Unbiased phosphoproteomic method identifies the initial effects of a methacrylic acid copolymer on macrophages Michael Dean Chamberlaina,1, Laura A. Wellsa,1,2, Alexandra Lisovskya, Hongbo Guob, Ruth Isserlinb, Ilana Talior-Volodarskya, Redouan Mahoua, Andrew Emilib, and Michael V. Seftona,c,3 aInstitute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada M5S 3G9; bDonnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3G9; and cDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada M5S 3G9 Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved July 21, 2015 (received for review May 5, 2015) An unbiased phosphoproteomic method was used to identify the potential to develop “rules of engagement” between cells and biomaterial-associated changes in the phosphorylation patterns of biomaterials. macrophage-like cells. The phosphorylation differences between This study investigated the effects of a methacrylic acid differentiated THP1 (dTHP1) cells treated for 10, 20, or 30 min with (MAA) copolymer on cells because these polymers have been a vascular regenerative methacrylic acid (MAA) copolymer or a shown to promote vascular regenerative responses in vivo (9, 10), control methyl methacrylate (MM) copolymer were determined by but the mechanism behind this response is unknown (11–13). MS. There were 1,470 peptides (corresponding to 729 proteins) Previous studies showed that 45% poly(MAA-co-methyl meth- that were differentially phosphorylated in dTHP1 cells treated acrylate [MM]) copolymer beads promoted vascularization and with the two materials with a greater cellular response to MAA improved wound healing in diabetic mice (10) or with skin grafts treatment. In addition to identifying pathways (such as integrin in rats (9). In vitro MAA beads altered the gene-expression signaling and cytoskeletal arrangement) that are well known to pattern in macrophage-differentiated THP1 cells (dTHP1) over change with cell–material interaction, previously unidentified path- 4 d (11). Recently, smooth copolymer films of isodecyl acrylate SCIENCES ways, such as apoptosis and mRNA splicing, were also discovered. (IDA) and MAA, or IDA and MM as a control, were studied APPLIED BIOLOGICAL (Fig. 1B); MAA films resulted in increased gene expression of phosphoproteomic | material–cell interactions | macrophage | IL-6, IL-1β, TNF-α, HIF-1α, and SDF-1α and decreased ex- methacrylic acid pression of osteopontin in dTHP1 cells (13), consistent with the previous bead work. conventional description of the interaction between cells Aand a material begins with protein adsorption (as affected Phosphorylation Changed with Time and Polymer Type by material chemistry or topography) that translates into the The dTHP1 cells were grown on porous transwell inserts and resulting effects on cell adhesion, migration, proliferation, or exposed to 40% MAA-co-IDA or 40% MM-co-IDA films by differentiation (1–3). This perspective is driven by the goal of placing polymer-coated coverslips on top of a monolayer of cells understanding biocompatibility (4) or of creating scaffolds for for 10, 20, or 30 min (Fig. 1C). The transwell insert allowed for tissue engineering. However, the complexity of these interactions nutrient and oxygen exchange between the cells and the medium limits the ability to understand and control the biological re- while in contact with the material; there were no obvious changes sponses to biomaterials, perhaps underscoring the occasional in cell morphology, as expected. After incubation with the failure of devices in the clinic (5). Furthermore, the metrics of the materials, the cells were lysed in urea buffer and the proteins conventional approach are often disconnected in time scale from the cellular response. Proteins adsorb in seconds, cell behavior Significance changes in hours, and biocompatibility is determined in days and weeks. The materials are agonists of the biological response, Cells interact with materials, such as those used in implants, whether it is an inflammatory response (6) or cell differentiation through an adsorbed protein layer that causes changes in cell (7, 8), but assays that focus on a particular time scale or pathway behavior and gene expression. We have identified the activa- risk missing the key determinants. The present study focuses on tion of signaling pathways in the cell by a material by unbiased the molecular changes that occur within 30 min of cell exposure to screening of changes in phosphorylation patterns in the cell a material, to identify the cell processes that happen between after material exposure to the material. These changes were recognition of protein adsorption and adaptation of the cell. apparent 10 min after exposure, filling the gap between the When cells contact a material (via adsorbed proteins), surface seconds of protein adsorption and the hours of gene expres- receptors are activated and downstream signaling cascades are sion and leading to the identification of hitherto unknown initiated. The activation of each receptor results in the initiation effects of materials on cells. of multiple signaling cascades that direct the biological response Author contributions: M.D.C., L.A.W., A.L., and M.V.S. designed research; M.D.C., L.A.W., A. of the cells to the material. Signaling cascades typically involve L., H.G., I.T.-V., and R.M. performed research; M.D.C., L.A.W., A.L., H.G., and R.I. analyzed kinases and phosphatases that change the phosphorylation patterns data; and M.D.C., L.A.W., A.L., A.E., and M.V.S. wrote the paper. of proteins to regulate downstream behaviors such as apoptosis, The authors declare no conflict of interest. gene expression, cytoskeletal rearrangement, and differentiation. This article is a PNAS Direct Submission. We hypothesized that the manner in which a material influences 1M.D.C. and L.A.W. contributed equally to this work. cell responses may be inferred by studying changes in the phos- 2Present address: Department of Chemical Engineering, Queen’s University, Kingston, ON, phorylation events of the intracellular signaling cascades (Fig. 1A). Canada K7L 3N6. This work used phosphoproteomics to detect, in an unbiased 3To whom correspondence should be addressed. Email: [email protected]. manner, actively phosphorylated proteins in cells exposed to two This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. different materials to identify novel interactions. Ultimately, we see 1073/pnas.1508826112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1508826112 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 A MAA biomaterial In vivo response Jak p granulation tissue PI3K p vessel Akt p MAA biomaterial Material properties: Biological response: Topography Gene expression Charge Protein expression Elasticity Cell viability Phosphorylation cascade Porosity Cell morphology Chemical groups Proliferation In vivo response B D T260 T303 S259 S301 S357 RAF1 protein T258 S296 Y341 S257 S295 Y340 S43 S252 S291 S339 S642 S29 S244 S289 S338 S621 RBD C1_1 Pkinase_Tyr zf-RING-like Digested S301 S301 S43 S252 S295 S296 S621 C Enriched by TiO2 magnetic S301 S301 dTHP1 Biomaterial S43 S252 S295 S296 S621 (10, 20 & 30 min) Cell lysate Known function Unknown funciton Ex. S259, S296, T491, T494 Ex. Y38, S295 p-Peptide analysis Activating Inhibiting Ex. T491, T494 Ex. S259, S296 1,470 p-Peptide changes 729 p-Proteins changes Fig. 1. Overview of phosphoproteomic method to assess the effects of materials on biological responses. (A) Biological responses (e.g., gene expression, cell proliferation, in vivo host response) are related to material properties (e.g., topography or chemistry) through phosphorylation signaling cascades. The properties of the material govern how proteins from the extracellular milieu adsorb to the surface of the biomaterial and are presented to the cell. These proteins activate the signaling phosphorylation cascades within the cell that drive the biological response to the material. (B) Films containing MAA or MM copolymerized with IDA were cast onto the underside of glass coverslips. (C) Coverslips coated with 40% MAA or 40% MM films were placed on serum- starved dTHP1 cells for 10, 20, or 30 min (with films lying atop cells in a Transwell insert), after which the cells were lysed with urea buffer. The cell lysate was digested with trypsin and the phosphorylated peptides were enriched from the cell lysate by TiO2 magnetic beads and analyzed by MS. (D) Phosphorylated peptides from proteins (RAF-1 is shown as an example) were enriched and identified by MS. Peptides were sorted based on the function of the phosphor- ylation site. Some proteins had a known function of the phosphorylation site that was further distinguished by literature review; other phosphorylation sites had no known function. digested with trypsin. The phosphopeptides were enriched from method for the enrichment of phosphopeptides. In general, there the total peptide mixture by TiO2 magnetic beads and identified were three types of phosphorylations that were identified in this by using an Orbitrap Velos mass spectrometer coupled with screen: (i) phosphorylations that are known to promote the ac- separation via an inline nano-HPLC (14). tivity of the protein, (ii) phosphorylations that are known to inhibit Peptide lists were generated from the MS data by using the activity of the protein,