Publications by authors named "Ole N Jensen"

194 Publications

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis.

Mol Cell 2021 Feb 23. Epub 2021 Feb 23.

Cell Biology Program and Center for Epigenetics, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen N 2200, Denmark; The Novo Nordisk Foundation Center for Stem Cell Biology (Danstem), University of Copenhagen, Copenhagen N 2200, Denmark. Electronic address:

Acetylation of lysine 16 on histone H4 (H4K16ac) is catalyzed by histone acetyltransferase KAT8 and can prevent chromatin compaction in vitro. Although extensively studied in Drosophila, the functions of H4K16ac and two KAT8-containing protein complexes (NSL and MSL) are not well understood in mammals. Here, we demonstrate a surprising complex-dependent activity of KAT8: it catalyzes H4K5ac and H4K8ac as part of the NSL complex, whereas it catalyzes the bulk of H4K16ac as part of the MSL complex. Furthermore, we show that MSL complex proteins and H4K16ac are not required for cell proliferation and chromatin accessibility, whereas the NSL complex is essential for cell survival, as it stimulates transcription initiation at the promoters of housekeeping genes. In summary, we show that KAT8 switches catalytic activity and function depending on its associated proteins and that, when in the NSL complex, it catalyzes H4K5ac and H4K8ac required for the expression of essential genes.
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http://dx.doi.org/10.1016/j.molcel.2021.02.012DOI Listing
February 2021

Selective Enrichment of Histidine Phosphorylated Peptides Using Molecularly Imprinted Polymers.

Anal Chem 2021 Mar 16;93(8):3857-3866. Epub 2021 Feb 16.

Department of Biomedical Science, Faculty of Health and Society, Malmö University, 205 06 Malmö, Sweden.

Protein histidine phosphorylation (pHis) is involved in molecular signaling networks in bacteria, fungi, plants, and higher eukaryotes including mammals and is implicated in human diseases such as cancer. Detailed investigations of the pHis modification are hampered due to its acid-labile nature and consequent lack of tools to study this post-translational modification (PTM). We here demonstrate three molecularly imprinted polymer (MIP)-based reagents, MIP1-MIP3, for enrichment of pHis peptides and subsequent characterization by chromatography and mass spectrometry (LC-MS). The combination of MIP1 and β-elimination provided some selectivity for improved detection of pHis peptides. MIP2 was amenable to larger pHis peptides, although with poor selectivity. Microsphere-based MIP3 exhibited improved selectivity and was amenable to enrichment and detection by LC-MS of pHis peptides in tryptic digests of protein mixtures. These MIP protocols do not involve any acidic solvents during sample preparation and enrichment, thus preserving the pHis modification. The presented proof-of-concept results will lead to new protocols for highly selective enrichment of labile protein phosphorylations using molecularly imprinted materials.
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http://dx.doi.org/10.1021/acs.analchem.0c04474DOI Listing
March 2021

Lipid molecular timeline profiling reveals diurnal crosstalk between the liver and circulation.

Cell Rep 2021 Feb;34(5):108710

Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Odense, Denmark; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany. Electronic address:

Diurnal regulation of whole-body lipid metabolism plays a vital role in metabolic health. Although changes in lipid levels across the diurnal cycle have been investigated, the system-wide molecular responses to both short-acting fasting-feeding transitions and longer-timescale circadian rhythms have not been explored in parallel. Here, we perform time-series multi-omics analyses of liver and plasma revealing that the majority of molecular oscillations are entrained by adaptations to fasting, food intake, and the postprandial state. By developing algorithms for lipid structure enrichment analysis and lipid molecular crosstalk between tissues, we find that the hepatic phosphatidylethanolamine (PE) methylation pathway is diurnally regulated, giving rise to two pools of oscillating phosphatidylcholine (PC) molecules in the circulation, which are coupled to secretion of either very low-density lipoprotein (VLDL) or high-density lipoprotein (HDL) particles. Our work demonstrates that lipid molecular timeline profiling across tissues is key to disentangling complex metabolic processes and provides a critical resource for the study of whole-body lipid metabolism.
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http://dx.doi.org/10.1016/j.celrep.2021.108710DOI Listing
February 2021

A conserved, buried cysteine near the P-site is accessible to cysteine modifications and increases ROS stability in the P-type plasma membrane H+-ATPase.

Biochem J 2021 Feb;478(3):619-632

Department of Plant and Environmental Sciences, University of Copenhagen, Denmark.

Sulfur-containing amino acid residues function in antioxidative responses, which can be induced by the reactive oxygen species generated by excessive copper and hydrogen peroxide. In all Na+/K+, Ca2+, and H+ pumping P-type ATPases, a cysteine residue is present two residues upstream of the essential aspartate residue, which is obligatorily phosphorylated in each catalytic cycle. Despite its conservation, the function of this cysteine residue was hitherto unknown. In this study, we analyzed the function of the corresponding cysteine residue (Cys-327) in the autoinhibited plasma membrane H+-ATPase isoform 2 (AHA2) from Arabidopsis thaliana by mutagenesis and heterologous expression in a yeast host. Enzyme kinetics of alanine, serine, and leucine substitutions were identical with those of the wild-type pump but the sensitivity of the mutant pumps was increased towards copper and hydrogen peroxide. Peptide identification and sequencing by mass spectrometry demonstrated that Cys-327 was prone to oxidation. These data suggest that Cys-327 functions as a protective residue in the plasma membrane H+-ATPase, and possibly in other P-type ATPases as well.
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http://dx.doi.org/10.1042/BCJ20200559DOI Listing
February 2021

Zirconium(IV)-IMAC Revisited: Improved Performance and Phosphoproteome Coverage by Magnetic Microparticles for Phosphopeptide Affinity Enrichment.

J Proteome Res 2021 Jan 23;20(1):453-462. Epub 2020 Nov 23.

Department of Biochemistry & Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Odense M DK-5230, Denmark.

Phosphopeptide enrichment is an essential step in large-scale, quantitative phosphoproteomics by mass spectrometry. Several phosphopeptide affinity enrichment techniques exist, such as immobilized metal-ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC). We compared zirconium(IV) IMAC (Zr-IMAC) magnetic microparticles to more commonly used titanium(IV) IMAC (Ti-IMAC) and TiO magnetic microparticles for phosphopeptide enrichment from simple and complex protein samples prior to phosphopeptide sequencing and characterization by mass spectrometry (liquid chromatography-tandem mass spectrometry, LC-MS/MS). We optimized sample-loading conditions to increase phosphopeptide recovery for Zr-IMAC-, Ti-IMAC-, and TiO-based workflows by 22, 24, and 35%, respectively. The optimized protocol resulted in improved performance of Zr-IMAC over Ti-IMAC and TiO as well as high-performance liquid chromatography-based Fe(III)-IMAC with up to 23% more identified phosphopeptides. The different enrichment chemistries showed a high degree of overlap but also differences in phosphopeptide selectivity and complementarity. We conclude that Zr-IMAC improves phosphoproteome coverage and recommend that this complementary and scalable affinity enrichment method is more widely used in biological and biomedical studies of cell signaling and the search for biomarkers. Data are available via ProteomeXchange with identifier PXD018273.
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http://dx.doi.org/10.1021/acs.jproteome.0c00508DOI Listing
January 2021

Histo-molecular differentiation of renal cancer subtypes by mass spectrometry imaging and rapid proteome profiling of formalin-fixed paraffin-embedded tumor tissue sections.

Oncotarget 2020 Nov 3;11(44):3998-4015. Epub 2020 Nov 3.

Department of Biochemistry & Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Odense, Denmark.

Pathology differentiation of renal cancer types is challenging due to tissue similarities or overlapping histological features of various tumor (sub) types. As assessment is often manually conducted outcomes can be prone to human error and therefore require high-level expertise and experience. Mass spectrometry can provide detailed histo-molecular information on tissue and is becoming increasingly popular in clinical settings. Spatially resolving technologies such as mass spectrometry imaging and quantitative microproteomics profiling in combination with machine learning approaches provide promising tools for automated tumor classification of clinical tissue sections. In this proof of concept study we used MALDI-MS imaging (MSI) and rapid LC-MS/MS-based microproteomics technologies (15 min/sample) to analyze formalin-fixed paraffin embedded (FFPE) tissue sections and classify renal oncocytoma (RO, = 11), clear cell renal cell carcinoma (ccRCC, = 12) and chromophobe renal cell carcinoma (ChRCC, = 5). Both methods were able to distinguish ccRCC, RO and ChRCC in cross-validation experiments. MSI correctly classified 87% of the patients whereas the rapid LC-MS/MS-based microproteomics approach correctly classified 100% of the patients. This strategy involving MSI and rapid proteome profiling by LC-MS/MS reveals molecular features of tumor sections and enables cancer subtype classification. Mass spectrometry provides a promising complementary approach to current pathological technologies for precise digitized diagnosis of diseases.
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http://dx.doi.org/10.18632/oncotarget.27787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646834PMC
November 2020

Combinatory Treatment of Canavanine and Arginine Deprivation Efficiently Targets Human Glioblastoma Cells via Pleiotropic Mechanisms.

Cells 2020 09 30;9(10). Epub 2020 Sep 30.

Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland.

Glioblastomas are the most frequent and aggressive form of primary brain tumors with no efficient cure. However, they often exhibit specific metabolic shifts that include deficiency in the biosynthesis of and dependence on certain exogenous amino acids. Here, we evaluated, in vitro, a novel combinatory antiglioblastoma approach based on arginine deprivation and canavanine, an arginine analogue of plant origin, using two human glioblastoma cell models, U251MG and U87MG. The combinatory treatment profoundly affected cell viability, morphology, motility and adhesion, destabilizing the cytoskeleton and mitochondrial network, and induced apoptotic cell death. Importantly, the effects were selective toward glioblastoma cells, as they were not pronounced for primary rat glial cells. At the molecular level, canavanine inhibited prosurvival kinases such as FAK, Akt and AMPK. Its effects on protein synthesis and stress response pathways were more complex and dependent on exposure time. We directly observed canavanine incorporation into nascent proteins by using quantitative proteomics. Although canavanine in the absence of arginine readily incorporated into polypeptides, no motif preference for such incorporation was observed. Our findings provide a strong rationale for further developing the proposed modality based on canavanine and arginine deprivation as a potential antiglioblastoma metabolic therapy independent of the blood-brain barrier.
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http://dx.doi.org/10.3390/cells9102217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600648PMC
September 2020

Yeast Ppz1 protein phosphatase toxicity involves the alteration of multiple cellular targets.

Sci Rep 2020 09 24;10(1):15613. Epub 2020 Sep 24.

Institut de Biotecnologia i Biomedicina & Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain.

Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.
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http://dx.doi.org/10.1038/s41598-020-72391-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519054PMC
September 2020

Phosphoproteomic Analysis across the Yeast Life Cycle Reveals Control of Fatty Acyl Chain Length by Phosphorylation of the Fatty Acid Synthase Complex.

Cell Rep 2020 08;32(6):108024

Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Odense, Denmark; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany. Electronic address:

The ability to remodel lipid metabolism under changing conditions is pivotal for cellular functionality and homeostasis. Here, we characterize the regulatory landscape of phosphorylation-based signaling events across the life cycle of Saccharomyces cerevisiae and determine its impact on the regulation of lipid metabolism. Our data show that 50 lipid metabolic proteins are differentially phosphorylated as cells transit between different physiological states. To identify functional phosphosites, we devised a strategy where multiple phosphosites are simultaneously mutated into phosphomimetic or phosphodeficient alleles and mutants are phenotyped by in-depth lipidomics flux analysis. This uncovers functional phosphosites in the phosphatidate cytidylyltransferase Cds1, the phosphatidylserine synthase Cho1, and Fas2, the α-subunit of the fatty acid synthase (FAS) complex. Furthermore, we show that the fatty acyl chain length produced by FAS is governed by phosphorylation. Overall, our work demonstrates a vital role for phosphoregulation of lipid metabolism and provides a resource to investigate its molecular underpinnings.
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http://dx.doi.org/10.1016/j.celrep.2020.108024DOI Listing
August 2020

Mutant FOXL2 Hijacks SMAD4 and SMAD2/3 to Drive Adult Granulosa Cell Tumors.

Cancer Res 2020 09 8;80(17):3466-3479. Epub 2020 Jul 8.

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen N, Denmark.

The mutant protein FOXL2 is expressed in at least 95% of adult-type ovarian granulosa cell tumors (AGCT) and is considered to be a driver of oncogenesis in this disease. However, the molecular mechanism by which FOXL2 contributes to tumorigenesis is not known. Here, we show that mutant FOXL2 acquires the ability to bind SMAD4, forming a FOXL2/SMAD4/SMAD2/3 complex that binds a novel hybrid DNA motif AGHCAHAA, unique to the FOXL2 mutant. This binding induced an enhancer-like chromatin state, leading to transcription of nearby genes, many of which are characteristic of epithelial-to-mesenchymal transition. FOXL2 also bound hybrid loci in primary AGCT. Ablation of SMAD4 or SMAD2/3 resulted in strong reduction of FOXL2 binding at hybrid sites and decreased expression of associated genes. Accordingly, inhibition of TGFβ mitigated the transcriptional effect of FOXL2. Our results provide mechanistic insight into AGCT pathogenesis, identifying FOXL2 and its interaction with SMAD4 as potential therapeutic targets to this condition. SIGNIFICANCE: FOXL2 hijacks SMAD4 and leads to the expression of genes involved in EMT, stemness, and oncogenesis in AGCT, making FOXL2 and the TGFβ pathway therapeutic targets in this condition. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3466/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0259DOI Listing
September 2020

PolySTest: Robust Statistical Testing of Proteomics Data with Missing Values Improves Detection of Biologically Relevant Features.

Mol Cell Proteomics 2020 08 18;19(8):1396-1408. Epub 2020 May 18.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.

Statistical testing remains one of the main challenges for high-confidence detection of differentially regulated proteins or peptides in large-scale quantitative proteomics experiments by mass spectrometry. Statistical tests need to be sufficiently robust to deal with experiment intrinsic data structures and variations and often also reduced feature coverage across different biological samples due to ubiquitous missing values. A robust statistical test provides accurate confidence scores of large-scale proteomics results, regardless of instrument platform, experimental protocol and software tools. However, the multitude of different combinations of experimental strategies, mass spectrometry techniques and informatics methods complicate the decision of choosing appropriate statistical approaches. We address this challenge by introducing PolySTest, a user-friendly web service for statistical testing, data browsing and data visualization. We introduce a new method, Miss test, that simultaneously tests for missingness and feature abundance, thereby complementing common statistical tests by rescuing otherwise discarded data features. We demonstrate that PolySTest with integrated Miss test achieves higher confidence and higher sensitivity for artificial and experimental proteomics data sets with known ground truth. Application of PolySTest to mass spectrometry based large-scale proteomics data obtained from differentiating muscle cells resulted in the rescue of 10-20% additional proteins in the identified molecular networks relevant to muscle differentiation. We conclude that PolySTest is a valuable addition to existing tools and instrument enhancements that improve coverage and depth of large-scale proteomics experiments. A fully functional demo version of PolySTest and Miss test is available via http://computproteomics.bmb.sdu.dk/Apps/PolySTest.
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http://dx.doi.org/10.1074/mcp.RA119.001777DOI Listing
August 2020

The Hypoxic Proteome and Metabolome of Barley ( L.) with and without Phytoglobin Priming.

Int J Mol Sci 2020 Feb 24;21(4). Epub 2020 Feb 24.

Department of Agroecology, Section of Crop Genetics and Biotechnology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark.

Overexpression of phytoglobins (formerly plant hemoglobins) increases the survival rate of plant tissues under hypoxia stress by the following two known mechanisms: (1) scavenging of nitric oxide (NO) in the phytoglobin/NO cycle and (2) mimicking ethylene priming to hypoxia when NO scavenging activates transcription factors that are regulated by levels of NO and O in the N-end rule pathway. To map the cellular and metabolic effects of hypoxia in barley ( L., cv. Golden Promise), with or without priming to hypoxia, we studied the proteome and metabolome of wild type (WT) and hemoglobin overexpressing (HO) plants in normoxia and after 24 h hypoxia (WT24, HO24). The WT plants were more susceptible to hypoxia than HO plants. The chlorophyll a + b content was lowered by 50% and biomass by 30% in WT24 compared to WT, while HO plants were unaffected. We observed an increase in ROS production during hypoxia treatment in WT seedlings that was not observed in HO seedlings. We identified and quantified 9694 proteins out of which 1107 changed significantly in abundance. Many proteins, such as ion transporters, Ca-signal transduction, and proteins related to protein degradation were downregulated in HO plants during hypoxia, but not in WT plants. Changes in the levels of histones indicates that chromatin restructuring plays a role in the priming of hypoxia. We also identified and quantified 1470 metabolites, of which the abundance of >500 changed significantly. In summary the data confirm known mechanisms of hypoxia priming by ethylene priming and N-end rule activation; however, the data also indicate the existence of other mechanisms for hypoxia priming in plants.
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http://dx.doi.org/10.3390/ijms21041546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073221PMC
February 2020

Visualization of the dynamics of histone modifications and their crosstalk using PTM-CrossTalkMapper.

Methods 2020 12 21;184:78-85. Epub 2020 Jan 21.

Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, DK-5230 Odense M, Denmark. Electronic address:

Visualization of large-scale, multi-dimensional omics data is a major challenge. Experimental study designs in proteomics research produce multiple data layers, e.g. relationships between hundreds of proteins, their interactions and abundances as a function of time or perturbation, as well as dynamics of post-translational modifications (PTMs) and allelic variants (proteoforms). These different levels and types of information of proteins and proteoforms complicate data analysis and generation of insightful and comprehensible graphic visualizations. Middle-down mass spectrometry of histone proteins now allows quantifying hundreds of histone proteoforms including co-existing methylation, acetylation and phosphorylation events at distinct amino acid residues within histone molecules. The histone PTM landscape plays a dominant role in the regulation of chromatin activity and transcriptional and epigenetic control. The dynamics of these reversible modifications are governed by reader, writer and eraser enzymes that cooperate to regulate molecular mechanisms that rely on multiple interdependent PTM marks in histones and nucleosomes in chromatin. This PTM crosstalk can be quantified and provides a detailed picture of the underlying rules for setting the histone PTM landscape and chromatin activity, and is available to the community via our CrosstalkDB platform. Here, we developed a new computational method, PTM-CrossTalkMapper, to visualize the dynamics of histone PTMs for different experimental conditions, replicates and proteoforms onto a landscape, thereby describing the crosstalk and interplay between PTMs in a more comprehensible manner. We show the power of combining different levels of information on such crosstalk maps for histone PTM dynamics in mouse organs during the aging process. The PTM-CrossTalkMapper toolkit provides flexible functions to create these maps in various scenarios of multi-dimensional experimental designs, including histone PTM patterns and PTM crosstalk. The source code is available at https://github.com/veitveit/CrossTalkMapper.
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http://dx.doi.org/10.1016/j.ymeth.2020.01.012DOI Listing
December 2020

Middle-Down Proteomic Analyses with Ion Mobility Separations of Endogenous Isomeric Proteoforms.

Anal Chem 2020 02 17;92(3):2364-2368. Epub 2020 Jan 17.

Department of Biochemistry & Molecular Biology and VILLUM Center for Bioanalytical Sciences , University of Southern Denmark , DK-5230 Odense M , Denmark.

Biological functions of many proteins are governed by post-translational modifications (PTMs). In particular, the rich PTM complement in histones controls the gene expression and chromatin structure with major health implications via a combinatoric language. Deciphering that "histone code" is the great challenge for proteomics given an astounding number of possible proteoforms, including isomers with different PTM positions. These must be disentangled on the top- or middle-down level to preserve the key PTM connectivity, which condensed-phase separations failed to achieve. We reported the capability of ion mobility spectrometry (IMS) methods to resolve such isomers for model histone tails. Here, we advance to biological samples, showing middle-down analyses of histones from mouse embryonic stem cells via online chromatography to fractionate proteoforms with distinct PTM sets, differential or field asymmetric waveform IMS (FAIMS) to resolve the isomers, and Orbitrap mass spectrometry with electron transfer dissociation to identify the resolved species.
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http://dx.doi.org/10.1021/acs.analchem.9b05011DOI Listing
February 2020

PRMT5 methylome profiling uncovers a direct link to splicing regulation in acute myeloid leukemia.

Nat Struct Mol Biol 2019 11 14;26(11):999-1012. Epub 2019 Oct 14.

Biotech Research and Innovation Centre, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Protein arginine methyltransferase 5 (PRMT5) has emerged as a promising cancer drug target, and three PRMT5 inhibitors are currently in clinical trials for multiple malignancies. In this study, we investigated the role of PRMT5 in human acute myeloid leukemia (AML). Using an enzymatic dead version of PRMT5 and a PRMT5-specific inhibitor, we demonstrated the requirement of the catalytic activity of PRMT5 for the survival of AML cells. We then identified PRMT5 substrates using multiplexed quantitative proteomics and investigated their role in the survival of AML cells. We found that the function of the splicing regulator SRSF1 relies on its methylation by PRMT5 and that loss of PRMT5 leads to changes in alternative splicing of multiple essential genes. Our study proposes a mechanism for the requirement of PRMT5 for leukemia cell survival and provides potential biomarkers for the treatment response to PRMT5 inhibitors.
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http://dx.doi.org/10.1038/s41594-019-0313-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858565PMC
November 2019

High-Resolution Differential Ion Mobility Separations/Orbitrap Mass Spectrometry without Buffer Gas Limitations.

Anal Chem 2019 05 9;91(10):6918-6925. Epub 2019 May 9.

Department of Chemistry , Wichita State University , 1845 Fairmount , Wichita , Kansas 67260 , United States.

Strong orthogonality between differential ion mobility spectrometry (FAIMS) and mass spectrometry (MS) makes their hybrid a powerful approach to separate isomers and isobars. Harnessing that power depends on high resolution in both dimensions. The ultimate mass resolution and accuracy are delivered by Fourier Transform MS increasingly realized in Orbitrap MS, whereas FAIMS resolution is generally maximized by buffers rich in He or H that elevate ion mobility and lead to prominent non-Blanc effects. However, turbomolecular pumps have lower efficiency for light gas molecules and their flow from the FAIMS stage complicates maintaining the ultrahigh vacuum (UHV) needed for Orbitrap operation. Here we address this challenge via two hardware modifications: (i) a differential pumping step between FAIMS and MS stages and (ii) reconfiguration of vacuum lines to isolate pumping of the high vacuum (HV) region. Either greatly ameliorates the pressure increases upon He or H aspiration. This development enables free optimization of FAIMS carrier gas without concerns about MS performance, maximizing the utility and flexibility of FAIMS/MS platforms.
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http://dx.doi.org/10.1021/acs.analchem.9b01309DOI Listing
May 2019

Differential Ion Mobility Separations/Mass Spectrometry with High Resolution in Both Dimensions.

Anal Chem 2019 01 27;91(2):1479-1485. Epub 2018 Dec 27.

Department of Chemistry , Wichita State University , 1845 Fairmount , Wichita , Kansas 67260 , United States.

Strong orthogonality to mass spectrometry makes differential ion mobility spectrometry (FAIMS) a powerful tool for isomer separations. However, high FAIMS resolution has been achieved overall only with buffers rich in He or H. That obstructed coupling to Fourier transform mass spectrometers operating under ultrahigh vacuum, but exceptional m/ z resolution and accuracy of FTMS are indispensable for frontline biological and environmental applications. By raising the waveform amplitude to 6 kV, we enabled high FAIMS resolution using solely N and thus straightforward integration with any MS platform: here Orbitrap XL with the electron transfer dissociation (ETD) option. The initial evaluation for complete histone tails (50 residues) with diverse post-translational modifications on alternative sites demonstrates a broad capability to separate and confidently identify the PTM localization variants in the middle-down range.
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http://dx.doi.org/10.1021/acs.analchem.8b04518DOI Listing
January 2019

FlashPack: Fast and Simple Preparation of Ultrahigh-performance Capillary Columns for LC-MS.

Mol Cell Proteomics 2019 02 29;18(2):383-390. Epub 2018 Oct 29.

From the Department of Biochemistry & Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark

Capillary ultrahigh-pressure liquid chromatography (cUHPLC) is essential for in-depth characterization of complex biomolecule mixtures by LC-MS. We developed a simple and fast method called for custom packing of capillary columns of 50-100 cm length with sub- 2 μm sorbent particles. uses high sorbent concentrations of 500-1,000 mg/ml for packing at relatively low pressure of 100 bar. Column blocking by sorbent aggregation is avoided during the packing by gentle mechanical tapping of the capillary proximal end by a slowly rotating magnet bar. Utilizing a standard 100-bar pressure bomb, allows for production of 15-25 cm cUHPLC columns within a few minutes and of 50 cm cUHPLC columns in less than an hour. Columns exhibit excellent reproducibility of back-pressure, retention time, and resolution (CV 8.7%). cUHPLC columns are inexpensive, robust and deliver performance comparable to commercially available cUHPLC columns. The FlashPack method is versatile and enables production of cUHPLC columns using a variety of sorbent materials.
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http://dx.doi.org/10.1074/mcp.TIR118.000953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356079PMC
February 2019

Phosphoproteomics in Microbiology: Protocols for Studying Streptomyces coelicolor Differentiation.

Methods Mol Biol 2018 ;1841:249-260

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

The extension and biological role of Ser/Thr/Tyr phosphorylation in prokaryotes have been only scarcely studied. In this chapter, we describe the state of the art of microbial phosphoproteomics, focusing on protocols used for studying the phosphoproteome of Streptomyces coelicolor, one of the bacteria encoding the largest number of eukaryote-like Ser/Thr/Tyr kinases.
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http://dx.doi.org/10.1007/978-1-4939-8695-8_17DOI Listing
April 2019

Maximizing Sequence Coverage in Top-Down Proteomics By Automated Multimodal Gas-Phase Protein Fragmentation.

Anal Chem 2018 11 10;90(21):12519-12526. Epub 2018 Oct 10.

Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical Sciences , University of Southern Denmark , DK-5230 Odense M , Denmark.

Intact protein sequencing by tandem mass spectrometry (MS/MS), known as top-down protein sequencing, relies on efficient gas-phase fragmentation at multiple experimental conditions to achieve extensive amino acid sequence coverage. We developed the "topdownr" R-package for automated construction of multimodal (i.e., involving CID, HCD, ETD, ETciD, EThcD, and UVPD) MS/MS fragmentation methods on an orbitrap instrument platform and systematic analysis of the resultant spectra. We used topdownr to generate and analyze thousands of MS/MS spectra for five intact proteins of 10-30 kDa. We achieved 90-100% coverage for the proteins tested and derived guiding principles for efficient sequencing of intact proteins. The data analysis workflow and statistical models of topdownr software and multimodal MS/MS experiments provide a framework for optimizing MS/MS sequencing for any intact protein. Refined topdownr software will be suited for comprehensive characterization of protein pharmaceuticals and eventually also for de novo sequencing and detailed characterization of intact proteins.
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http://dx.doi.org/10.1021/acs.analchem.8b02344DOI Listing
November 2018

Distinct Roles of Two Histone Methyltransferases in Transmitting H3K36me3-Based Epigenetic Memory Across Generations in .

Genetics 2018 11 14;210(3):969-982. Epub 2018 Sep 14.

Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California 95064

Epigenetic information contributes to proper gene expression and development, and can be transmitted not only through mitotic divisions but also from parents to progeny. We investigated the roles in epigenetic inheritance of MES-4 and MET-1, the two enzymes that methylate H3K36 (histone H3 Lys 36). Mass spectrometry analysis confirmed immunostaining results showing that both MES-4 and MET-1 catalyze H3K36me3. In the adult germline, MES-4 is enriched in the distal mitotic zone and MET-1 is enriched in the meiotic pachytene zone. Embryos inherit H3K36me3-marked chromosomes from both the oocyte and sperm, and a maternal load of MES-4 and MET-1 Maternal MES-4 quickly associates with sperm chromosomes; that association requires that the sperm chromosomes bear H3K36me3, suggesting that MES-4 is recruited to chromosomes by preexisting H3K36me3. In embryos that inherit H3K36me3-positive oocyte chromosomes and H3K36me3-negative sperm chromosomes, MES-4 and H3K36me3 are maintained on only a subset of chromosomes until at least the 32-cell stage, likely because MES-4 propagates H3K36me3 on regions of the genome with preexisting H3K36me3. In embryos lacking MES-4, H3K36me3 levels on chromosomes drop precipitously postfertilization. In contrast to the relatively high levels of MES-4 in early-stage embryos, MET-1 levels are low at early stages and start increasing by the ∼26-cell stage, consistent with expression from the zygotic genome. Our findings support the model that MET-1 mediates transcription-coupled H3K36me3 in the parental germline and transcriptionally active embryos, and that MES-4 transmits an epigenetic memory of H3K36me3 across generations and through early embryo cell divisions by maintaining inherited patterns of H3K36me3.
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http://dx.doi.org/10.1534/genetics.118.301353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218224PMC
November 2018

Extensive Characterization of Heavily Modified Histone Tails by 193 nm Ultraviolet Photodissociation Mass Spectrometry via a Middle-Down Strategy.

Anal Chem 2018 09 16;90(17):10425-10433. Epub 2018 Aug 16.

Department of Chemistry , University of Texas at Austin , Austin , Texas 78712 , United States.

The ability to map combinatorial patterns of post-translational modifications (PTMs) of proteins remains challenging for traditional bottom-up mass spectrometry workflows. There are also hurdles associated with top-down approaches related to limited data analysis options for heavily modified proteoforms. These shortcomings have accelerated interest in middle-down MS methods that focus on analysis of large peptides generated by specific proteases in conjunction with validated bioinformatics strategies to allow quantification of isomeric histoforms. Mapping multiple PTMs simultaneously requires the ability to obtain high sequence coverage to allow confident localization of the modifications, and 193 nm ultraviolet photodissociation (UVPD) has been shown to cause extensive fragmentation for large peptides and proteins. Histones are an ideal system to test the ability of UVPD to characterize multiple modifications, as the combinations of PTMs are the underpinning of the biological significance of histones and at the same time create an imposing challenge for characterization. The present study focuses on applying 193 nm UVPD to the identification and localization of PTMs on histones by UVPD and comparison to a popular alternative, electron-transfer dissociation (ETD), via a high-throughput middle-down LC/MS/MS strategy. Histone Coder and IsoScale, bioinformatics tools for verification of PTM assignments and quantification of histone peptides, were adapted for UVPD data and applied in the present study. In total, over 300 modified forms were identified, and the distributions of PTMs were quantified between UVPD and ETD. Significant differences in patterns of PTMs were found for histones from HeLa cells prior to and after treatment with a deacetylase inhibitor. Additional fragment ion types generated by UVPD proved essential for extensive characterization of the most heavily modified forms (>5 PTMs).
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http://dx.doi.org/10.1021/acs.analchem.8b02320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383154PMC
September 2018

The Tumor Suppressor CIC Directly Regulates MAPK Pathway Genes via Histone Deacetylation.

Cancer Res 2018 08 29;78(15):4114-4125. Epub 2018 May 29.

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.

Oligodendrogliomas are brain tumors accounting for approximately 10% of all central nervous system cancers. CIC is a transcription factor that is mutated in most patients with oligodendrogliomas; these mutations are believed to be a key oncogenic event in such cancers. Analysis of the ortholog of CIC, Capicua, indicates that CIC loss phenocopies activation of the EGFR/RAS/MAPK pathway, and studies in mammalian cells have demonstrated a role for CIC in repressing the transcription of the PEA3 subfamily of ETS transcription factors. Here, we address the mechanism by which CIC represses transcription and assess the functional consequences of CIC inactivation. Genome-wide binding patterns of CIC in several cell types revealed that CIC target genes were enriched for MAPK effector genes involved in cell-cycle regulation and proliferation. CIC binding to target genes was abolished by high MAPK activity, which led to their transcriptional activation. CIC interacted with the SIN3 deacetylation complex and, based on our results, we suggest that CIC functions as a transcriptional repressor through the recruitment of histone deacetylases. Independent single amino acid substitutions found in oligodendrogliomas prevented CIC from binding its target genes. Taken together, our results show that CIC is a transcriptional repressor of genes regulated by MAPK signaling, and that ablation of CIC function leads to increased histone acetylation levels and transcription at these genes, ultimately fueling mitogen-independent tumor growth. Inactivation of CIC inhibits its direct repression of MAPK pathway genes, leading to their increased expression and mitogen-independent growth. http://cancerres.aacrjournals.org/content/canres/78/15/4114/F1.large.jpg .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6076439PMC
August 2018

Quantitative Proteome and Phosphoproteome Analyses of Reveal Proteins and Phosphoproteins Modulating Differentiation and Secondary Metabolism.

Mol Cell Proteomics 2018 08 21;17(8):1591-1611. Epub 2018 May 21.

From the ‡Área de Microbiología, Departamento de Biología Funcional e IUOPA, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain;

Streptomycetes are multicellular bacteria with complex developmental cycles. They are of biotechnological importance as they produce most bioactive compounds used in biomedicine, antibiotic, antitumoral and immunosupressor compounds. genomes encode many Ser/Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of We identified and quantified 3461 proteins corresponding to 44.3% of the proteome across three developmental stages: vegetative hypha (first mycelium); secondary metabolite producing hyphae (second mycelium); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signaling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism. Phosphoproteomics revealed 85 unique protein phosphorylation sites, 58 of them differentially phosphorylated during differentiation. Computational analysis suggested that these regulated protein phosphorylation events are implicated in important cellular processes, including cell division, differentiation, regulation of secondary metabolism, transcription, protein synthesis, protein folding and stress responses. We discovered a novel regulated phosphorylation site in the key bacterial cell division protein FtsZ (pSer319) that modulates sporulation and regulates actinorhodin antibiotic production. We conclude that manipulation of distinct protein phosphorylation events may improve secondary metabolite production in industrial streptomycetes, including the activation of cryptic pathways during the screening for new secondary metabolites from streptomycetes.
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http://dx.doi.org/10.1074/mcp.RA117.000515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072539PMC
August 2018

VSClust: feature-based variance-sensitive clustering of omics data.

Bioinformatics 2018 09;34(17):2965-2972

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.

Motivation: Data clustering is indispensable for identifying biologically relevant molecular features in large-scale omics experiments with thousands of measurements at multiple conditions. Optimal clustering results yield groups of functionally related features that may include genes, proteins and metabolites in biological processes and molecular networks. Omics experiments typically include replicated measurements of each feature within a given condition to statistically assess feature-specific variation. Current clustering approaches ignore this variation by averaging, which often leads to incorrect cluster assignments.

Results: We present VSClust that accounts for feature-specific variance. Based on an algorithm derived from fuzzy clustering, VSClust unifies statistical testing with pattern recognition to cluster the data into feature groups that more accurately reflect the underlying molecular and functional behavior. We apply VSClust to artificial and experimental datasets comprising hundreds to >80 000 features across 6-20 different conditions including genomics, transcriptomics, proteomics and metabolomics experiments. VSClust avoids arbitrary averaging methods, outperforms standard fuzzy c-means clustering and simplifies the data analysis workflow in large-scale omics studies.

Availability And Implementation: Download VSClust at https://bitbucket.org/veitveit/vsclust or access it through computproteomics.bmb.sdu.dk/Apps/VSClust.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty224DOI Listing
September 2018

Accurate H3K27 methylation can be established de novo by SUZ12-directed PRC2.

Nat Struct Mol Biol 2018 03 26;25(3):225-232. Epub 2018 Feb 26.

Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Polycomb repressive complex 2 (PRC2) catalyzes methylation on lysine 27 of histone H3 (H3K27) and is required for maintaining transcriptional patterns and cellular identity, but the specification and maintenance of genomic PRC2 binding and H3K27 methylation patterns remain incompletely understood. Epigenetic mechanisms have been proposed, wherein pre-existing H3K27 methylation directs recruitment and regulates the catalytic activity of PRC2 to support its own maintenance. Here we investigate whether such mechanisms are required for specifying H3K27 methylation patterns in mouse embryonic stem cells (mESCs). Through re-expression of PRC2 subunits in PRC2-knockout cells that have lost all H3K27 methylation, we demonstrate that methylation patterns can be accurately established de novo. We find that regional methylation kinetics correlate with original methylation patterns even in their absence, and specification of the genomic PRC2 binding pattern is retained and specifically dependent on the PRC2 core subunit SUZ12. Thus, the H3K27 methylation patterns in mESCs are not dependent on self-autonomous epigenetic inheritance.
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http://dx.doi.org/10.1038/s41594-018-0036-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842896PMC
March 2018

How many human proteoforms are there?

Nat Chem Biol 2018 02;14(3):206-214

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.
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http://dx.doi.org/10.1038/nchembio.2576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837046PMC
February 2018

Linear and Differential Ion Mobility Separations of Middle-Down Proteoforms.

Anal Chem 2018 02 6;90(4):2918-2925. Epub 2018 Feb 6.

Department of Chemistry, Wichita State University , 1845 Fairmount, Wichita, Kansas 67260, United States.

Comprehensive characterization of proteomes comprising the same proteins with distinct post-translational modifications (PTMs) is a staggering challenge. Many such proteoforms are isomers (localization variants) that require separation followed by top-down or middle-down mass spectrometric analyses, but condensed-phase separations are ineffective in those size ranges. The variants for "middle-down" peptides were resolved by differential ion mobility spectrometry (FAIMS), relying on the mobility increment at high electric fields, but not previously by linear IMS on the basis of absolute mobility. We now use complete histone tails with diverse PTMs on alternative sites to demonstrate that high-resolution linear IMS, here trapped IMS (TIMS), broadly resolves the variants of ∼50 residues in full or into binary mixtures quantifiable by tandem MS, largely thanks to orthogonal separations across charge states. Separations using traveling-wave (TWIMS) and/or involving various time scales and electrospray ionization source conditions are similar (with lower resolution for TWIMS), showing the transferability of results across linear IMS instruments. The linear IMS and FAIMS dimensions are substantially orthogonal, suggesting FAIMS/IMS/MS as a powerful platform for proteoform analyses.
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http://dx.doi.org/10.1021/acs.analchem.7b05224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366606PMC
February 2018

Organic matter processing by microbial communities throughout the Atlantic water column as revealed by metaproteomics.

Proc Natl Acad Sci U S A 2018 01 18;115(3):E400-E408. Epub 2017 Dec 18.

Department of Limnology and Bio-Oceanography, University of Vienna, A-1090 Vienna, Austria.

The phylogenetic composition of the heterotrophic microbial community is depth stratified in the oceanic water column down to abyssopelagic layers. In the layers below the euphotic zone, it has been suggested that heterotrophic microbes rely largely on solubilized particulate organic matter as a carbon and energy source rather than on dissolved organic matter. To decipher whether changes in the phylogenetic composition with depth are reflected in changes in the bacterial and archaeal transporter proteins, we generated an extensive metaproteomic and metagenomic dataset of microbial communities collected from 100- to 5,000-m depth in the Atlantic Ocean. By identifying which compounds of the organic matter pool are absorbed, transported, and incorporated into microbial cells, intriguing insights into organic matter transformation in the deep ocean emerged. On average, solute transporters accounted for 23% of identified protein sequences in the lower euphotic and ∼39% in the bathypelagic layer, indicating the central role of heterotrophy in the dark ocean. In the bathypelagic layer, substrate affinities of expressed transporters suggest that, in addition to amino acids, peptides and carbohydrates, carboxylic acids and compatible solutes may be essential substrates for the microbial community. Key players with highest expression of solute transporters were Alphaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria, accounting for 40%, 11%, and 10%, respectively, of relative protein abundances. The in situ expression of solute transporters indicates that the heterotrophic prokaryotic community is geared toward the utilization of similar organic compounds throughout the water column, with yet higher abundances of transporters targeting aromatic compounds in the bathypelagic realm.
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http://dx.doi.org/10.1073/pnas.1708779115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5776962PMC
January 2018

Phosphotyrosine Biased Enrichment of Tryptic Peptides from Cancer Cells by Combining pY-MIP and TiO Affinity Resins.

Anal Chem 2017 11 25;89(21):11332-11340. Epub 2017 Oct 25.

Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark , DK-5230 Odense M, Denmark.

Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific, and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY-imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells. The combination of pY-MIP- and TiO-based phosphopeptide enrichment provided more than 90% selectivity for phosphopeptides. Mass spectrometry signal intensities were enhanced for most pY-phosphopeptides (approximately 70%) when using the pY-MIP-TiO combination as compared to TiO alone. pY constituted up to 8% of the pY-MIP-TiO-enriched phosphopeptide fractions. The pY-MIP-TiO and the TiO protocols yielded comparable numbers of distinct phosphopeptides, 1693 and 1842, respectively, from microgram levels of peptide samples. Detailed analysis of physicochemical properties of pY-MIP-TiO-enriched phosphopeptides demonstrated that this protocol retrieved phosphopeptides that tend to be smaller (<24 residues), less acidic, and almost exclusively monophosphorylated, as compared to TiO alone. These unique properties render the pY-MIP-based phosphopeptide enrichment technique an attractive alternative for applications in phosphoproteomics research.
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http://dx.doi.org/10.1021/acs.analchem.7b02091DOI Listing
November 2017