Bacterial Cellulose Shifts Transcriptome and Proteome of Cultured Endothelial Cells Towards Native Differentiation
2017; Elsevier BV; Volume: 16; Issue: 9 Linguagem: Inglês
10.1074/mcp.ra117.000001
ISSN1535-9484
AutoresGerhard Feil, Ralf Horres, Julia Schulte, Andreas F. Mack, Svenja Wiechmann, Caroline Arnold, Chen Meng, Lukas Jost, Jochen Boxleitner, Nicole Kiessling-Wolf, Ender Serbest, Dominic Helm, Bernhard Küster, Isabel Hartmann, Thomas Korff, Hannes Hahne,
Tópico(s)Tissue Engineering and Regenerative Medicine
ResumoPreserving the native phenotype of primary cells in vitro is a complex challenge. Recently, hydrogel-based cellular matrices have evolved as alternatives to conventional cell culture techniques. We developed a bacterial cellulose-based aqueous gel-like biomaterial, dubbed Xellulin, which mimics a cellular microenvironment and seems to maintain the native phenotype of cultured and primary cells. When applied to human umbilical vein endothelial cells (HUVEC), it allowed the continuous cultivation of cell monolayers for more than one year without degradation or dedifferentiation. To investigate the impact of Xellulin on the endothelial cell phenotype in detail, we applied quantitative transcriptomics and proteomics and compared the molecular makeup of native HUVEC, HUVEC on collagen-coated Xellulin and collagen-coated cell culture plastic (polystyrene).Statistical analysis of 12,475 transcripts and 7831 proteins unveiled massive quantitative differences of the compared transcriptomes and proteomes. K-means clustering followed by network analysis showed that HUVEC on plastic upregulate transcripts and proteins controlling proliferation, cell cycle and protein biosynthesis. In contrast, HUVEC on Xellulin maintained, by and large, the expression levels of genes supporting their native biological functions and signaling networks such as integrin, receptor tyrosine kinase MAP/ERK and PI3K signaling pathways, while decreasing the expression of proliferation associated proteins. Moreover, CD34-an endothelial cell differentiation marker usually lost early during cell culture - was re-expressed within 2 weeks on Xellulin but not on plastic. And HUVEC on Xellulin showed a significantly stronger functional responsiveness to a prototypic pro-inflammatory stimulus than HUVEC on plastic.Taken together, this is one of the most comprehensive transcriptomic and proteomic studies of native and propagated HUVEC, which underscores the importance of the morphology of the cellular microenvironment to regulate cellular differentiation, and demonstrates, for the first time, the potential of Xellulin as versatile tool promoting an in vivo-like phenotype in primary and propagated cell culture. Preserving the native phenotype of primary cells in vitro is a complex challenge. Recently, hydrogel-based cellular matrices have evolved as alternatives to conventional cell culture techniques. We developed a bacterial cellulose-based aqueous gel-like biomaterial, dubbed Xellulin, which mimics a cellular microenvironment and seems to maintain the native phenotype of cultured and primary cells. When applied to human umbilical vein endothelial cells (HUVEC), it allowed the continuous cultivation of cell monolayers for more than one year without degradation or dedifferentiation. To investigate the impact of Xellulin on the endothelial cell phenotype in detail, we applied quantitative transcriptomics and proteomics and compared the molecular makeup of native HUVEC, HUVEC on collagen-coated Xellulin and collagen-coated cell culture plastic (polystyrene). Statistical analysis of 12,475 transcripts and 7831 proteins unveiled massive quantitative differences of the compared transcriptomes and proteomes. K-means clustering followed by network analysis showed that HUVEC on plastic upregulate transcripts and proteins controlling proliferation, cell cycle and protein biosynthesis. In contrast, HUVEC on Xellulin maintained, by and large, the expression levels of genes supporting their native biological functions and signaling networks such as integrin, receptor tyrosine kinase MAP/ERK and PI3K signaling pathways, while decreasing the expression of proliferation associated proteins. Moreover, CD34-an endothelial cell differentiation marker usually lost early during cell culture - was re-expressed within 2 weeks on Xellulin but not on plastic. And HUVEC on Xellulin showed a significantly stronger functional responsiveness to a prototypic pro-inflammatory stimulus than HUVEC on plastic. Taken together, this is one of the most comprehensive transcriptomic and proteomic studies of native and propagated HUVEC, which underscores the importance of the morphology of the cellular microenvironment to regulate cellular differentiation, and demonstrates, for the first time, the potential of Xellulin as versatile tool promoting an in vivo-like phenotype in primary and propagated cell culture. Primary cells cultured in vitro are well-established model systems to analyze distinct cellular responses under defined experimental conditions to investigate disease mechanisms and to identify putative therapeutic options. However, primary cell culture is still subject to biological as well as experimental constraints. Conventional culture of primary cells is almost always associated with culture conditions stimulating proliferation and cellular activity. As a result, the phenotypes may be unstable and promote dedifferentiation of primary cells upon repeated propagation in culture (1Reischl J. Prelle K. Schol H. Neumuller C. Einspanier R. Sinowatz F. Wolf E. Factors affecting proliferation and dedifferentiation of primary bovine oviduct epithelial cells in vitro.Cell Tissue Res. 1999; 296: 371-383Crossref PubMed Scopus (59) Google Scholar, 2Zhang Y. Li T.S. Lee S.T. Wawrowsky K.A. Cheng K. Galang G. Malliaras K. Abraham M.R. Wang C. Marban E. Dedifferentiation and proliferation of mammalian cardiomyocytes.PLoS ONE. 2010; 5: e12559Crossref PubMed Scopus (152) Google Scholar). In addition, cellular differentiation is also modulated through the mechanical properties of the in vivo microenvironment (3Wells R.G. The role of matrix stiffness in regulating cell behavior.Hepatology. 2008; 47: 1394-1400Crossref PubMed Scopus (749) Google Scholar, 4Cohn J.N. 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A. 2010; 94: 1124-1132PubMed Google Scholar), is bacterial cellulose (BC) 1The abbreviations used are: BC, Bacterial cellulose; BH, Benjamini-Hochberg; DAPI, 4′,6-diamidino-2-phenylindole; FCS, Fetal calf serum; FDR, False discovery rate; GSEA, Gene set enrichment analysis; HUVEC, Human umbilical vein endothelial cells; LC-MS/MS, Liquid chromatography tandem mass spectrometry; MACE, Massive Analysis of cDNA Ends; PCA, Principle component analysis; ROS, Reactive oxygen species; TMT, Tandem mass tags; TPM, Tags per million. 1The abbreviations used are: BC, Bacterial cellulose; BH, Benjamini-Hochberg; DAPI, 4′,6-diamidino-2-phenylindole; FCS, Fetal calf serum; FDR, False discovery rate; GSEA, Gene set enrichment analysis; HUVEC, Human umbilical vein endothelial cells; LC-MS/MS, Liquid chromatography tandem mass spectrometry; MACE, Massive Analysis of cDNA Ends; PCA, Principle component analysis; ROS, Reactive oxygen species; TMT, Tandem mass tags; TPM, Tags per million.. This exopolysaccharide, consisting of linear d-glucose molecules linked by β(1–4) glycosidic bonds and synthesized by Gluconacetobacter xylinus, is pure and exhibits mechanical strength, porosity, biocompatibility, and noninherent biodegradability in vivo (10Rajwade J.M. Paknikar K.M. Kumbhar J.V. Applications of bacterial cellulose and its composites in biomedicine.Appl. Microbiol. Biotechnol. 2015; 99: 2491-2511Crossref PubMed Scopus (220) Google Scholar, 15Ullah M.W. Ul-Islam M. Khan S. Kim Y. Park J.K. Structural and physico-mechanical characterization of bio-cellulose produced by a cell-free system.Carbohydr. Polym. 2016; 136: 908-916Crossref PubMed Scopus (107) Google Scholar). To assess the potential of BC in cell culture, we have developed a BC-based hydrological biomaterial as cell culture support, dubbed Xellulin, and obtained cell cultures on Xellulin for a variety of cell types. Given the significance of endothelial cells for physiological and pharmacological investigations of (tumor-associated) angiogenesis, coagulation, inflammation, and other processes in endothelia (16Bevilacqua M.P. Pober J.S. Majeau G.R. Cotran R.S. Gimbrone Jr., M.A. Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells.J. Exp. Med. 1984; 160: 618-623Crossref PubMed Scopus (703) Google Scholar, 17Klingberg H. Loft S. Oddershede L.B. Moller P. The influence of flow, shear stress and adhesion molecule targeting on gold nanoparticle uptake in human endothelial cells.Nanoscale. 2015; 7: 11409-11419Crossref PubMed Google Scholar), we consequently investigated the differentiation of human umbilical vein endothelial cells (HUVEC) on Xellulin to exemplary showcase the versatility of this BC for cell culture purposes. Here we demonstrate that the transcriptomic and proteomic phenotype of propagated HUVEC cultured on collagen-coated Xellulin preserves important features of the native phenotype, including reduced expression of proliferation associated proteins, and sustained expression of integrin and receptor tyrosine kinase signaling, MAP/ERK and PI3K signaling pathways, and ELK, MEF, and NFAT transcription factors. Our studies also revealed that collagen-coated Xellulin induces the re-expression of the endothelial differentiation marker CD34 and that the quiescent HUVEC monolayers may be stimulated to acquire an activated phenotype on Xellulin. These results indicate the potential of BC in general, and Xellulin in particular, as versatile cell culture support promoting an in vivo-like phenotype in primary and propagated cell culture. Xellulin is a natural polymer based on BC. This exopolysaccharide is synthesized by authentic cultures derived from strains of Gluconacetobacter xylinus (Leibniz-Institut DSMZ - Deutsche Sammlung Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, DSM 2325). Xellulin is produced in flat-bed bioreactors in a modified process according to Hofinger et al. (18Hofinger M. Bertholdt G. Weuster-Botz D. Microbial production of homogeneously layered cellulose pellicles in a membrane bioreactor.Biotechnol. Bioeng. 2011; 108: 2237-2240Crossref PubMed Scopus (17) Google Scholar). Harvested Xellulin sheets are cleaned in 0.2 m NaOH (Carl Roth, Karlsruhe, Germany) and then washed in distilled water. Discs are cut out from thin Xellulin sheets and mounted on frames. These so called Xell-Discs fit to 6-well and 24-well cell culture plate formats, and Xell-Discs in the 6-well format were used as cell culture support for HUVEC in this study. Xellulin was examined using scanning electron microscopy (S.E.) as described previously by Schulte et al. (19Schulte J. Friedrich A. Hollweck T. König F. Eblenkamp M. Beiras-Fernandez A. Fano C. Hagl C. Akra B. A novel seeding and conditioning bioreactor for vascular tissue engineering.Processes. 2014; 2: 526Crossref Scopus (6) Google Scholar). Investigations with human umbilical vein endothelial cells (HUVEC) were performed with written informed consent from six paras and following ethical guidelines. Umbilical cords were collected after birth and stored at 2–8 °C. HUVEC were isolated with pre-warmed dispase solution (3.6–3.8 U/ml; Thermo Fisher Scientific, Darmstadt, Germany) within one to four hours after birth. The obtained HUVEC were propagated in cell culture flasks. HUVEC passage 1 were seeded at a density of 3.8 × 104 cells/cm2 on Xell-Discs (Xellutec GmbH, Neuried, Germany) that had been coated with 0.5 ml Cellovations® rat tail (type I) collagen solution (750 μg/ml in 0.1% acetic acid; PELOBiotech GmbH, Martinsried, Germany). HUVEC were cultivated in complete endothelial cell growth medium (PromoCell, Heidelberg, Germany) supplemented with 1% fetal bovine serum (FBS; Thermo Fisher Scientific). After 3 weeks and 6 weeks, respectively, HUVEC were detached with collagenase type IV (500 U/ml; Thermo Fisher Scientific). For standard cell culture HUVEC passage 1 were seeded with a density of 2.2 × 104 cells/cm2 in 6-well cell culture plates (Techno Plastic Products AG, Trasadingen, Switzerland) coated with 1 ml Cellovations® rat tail (type I) collagen solution. The HUVEC were fed with complete endothelial cell growth medium supplemented with a total of 1% or 5% FBS. Confluent monolayers were harvested 2 days after cell seeding (details are given in supplementary Material). Native HUVEC were freshly isolated from six umbilical cords and then propagated (passage 0). Ten samples HUVEC passage 1 were cultured on Xellulin and twelve samples HUVEC passage 1 in standard plastic cell culture. An overview of the experimental design is given in supplemental Table S1. Additionally, HUVEC cultivated on Xellulin for one year and for two months, respectively, were detached, propagated on plasticware and then re-seeded on Xellulin as described above. To assess the physiological potential of quiescent HUVEC on Xellulin, HUVEC monolayers cultured on collagen-coated Xellulin were injured by a scratch with a 100 μl pipette tip. As control, HUVEC monolayers cultured on collagen-coated culture plates were scratched as well. Cell behavior was monitored for 2 days. Tissue samples were fixed and embedded for cryostat, or for paraffin sectioning using standard methods. Paraffin sections were deparaffinized, rehydrated, and then stained histologically with Azan stain according to standard protocols (supplementary Material). Cryosections were incubated with blocking/permeabilizing solution. Afterward, primary antibodies against platelet/endothelial cell adhesion molecule PECAM-1 (CD31) and zonula occludens protein 1 (ZO-1) were applied. Antibodies were detected by incubation with goat anti-mouse IgG Alexa Fluor 546, and goat anti-rabbit IgG Alexa Fluor 633 secondary antibodies followed by incubation with SYTOX green nucleic acid stain (supplementary Material). Xellulin with HUVEC monolayers was fixed and then stained immunocytochemically for CD31, von Willebrand factor (vWF), or for CD 34, respectively, with antigen-specific primary antibodies (supplementary Material). Bound antibodies were visualized by subsequent incubation with Alexa Fluor 594 goat anti-mouse IgG antibodies, anti-rabbit IgG-FITC antibodies, or Alexa Fluor 488 goat anti-mouse IgG antibodies, respectively. Nuclei were stained with DAPI. To characterize the phenotype of HUVEC seeded on Xellulin, HUVEC passage 1 from the same origin were additionally seeded into ibiTreat 8-well μ-Slides (ibidi, Martinsried, Germany). Chamber slide cultures were fixed and stained for von Willebrand Factor (vWF) and CD31 (supplementary Material). For comparison to umbilical vein sections stained immunohistologically against CD31 and ZO-1 (see above), HUVEC on Xellulin were fixed and then incubated with blocking/permeabilizing solution. Subsequently, primary antibodies against CD31 and ZO-1 were applied in cocktails. Bound antibodies were detected by goat anti-rabbit Cy3, or goat anti-mouse Cy5 secondary antibodies followed by incubation with SYTOX green nucleic acid stain (supplementary Material). Images of HUVEC both on plastic and Xellulin stained immunologically for CD31, vWF, and CD34 were acquired with a phase contrast and fluorescence microscope (Axio Observer.Z1, Carl Zeiss) and taken with a digital microscope camera. Images of human umbilical cord sections and HUVEC on Xellulin stained immunologically for CD31, ZO-1, CD34, vWF, VE-Cadherin and Angiopoietin were acquired with a confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Jena, Germany) using sequential scans and appropriate filter sets (Supplementary Material). Images were exported and figure panels assembled using Adobe Photoshop (San Jose, CA). The experimental design is summarized in Fig. 1A and supplemental Table S1. A total of 28 HUVEC samples from six umbilical veins were analyzed corresponding to native HUVEC from six different donors, 2 × 5 propagated HUVEC (passage 1) cultivated on Xellulin for 3 and 6 weeks, respectively, and 2 × 6 propagated HUVEC (passage 1) cultivated on plastic with 1 and 5% FCS, respectively. We profiled transcriptomes of all 28 samples corresponding to at least 5 biological replicates per condition using the MACE technology. We profiled proteomes of a subset of 12 samples corresponding to biological triplicates of donor-matched propagated HUVEC on Xellulin for 3 and 6 weeks, and propagated HUVEC on plastic with 1 and 5% FCS using a TMT10plex-based quantification approach. Because of limited sample amounts, we did not include native HUVEC in the proteome study. In addition, we analyzed the proteomes of pooled HUVEC samples from Xellulin and plastic culture (pools of the same samples used for TMT10plex quantification; no replicates) using a label-free intensity based proteomics approach. For RNA isolation from native HUVEC, the umbilical vein was washed with DPBS (1x) w/o Ca & Mg and thereafter incubated with 5 to 7 ml RNAprotect cell reagent (Qiagen, Hilden, Germany, 76526) for 2 mins. The liquid was transferred to a centrifuge tube. After another washing step with DPBS (1×) w/o Ca & Mg, HUVEC were isolated by incubation with pre-warmed (37 °C) GibcoTM dispase solution (3.6–3.8 U/ml) for one hour. All liquids were collected in one centrifuge tube. The cells were pelleted (5 min., 5000 × g) and the supernatant was discarded. For extracting RNA from HUVEC passage 1, the cells were washed with DPBS (1x) w/o Ca & Mg and then incubated with 600 μl RNAprotect cell reagent for 2 mins. Cells were detached with a cell scraper and transferred into a centrifuge tube. For maximization of the cell yield, residual HUVEC on Xellulin and plastic surface, respectively, were washed with an additional ml of DPBS (1×) w/o Ca & Mg and the liquid was transferred to the centrifuge tube used previously. The detached cells were pelleted. RNA was isolated and purified using the RNeasy Mini Kit (Qiagen, 74104) according to the manufacturer's protocol. The eluates were stored at −20 °C until further processing. A DNase I digestion of all RNA samples was made with Baseline-Zero™ DNase (Epicenter, Madison, WI; provided by Biozym Scientific GmbH, Hessisch Oldendorf, Germany) in solution to ensure that the samples were completely free of DNA. RNA samples were subsequently purified using RNA Clean & ConcentratorTM-5 Kit (Zymo Research Europe, Freiburg Germany). Total RNA concentration was measured with Qubit1 2.0 Fluorometer and Qubit1 RNA HS Assay Kit (Life Technologies GmbH, Darmstadt, Germany). RNA quality was estimated in a dilution series with the LabChip GX by PerkinElmer Rodgau, Germany (Software Version 4.2.1745.0). The RNA quality of all samples was estimated using the RNA Integrity Number (RIN) (20Schroeder A. Mueller O. Stocker S. Salowsky R. Leiber M. Gassmann M. Lightfoot S. Menzel W. Granzow M. Ragg T. The RIN: an RNA integrity number for assigning integrity values to RNA measurements.BMC Mol. Biol. 2006; 7: 3Crossref PubMed Scopus (1807) Google Scholar) and RIN scores between 6.4 and 10 were obtained. The transcriptome of HUVEC samples was sequenced and quantified using the Massive Analysis of cDNA Ends (MACE) technology in combination with the TrueQuant method, eliminates and corrects for PCR introduced copies, thereby enabling a more accurate transcriptome quantification (21Zajac B.K. Amendt J. Horres R. Verhoff M.A. Zehner R. De novo transcriptome analysis and highly sensitive digital gene expression profiling of Calliphora vicina (Diptera: Calliphoridae) pupae using MACE (Massive Analysis of cDNA Ends).Forensic Sci. Int. Genet. 2015; 15: 137-146Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). From each RNA sample a MACE cDNA library was constructed targeting sequences near the cDNA 3′-ends. MACE was conducted by GenXPro GmbH as described (22Kahl G. Molina C. Rotter B. Jüngling R. Frank A. Krezdorn N. Hoffmeier K. Winter P. Reduced representation sequencing of plant stress transcriptomes.J. Plant Biochem. Biotechnol. 2012; 21: 119-127Crossref Google Scholar, 23Zawada A.M. Rogacev K.S. Muller S. Rotter B. Winter P. Fliser D. Heine G.H. Massive analysis of cDNA Ends (MACE) and miRNA expression profiling identifies proatherogenic pathways in chronic kidney disease.Epigenetics. 2014; 9: 161-172Crossref PubMed Scopus (83) Google Scholar). Briefly, the polyadenylated mRNA was reverse transcribed and the resulting cDNA was immobilized with biotinylated oligo(dT) primers via streptavidin coated magnetic beads after random fragmentation with directed ultra sound (Bioruptor, Diagenode, Belgium). The remaining fragments were discarded. 50–600 bp long fragments before the Poly-A tail starting from the 5′-ends were sequenced (single-read) using the Illumina Next Seq500 platform (Illumina Inc., San Diego, CA), generating 75bp long reads. Illumina's Control Software was used for sequencing and real-time analysis followed by Illumina Consensus Assessment of Sequence and Variation procedure for base calling and demultiplexing. To prevent PCR-biased quantification by Illumina sequencing, the described TrueQuant method was applied. HUVEC in passage 2 were seeded on collagen I-coated plastic or Xellulin discs. After ∼2 days, the cells reached confluency and were stimulated with 5 ng/ml human recombinant TNF alpha (hrTNFa, Biomol #50435, Lot#2214–1) for 3 h. Subsequently, cells were washed once in ice-cold HBSS w/Ca2+/Mg2+ and lysed in 300 μl RLT-Lysis Buffer/1% beta-mercaptoethanol (Qiagen) and snap frozen in liquid nitrogen. RNA was isolated according to manufacturer's instructions (RNeasy MiniKit, Qiagen Cat. No.: 74106). For cDNA synthesis, 100 ng RNA were used according to manufacturer's protocol (Omniscript RT Kit, Qiagen, Cat. No.: 205113). The PCR reaction was performed using 2.5 μl cDNA using the following primers and conditions: hRPL32: forward 5′GTTCATCCGGCACCAGTCAG3′, reverse 5′ACGTGCACATGAGCTGCCTAC3′, 5 min 95 °C, cycles: 30 s, 95 °C, 60 °C, 45 s, 72 °C, 1 min, 24 cycles, 72 °C 5 min, 369 bp, hE-Selectin: forward 5′TTCGCCTGTCCTGAAGGATG3′, reverse 5′TCAGTTGAAGGCCGTCCTTG3′, 5 min 95 °C, cycles: 30 s, 95 °C, 55 °C, 45 s, 72 °C, 1 min, 33 cycles, 72 °C 5 min, 474 bp. The PCR products were seperated on a 1% agarose gel and documented using the Gel Doc XR+ system and the Quantity One Software, Version 4.6.9 (Bio-Rad). Gray intensities were quantified using Image J Software (Version 1.49a). HUVEC were lysed using lysis buffer containing 8 m urea, 40 mm Tris/HCl (pH 7.6), 1× EDTA-free protease inhibitor mixture (Complete Mini, Roche). The protein concentration was determined using the Bradford method (Coomassie Protein Assay Kit, Thermo Scientific). Proteins were reduced and alkylated with 10 mm tris(2-carboxyethyl)phosphin (TCEP) at 37 °C for 1 h followed by 25 mm chloro-acetamide for 45 min at room temperature in the dark. The protein mixture was diluted with 40 mm Tris/HCl to a final urea concentration of 1.6 m. Protein aliquots of 50 μg were digested by adding sequencing-grade trypsin (Promega, Mannheim, Germany; 1:100 enzyme/substrate ratio) and samples were incubated at 37 °C for 4 h. Another 1:100 aliquot of trypsin was added for overnight digestion at 37 °C. Samples were acidified with formic acid to pH 2 and subsequently cleaned up and concentrated using C18 StageTips essentially as described (24Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2569) Google Scholar). To compare and normalize Tandem Mass tag (TMT) reporter intensities between different TMT10-plex LC-MS/MS experiments, identical samples representing a pooled digest of all samples were used as common reference. For the common reference pool, 15 μg of peptide digest per sample were combined. Peptide aliquots were dried in vacuo, reconstituted in 40 μl 50 mm triethyl ammonium bicarbonate and incubated at room temperature for 15min. TMT10-plex reagents (Thermo Fisher Scientific, Dreieich) were dissolved in 42 μl of water-free acetonitrile and 10 μl of TMT was added per sample. After incubation at room temperature for 1 h, the labeling reaction was quenched by adding hydroxylamine to a final concentration of 0.2% (v/v). Labeled peptides were mixed in equal amounts, acidified with formic acid, and desalted using C18 SepPak cartridges according to the manufacturer's instructions (C18 cartridges, Sep-Pak Vac, 1 cc, 50 mg, Waters Corp., Eschborn, Germany). Eluates were dried down and stored at −80 °C. Samples were further fractionated into 24 fractions using hydrophilic strong anion exchange chromatography (hSAX) as described previously (25Ruprecht B. Koch H. Medard G. Mundt M. Kuster B. Lemeer S. Comprehensive and reproducible phosphopeptide enrichment using iron immobilized metal ion affinity chromatography (Fe-IMAC) columns.Mol. Cell. Proteomics. 2015; 14: 205-215Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and peptides were desalted and concentrated using C18 StageTips. Peptide aliquots of six Xellulin and six plastic samples were pooled separately (see also supplemental Table S1), desalted and concentrated using C18 StageTips and fractionated in StageTips as described (26Chen W. Wang S. Adhikari S. Deng Z. Wang L. Chen L. Ke M. Yang P. Tian R. Simple and integrated spintip-based technology applied for deep proteome profiling.Anal. Chem. 2016; 88: 4864-4871Crossref PubMed Scopus (85) Google Scholar) using a stepwise gradient of ACN in 25 mm ammonium formiate (flowthrough, 5%, 10%, 15%, 17.5%, 50%) before fraction 1 and 5 and 2 and 6 were pooled. Nanoflow LC-MS/MS analysis of peptide samples was performed on an UltiMate 3000 RSLCnano System (Thermo Scientific, Dreieich, Germany) coupled to a Q-Exactive HF mass spectrometer (Thermo Scientific, Bremen, Germany). Peptides were delivered to a trap column (100-μm inner diameter × 2 cm, packed with 5-μm C18 resin (Reprosil GOLD, Dr. Maisch, Germany) at a flow rate of 5 μl/min for 10 min in 100% solvent A (0.1% FA in HPLC-grade water). After loading and washing, peptides were transferred to an analytical column (75 μm × 40 cm C18 column; Rerosil GOLD, 3 μm, Dr. Maisch, Germany) and separated using a linear gradient of 110 min ramping from 4% to 32% solvent B (0.1% FA, 5% dimethyl sulfoxide in ACN) at a flow rate of 300 nL/min. 5% (v/v) DMSO were used in solvent A and B to boost the nanoelectrospray response (27Hahne H. Pachl F. Ruprecht B. Maier S.K. Klaeger S. Helm D. Medard G. Wilm M. Lemeer S. Kuster B. DMSO enhances electrospray response, boosting sensitivity of proteomic experiments.Nat. Methods. 2013; 10: 989-991Crossref PubMed Scopus (175) Google Scholar). Peptides were ionized using a 2.2-kV electrospray voltage and a capillary temperature of 275 °C. The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS2. Full-scan MS spectra (m/z
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