulation: Time to Harmonize Law and Research Ethics?

Hypertension 2021 04 15;77(4):1029-1035. Epub 2021 Feb 15.

Urologie 24, Nuremberg, and Department of Urology, Friedrich-Alexander University of Erlangen, Germany (B.J.S-D).

The General Data Protection Regulation (GDPR) became binding law in the European Union Member States in 2018, as a step toward harmonizing personal data protection legislation in the European Union. The Regulation governs almost all types of personal data processing, hence, also, those pertaining to biomedical research. The purpose of this article is to highlight the main practical issues related to data and biological sample sharing that biomedical researchers face regularly, and to specify how these are addressed in the context of GDPR, after consulting with ethics/legal experts. We identify areas in which clarifications of the GDPR are needed, particularly those related to consent requirements by study participants. Amendments should target the following: (1) restricting exceptions based on national laws and increasing harmonization, (2) confirming the concept of broad consent, and (3) defining a roadmap for secondary use of data. These changes will be achieved by acknowledged learned societies in the field taking the lead in preparing a document giving guidance for the optimal interpretation of the GDPR, which will be finalized following a period of commenting by a broad multistakeholder audience. In parallel, promoting engagement and education of the public in the relevant issues (such as different consent types or residual risk for re-identification), on both local/national and international levels, is considered critical for advancement. We hope that this article will open this broad discussion involving all major stakeholders, toward optimizing the GDPR and allowing a harmonized transnational research approach.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.120.16340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968961PMC
April 2021

An antibody-free LC-MS/MS method for the quantification of intact insulin-like growth factors 1 and 2 in human plasma.

Anal Bioanal Chem 2021 Mar 10;413(8):2035-2044. Epub 2021 Feb 10.

Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands.

Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) are important biomarkers in research and diagnosis of growth disorders. Quantitative analysis is performed using various ligand-binding assays or enzymatic digestion LC-MS/MS methods, whose widespread adoption is hampered by time-consuming sample preparation procedures. We present a simple and fast antibody-free LC-MS/MS method for the quantification of intact IGF-1 and IGF-2 in human plasma. The method requires 50 μL of plasma and uses fully N-labelled IGF-1 as internal standard. It features trifluoroethanol (TFE)-based IGF/IGF-binding protein complex dissociation and a two-step selective protein precipitation workflow, using 5% acetic acid in 80/20 acetone/acetonitrile (precipitation 1) and ice-cold ethanol (precipitation 2). Detection of intact IGF-1 and IGF-2 is performed by means of a Waters XEVO TQ-S triple quadrupole mass spectrometer in positive electrospray ionisation (ESI+) mode. Lower limits of quantification were 5.9 ng/mL for IGF-1 and 8.4 ng/mL for IGF-2. Intra-assay imprecision was below 4.5% and inter-assay imprecision was below 5.8% for both analytes. An excellent correlation was found between nominal and measured concentrations of the WHO reference standard for IGF-1. Comparison with the IDS-iSYS IGF-1 immunoassay showed good correlation (R > 0.97), although a significant bias was observed with the immunoassay giving substantially higher concentrations. The LC-MS/MS method described here allows for reliable and simultaneous quantification of IGF-1 and IGF-2 in plasma, without the need for enzymatic digestion. The method can be readily implemented in clinical mass spectrometry laboratories and has the potential to be adapted for the analysis of different similarly sized peptide hormones.
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http://dx.doi.org/10.1007/s00216-021-03185-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943504PMC
March 2021

A Versatile Isobaric Tag Enables Proteome Quantification in Data-Dependent and Data-Independent Acquisition Modes.

Anal Chem 2020 12 30;92(24):16149-16157. Epub 2020 Nov 30.

Department of Analytical Biochemistry and Interfaculty Mass Spectrometry Center, Groningen Research Institute of Pharmacy, University of Groningen, A. Deusinglaan 16, 9713 AV Groningen, The Netherlands.

Quantifying proteins based on peptide-coupled reporter ions is a multiplexed quantitative strategy in proteomics that alleviates the problem of ratio distortion caused by peptide cofragmentation, as commonly observed in other reporter-ion-based approaches, such as TMT and iTRAQ. Data-independent acquisition (DIA) is an attractive alternative to data-dependent acquisition (DDA) due to its better reproducibility. While multiplexed labeling is widely used in DDA, it is rarely used in DIA, presumably because current approaches lead to more complex MS2 spectra, severe ratio distortion, or to a reduction in quantification accuracy and precision. Herein, we present a versatile acetyl-alanine-glycine (Ac-AG) tag that conceals quantitative information in isobarically labeled peptides and reveals it upon tandem MS in the form of peptide-coupled reporter ions. Since the peptide-coupled reporter ion is precursor-specific while fragment ions of the peptide backbone originating from different labeling channels are identical, the Ac-AG tag is compatible with both DDA and DIA. By isolating the monoisotopic peak of the precursor ion in DDA, intensities of the peptide-coupled reporter ions represent the relative ratios between constituent samples, whereas in DIA, the ratio can be inferred after deconvoluting the peptide-coupled reporter ion isotopes. The proteome quantification capability of the Ac-AG tag was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. Within this complex proteomics background, BSA spiked at 1:5:10 ratios was detected at ratios of 1.00:4.87:10.13 in DDA and 1.16:5.20:9.64 in DIA.
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http://dx.doi.org/10.1021/acs.analchem.0c03858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745205PMC
December 2020

A Collision-Induced Dissociation Cleavable Isobaric Tag for Peptide Fragment Ion-Based Quantification in Proteomics.

J Proteome Res 2020 09 17;19(9):3817-3824. Epub 2020 Aug 17.

Department of Analytical Biochemistry and Interfaculty Mass Spectrometry Center, Groningen Research Institute of Pharmacy, University of Groningen, Groningen 9713 AV, The Netherlands.

Quantifying peptides based on unique peptide fragment ions avoids the issue of ratio distortion that is commonly observed for reporter ion-based quantification approaches. Herein, we present a collision-induced dissociation-cleavable, isobaric acetyl-isoleucine-proline-glycine (Ac-IPG) tag, which conserves the merits of quantifying peptides based on unique fragments while reducing the complexity of the b-ion series compared to conventional fragment ion-based quantification methods thus facilitating data processing. Multiplex labeling is based on selective N-terminal dimethylation followed by derivatization of the ε-amino group of the C-terminal Lys residue of LysC peptides with isobaric Ac-IPG tags having complementary isotope distributions on Pro-Gly and Ac-Ile. Upon fragmentation between Ile and Pro, the resulting y ions, with the neutral loss of Ac-Ile, can be distinguished between the different labeling channels based on different numbers of isotope labels on the Pro-Gly part and thus contain the information for relative quantification, while b ions of different labeling channels have the same / values. The proteome quantification capability of this method was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. With the yeast proteins as the background, BSA was detected at ratios of 1.14:5.06:9.78 when spiked at 1:5:10 ratios. The raw mass data is available on the ProteomeXchange with the identifier PXD 018790.
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http://dx.doi.org/10.1021/acs.jproteome.0c00371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476077PMC
September 2020

In Matrix Derivatization Combined with LC-MS/MS Results in Ultrasensitive Quantification of Plasma Free Metanephrines and Catecholamines.

Anal Chem 2020 07 12;92(13):9072-9078. Epub 2020 Jun 12.

Analytical Biochemistry, Department of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Plasma-free metanephrines and catecholamines are essential markers in the biochemical diagnosis and follow-up of neuroendocrine tumors and inborn errors of metabolism. However, their low circulating concentrations (in the nanomolar range) and poor fragmentation characteristics hinder facile simultaneous quantification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we present a sensitive and simple matrix derivatization procedure using propionic anhydride that enables simultaneous quantification of unconjugated l-DOPA, catecholamines, and metanephrines in plasma by LC-MS/MS. Dilution of propionic anhydride 1:4 (v/v) in acetonitrile in combination with 50 μL of plasma resulted in the highest mass spectrometric response. In plasma, derivatization resulted in stable derivatives and increased sensitivity by a factor of 4-30 compared with a previous LC-MS/MS method for measuring plasma metanephrines in our laboratory. Furthermore, propionylation increased specificity, especially for 3-methoxytyramine, by preventing interference from antihypertensive medication (β-blockers). The method was validated according to international guidelines and correlated with a hydrophilic interaction LC-MS/MS method for measuring plasma metanephrines ( > 0.99) and high-performance liquid chromatography with an electrochemical detection method for measuring plasma catecholamines ( > 0.85). Reference intervals for l-DOPA, catecholamines, and metanephrines in = 115 healthy individuals were established. Our work shows that analytes in the subnanomolar range in plasma can be derivatized in situ without any preceding sample extraction. The developed method shows improved sensitivity and selectivity over existing methods and enables simultaneous quantification of several classes of amines.
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http://dx.doi.org/10.1021/acs.analchem.0c01263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349590PMC
July 2020

Targeted imaging of integrins in cancer tissues using photocleavable Ru(ii) polypyridine complexes as mass-tags.

Chem Commun (Camb) 2020 Jun 29;56(44):5941-5944. Epub 2020 Apr 29.

Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Targeted epitope-based mass spectrometry imaging (MSI) utilizes laser cleavable mass-tags bound to targeting moieties for detecting proteins in tissue sections. Our work constitutes the first proof-of-concept of a novel laser desorption ionization (LDI)-MSI strategy using photocleavable Ru(ii) polypyridine complexes as mass-tags for imaging of integrins αvβ3 in human cancer tissues.
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http://dx.doi.org/10.1039/d0cc00774aDOI Listing
June 2020

Selective Maleylation-Directed Isobaric Peptide Termini Labeling for Accurate Proteome Quantification.

Anal Chem 2020 06 12;92(11):7836-7844. Epub 2020 May 12.

Department of Pediatrics, University Medical Center Groningen, University of Groningen, Hanzeplein 1, Groningen 9713 GZ, The Netherlands.

Isobaric peptide termini labeling (IPTL) is an attractive protein quantification method because it provides more accurate and reliable quantification information than traditional isobaric labeling methods (e.g., TMT and iTRAQ) by making use of the entire fragment-ion series instead of only a single reporter ion. The multiplexing capacity of published IPTL implementations is, however, limited to three. Here, we present a selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) approach for quantitative proteomics of LysC protein digestion. SMD-IPTL extends the multiplexing capacity to 4-plex with the potential for higher levels of multiplexing using commercially available C/N labeled amino acids. SMD-IPTL is achieved in a one-pot reaction in three consecutive steps: (1) selective maleylation at the N-terminus; (2) labeling at the ε-NH group of the C-terminal Lys with isotopically labeled acetyl-alanine; (3) thiol Michael addition of an isotopically labeled acetyl-cysteine at the maleylated N-terminus. The isobarically labeled peptides are fragmented into sets of b- and y-ion clusters upon LC-MS/MS, which convey not only sequence information but also quantitative information for every labeling channel and avoid the issue of ratio distortion observed with reporter-ion-based approaches. We demonstrate the SMD-IPTL approach with a 4-plex labeled sample of bovine serum albumin (BSA) and yeast lysates mixed at different ratios. With the use of SMD-IPTL for labeling and a narrow precursor isolation window of 0.8 Th with an offset of -0.2 Th, accurate ratios were measured across a 10-fold mixing range of BSA in a background of yeast proteome. With the yeast proteins mixed at ratios of 1:5:1:5, BSA was detected at ratios of 0.94:2.46:4.70:9.92 when spiked at 1:2:5:10 ratios with an average standard deviation of peptide ratios of 0.34.
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http://dx.doi.org/10.1021/acs.analchem.0c01059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271076PMC
June 2020

Intact protein quantification in biological samples by liquid chromatography - high-resolution mass spectrometry: somatropin in rat plasma.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 May 23;1144:122079. Epub 2020 Mar 23.

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands; Bioanalytical Laboratory, PRA Health Sciences, Amerikaweg 18, 9407 TK Assen, the Netherlands. Electronic address:

The quantitative determination of intact proteins in biological samples by LC with high-resolution MS detection can be a useful alternative to ligand-binding assays or LC-MS-based quantification of a surrogate peptide after protein digestion. The 22-kDa biopharmaceutical protein somatropin (recombinant human growth hormone) was quantified down to 10 ng/mL (0.45 nM) in 75 μL of rat plasma by the combination of an immunocapture step using an anti-somatropin antibody and LC-MS on a quadrupole-time of flight instrument. Accuracy and precision of the method as well as its selectivity and sensitivity did not depend on the width of the mass extraction window nor on whether only one or a summation of multiple charge states of the protein analyte were used as the detection response. Quantification based on deconvoluted mass spectra showed equally acceptable method performance but with a less favorable lower limit of quantification of 30 ng/mL. Concentrations in plasma after dosing of somatropin to rats correlated well for the deconvolution approach and the quantification based on the summation of the response of the four most intense charge states (14 to 17) of somatropin.
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http://dx.doi.org/10.1016/j.jchromb.2020.122079DOI Listing
May 2020

Cigarette smoking prior to blood sampling acutely affects serum levels of the chronic obstructive pulmonary disease biomarker surfactant protein D.

Clin Chem Lab Med 2020 07;58(8):e138-e141

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Groningen, The Netherlands.

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http://dx.doi.org/10.1515/cclm-2019-1246DOI Listing
July 2020

Female Specific Association of Low Insulin-Like Growth Factor 1 (IGF1) Levels with Increased Risk of Premature Mortality in Renal Transplant Recipients.

J Clin Med 2020 Jan 21;9(2). Epub 2020 Jan 21.

Department of Internal Medicine, Division of Nephrology, University Medical Center Groningen, University of Groningen, 9700 RB Groningen, The Netherlands.

Associations between insulin-like growth factor 1 (IGF1) and mortality have been reported to be female specific in mice and in human nonagenarians. Intervention in the growth hormone (GH)-IGF1 axis may particularly benefit patients with high risk of losing muscle mass, including renal transplant recipients (RTR). We investigated whether a potential association of circulating IGF1 with all-cause mortality in stable RTR could be female specific and mediated by variation in muscle mass. To this end, plasma IGF1 levels were measured in 277 female and 343 male RTR by mass spectrometry, and their association with mortality was assessed by Cox regression. During a median follow-up time of 5.4 years, 56 female and 77 male RTR died. In females, IGF1 was inversely associated with risk (hazard ratio (HR) per 1-unit increment in log2-transformed (doubling of) IGF1 levels, 95% confidence interval (CI)) of mortality (0.40, 0.24-0.65; < 0.001), independent of age and the estimated Glomerular filtration rate (eGFR). In equivalent analyses, no significant association was observed for males (0.85, 0.56-1.29; = 0.44), for which it should be noted that in males, age was negatively and strongly associated with IGF1 levels. The association for females remained materially unchanged upon adjustment for potential confounders and was furthermore found to be mediated for 39% by 24 h urinary creatinine excretion. In conclusion, low IGF1 levels associate with an increased risk of all-cause mortality in female RTR, which may link to conditions of low muscle mass that are known to be associated with poor outcomes in transplantation patients. For males, the strongly negative association of age with IGF1 levels may explain why low IGF1 levels were not found to be associated with an increased risk of all-cause mortality.
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http://dx.doi.org/10.3390/jcm9020293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073643PMC
January 2020

Integrated proteogenomic approach identifying a protein signature of COPD and a new splice variant of SORBS1.

Thorax 2020 02 14;75(2):180-183. Epub 2020 Jan 14.

Department of Analytical Biochemistry, University of Groningen, Groningen Research Institute of Pharmacy, Groningen, the Netherlands.

Translation of genomic alterations to protein changes in chronic obstructive pulmonary disease (COPD) is largely unexplored. Using integrated proteomic and RNA sequencing analysis of COPD and control lung tissues, we identified a protein signature in COPD characterised by extracellular matrix changes and a potential regulatory role for SUMO2. Furthermore, we identified 61 differentially expressed novel, non-reference, peptides in COPD compared with control lungs. This included two peptides encoding for a new splice variant of SORBS1, of which the transcript usage was higher in COPD compared with control lungs. These explorative findings and integrative proteogenomic approach open new avenues to further unravel the pathology of COPD.
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http://dx.doi.org/10.1136/thoraxjnl-2019-213200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029225PMC
February 2020

Mass spectrometric analysis of PTM dynamics using stable isotope labeled metabolic precursors in cell culture.

Analyst 2019 Nov;144(23):6812-6833

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Biological organisms represent highly dynamic systems, which are continually exposed to environmental factors and always strive to restore steady-state homeostasis. Posttranslational modifications are key regulators with which biological systems respond to external stimuli. To understand how homeostasis is restored, it is important to study the kinetics of posttranslational modifications. In this review we discuss proteomic approaches using stable isotope labeled metabolic precursors to study dynamics of posttranslational modifications in cell culture.
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http://dx.doi.org/10.1039/c9an01258cDOI Listing
November 2019

Targeted Proteomics to Study Mitochondrial Biology.

Adv Exp Med Biol 2019 ;1158:101-117

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.

Targeted mass spectrometry in the selected or parallel reaction monitoring (SRM or PRM) mode is a widely used methodology to quantify proteins based on so-called signature or proteotypic peptides. SRM has the advantage of being able to quantify a range of proteins in a single analysis, for example, to measure the level of enzymes comprising a biochemical pathway. In this chapter, we will detail how to set up an SRM assay on the example of the mitochondrial protein succinate dehydrogenase [ubiquinone] flavoprotein subunit (mouse UniProt-code Q8K2B3). First, we will outline the in silico assay design including the choice of peptides based on a range of properties. We will further delineate different quantification strategies and introduce the reader to LC-MS assay development including the selection of the optimal peptide charge state and fragment ions as well as a discussion of the dynamic range of detection. The chapter will close with an application from the area of mitochondrial biology related to the quantification of a set of proteins isolated from mouse liver mitochondria in a study on mitochondrial respiratory flux decline in aging mouse muscle.
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http://dx.doi.org/10.1007/978-981-13-8367-0_7DOI Listing
September 2019

Separation of deamidated peptides with mixed-mode chromatography using phospholipid-functionalized monolithic stationary phases.

J Chromatogr A 2019 Oct 28;1603:417-421. Epub 2019 May 28.

Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China; Department of Pharmacy and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, Jinan University, Guangzhou, 510632, China. Electronic address:

Deamidation of asparagine (Asn) residues of monoclonal antibodies (mAbs) plays a pivotal role in the in vivo/vitro degradation or efficacy loss of biopharmaceuticals. However, a major challenge for MS analysis of deamidation of Asn-containing peptides in mAbs, is due to the fact that there is only a 1 Da mass shift between the native form (Asn residues) and deamidated forms (n-aspartyl (n-Asp) and isoaspartyl (isoAsp) residues with identical mass). Therefore, a chromatographic separation of the deamidated proteins and/or the peptides derived therefrom is needed prior to MS analysis. In this study, the monolithic column with various stationary phases, including reverse phase (RP), single phospholipid-functionalized and mixed phospholipid-functionalized monoliths, were prepared for the separation of the deamidation-sensitive signature peptide (IYPTNGYTR) of trastuzumab and its two deamidated products, n-Asp55 residue IYPTDGYTR and isoAsp55 residue IYPTisoDGYTR. Compared to the RP monolith, the phospholipid-functionalized monoliths provided mixed-mode interactions and exhibited better peak shape and separation selectivity. The effect of the parameters, including the type and concentration of buffer, temperature and pH value on the separation performance were investigated. Under the optimal conditions, the three peptides were fully separated on a mixed phosphocholine (PC) / phosphatidic acid (PA) functionalized monolith (poly (MDPCPA-co-EDMA)) due to the joint contribution of hydrophobic and electrostatic interactions. Therefore, the novel method based on the mixed phospholipids-functionalized monolithic column exhibited good potential for the analysis of deamidated peptides, which will be useful for the in-depth study of post-translational modifications of mAbs.
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http://dx.doi.org/10.1016/j.chroma.2019.05.053DOI Listing
October 2019

Quantification of surfactant protein D (SPD) in human serum by liquid chromatography-mass spectrometry (LC-MS).

Talanta 2019 Sep 6;202:507-513. Epub 2019 May 6.

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713, AV Groningen, Netherlands. Electronic address:

Quantification of intact proteins in complex biological matrices by liquid chromatography-mass spectrometry (LC-MS) is a promising analytical strategy but is technically challenging, notably for concentrations at or below the ng/mL level. Therefore, MS-based protein quantification is mostly based on measuring protein-specific peptides, so-called 'surrogate peptides', that are released through proteolysis. While quantitative protein bioanalysis based on peptide LC-MS is much more sensitive, not every peptide is suitable in this respect. For example, some peptides are too small to be unique for a protein while others are too large to be measured with sufficient sensitivity, so careful selection of appropriate peptides is essential. Here we present a validated LC-MS method for quantification of surfactant protein D (SPD) at clinically relevant levels between 5 and 500 ng/mL using 50 μL of serum. This method targets two SPD-specific peptides in the C-type lectin, ligand binding domain of the SPD protein. One of these peptides contains a methionine residue which would typically be avoided because of its unstable nature. Some quantitative methods do target methionine-containing peptides, and corresponding workflows feature an oxidation step at the peptide level using hydrogen peroxide (HO) to convert all methionine residues to more stable methionine sulfoxides. For our method, such a procedure was associated with peptide loss, hence we developed an oxidation procedure at the protein level using HO to oxidize methionine residues and the enzyme catalase to quench excess HO. This procedure may be applicable to other quantitative methods based on a surrogate peptide-based approach and may potentially also be useful for MS-based workflows targeting intact proteins.
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http://dx.doi.org/10.1016/j.talanta.2019.05.028DOI Listing
September 2019

Exact hypothesis testing for shrinkage-based Gaussian graphical models.

Bioinformatics 2019 12;35(23):5011-5017

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy.

Motivation: One of the main goals in systems biology is to learn molecular regulatory networks from quantitative profile data. In particular, Gaussian graphical models (GGMs) are widely used network models in bioinformatics where variables (e.g. transcripts, metabolites or proteins) are represented by nodes, and pairs of nodes are connected with an edge according to their partial correlation. Reconstructing a GGM from data is a challenging task when the sample size is smaller than the number of variables. The main problem consists in finding the inverse of the covariance estimator which is ill-conditioned in this case. Shrinkage-based covariance estimators are a popular approach, producing an invertible 'shrunk' covariance. However, a proper significance test for the 'shrunk' partial correlation (i.e. the GGM edges) is an open challenge as a probability density including the shrinkage is unknown. In this article, we present (i) a geometric reformulation of the shrinkage-based GGM, and (ii) a probability density that naturally includes the shrinkage parameter.

Results: Our results show that the inference using this new 'shrunk' probability density is as accurate as Monte Carlo estimation (an unbiased non-parametric method) for any shrinkage value, while being computationally more efficient. We show on synthetic data how the novel test for significance allows an accurate control of the Type I error and outperforms the network reconstruction obtained by the widely used R package GeneNet. This is further highlighted in two gene expression datasets from stress response in Eschericha coli, and the effect of influenza infection in Mus musculus.

Availability And Implementation: https://github.com/V-Bernal/GGM-Shrinkage.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901079PMC
December 2019
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