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Proteomics, Glycomics, and Glycoproteomics of Matrisome Molecules*

Open AccessPublished:August 30, 2019DOI:https://doi.org/10.1074/mcp.R119.001543
      The most straightforward applications of proteomics database searching involve intracellular proteins. Although intracellular gene products number in the thousands, their well-defined post-translational modifications (PTMs) makes database searching practical. By contrast, cell surface and extracellular matrisome proteins pass through the secretory pathway where many become glycosylated, modulating their physicochemical properties, adhesive interactions, and diversifying their functions. Although matrisome proteins number only a few hundred, their high degree of complex glycosylation multiplies the number of theoretical proteoforms by orders of magnitude. Given that extracellular networks that mediate cell-cell and cell-pathogen interactions in physiology depend on glycosylation, it is important to characterize the proteomes, glycomes, and glycoproteomes of matrisome molecules that exist in a given biological context. In this review, we summarize proteomics approaches for characterizing matrisome molecules, with an emphasis on applications to brain diseases. We demonstrate the availability of methods that should greatly increase the availability of information on matrisome molecular structure associated with health and disease.

      Graphical Abstract

      Viewed in evolutionary terms, the diversification of genes for intracellular versus extracellular proteins has separate drivers (
      • Demetriou M.
      • Nabi I.R.
      • Dennis J.W.
      Galectins as adaptors: linking glycosylation and metabolism with extracellular cues.
      ,
      • Dennis J.W.
      Genetic code asymmetry supports diversity through experimentation with posttranslational modifications.
      ,
      • Williams R.
      • Ma X.
      • Schott R.K.
      • Mohammad N.
      • Ho C.Y.
      • Li C.F.
      • Chang B.S.
      • Demetriou M.
      • Dennis J.W.
      Encoding asymmetry of the N-glycosylation motif facilitates glycoprotein evolution.
      ). Evolution of intracellular biology focuses on the regulation of signaling events, transcription and translation, through phosphorylation and other post-translational modifications (PTMs)
      The abbreviation used is:
      PTMs
      post-translational modifications.
      1The abbreviation used is:PTMs
      post-translational modifications.
      that influence allosteric enzyme regulation and signaling cascades through activation/deactivation of recognition domains, for example, SH2, SH3, bromo-, chromo- and tudor domains (
      • Deribe Y.L.
      • Pawson T.
      • Dikic I.
      Post-translational modifications in signal integration.
      ,
      • Reinhardt H.C.
      • Yaffe M.B.
      Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.
      ). Significantly, many of the PTMs that occur inside the cell produce well-defined molecular additions (phosphorylation, acetylation, acylation and methylation) that are compatible with the established database searching workflows (
      • Reinhardt H.C.
      • Yaffe M.B.
      Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.
      ,
      • Rogers L.D.
      • Overall C.M.
      Proteolytic post-translational modification of proteins: proteomic tools and methodology.
      ,
      • Rahimi N.
      • Costello C.E.
      Emerging roles of post-translational modifications in signal transduction and angiogenesis.
      ). Ubiquitination leaves a recognizable peptide tag after tryptic digestion that is amenable to proteomics approaches (
      • Rose C.M.
      • Isasa M.
      • Ordureau A.
      • Prado M.A.
      • Beausoleil S.A.
      • Jedrychowski M.P.
      • Finley D.J.
      • Harper J.W.
      • Gygi S.P.
      Highly multiplexed quantitative mass spectrometry analysis of ubiquitylomes.
      ,
      • Udeshi N.D.
      • Mertins P.
      • Svinkina T.
      • Carr S.A.
      Large-scale identification of ubiquitination sites by mass spectrometry.
      ,
      • Udeshi N.D.
      • Mani D.R.
      • Eisenhaure T.
      • Mertins P.
      • Jaffe J.D.
      • Clauser K.R.
      • Hacohen N.
      • Carr S.A.
      Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition.
      ). Evolution of such PTMs arose through the need for complex control of gene expression through regulated signaling networks. By contrast, complex glycosylation reflects the evolutionary response to pathogen pressure and the evolving need for multicellular complexity (
      • Dennis J.W.
      Genetic code asymmetry supports diversity through experimentation with posttranslational modifications.
      ,
      • Williams R.
      • Ma X.
      • Schott R.K.
      • Mohammad N.
      • Ho C.Y.
      • Li C.F.
      • Chang B.S.
      • Demetriou M.
      • Dennis J.W.
      Encoding asymmetry of the N-glycosylation motif facilitates glycoprotein evolution.
      ). Organisms need a multicellular organization with the ability to distinguish self from non-self and orchestrated responses to infection and regulated tissue plasticity. Therefore, much of cellular biology responds to signals received from the extracellular environment. Complex glycosylation is heterogeneous as a rule at each protein site, multiplying the number of molecular forms and requiring specialized proteomics methods.
      As summarized in recent reviews (
      • Sethi M.K.
      • Zaia J.
      Extracellular matrix proteomics in schizophrenia and Alzheimer's disease.
      ,
      • Frantz C.
      • Stewart K.M.
      • Weaver V.M.
      The extracellular matrix at a glance.
      ,
      • Iozzo R.V.
      • Gubbiotti M.A.
      Extracellular matrix: The driving force of mammalian diseases.
      ), the mechanisms of tissue homeostasis and most diseases include interactions with the extracellular microenvironment. The matrisome constitutes the non-cellular components that control biochemical and biomechanical cues, growth factor and morphogen gradients, and physical scaffolds that define tissue phenotypes including morphogenesis, differentiation, and homeostasis (
      • Frantz C.
      • Stewart K.M.
      • Weaver V.M.
      The extracellular matrix at a glance.
      ). Although each tissue has a unique extracellular environment, the number of gene products that code matrisome proteins in the entire body are limited (
      • Chautard E.
      • Fatoux-Ardore M.
      • Ballut L.
      • Thierry-Mieg N.
      • Ricard-Blum S.
      MatrixDB, the extracellular matrix interaction database.
      ,
      • Chautard E.
      • Ballut L.
      • Thierry-Mieg N.
      • Ricard-Blum S.
      MatrixDB, a database focused on extracellular protein-protein and protein-carbohydrate interactions.
      ,
      • Hynes R.O.
      • Naba A.
      Overview of the matrisome–an inventory of extracellular matrix constituents and functions.
      ,
      • Naba A.
      • Clauser K.R.
      • Ding H.
      • Whittaker C.A.
      • Carr S.A.
      • Hynes R.O.
      The extracellular matrix: Tools and insights for the “omics” era.
      ,
      • Naba A.
      • Clauser K.R.
      • Hoersch S.
      • Liu H.
      • Carr S.A.
      • Hynes R.O.
      The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices.
      ). Cell surface receptors regulate adhesion and cytoskeletal connections to the matrisome. The structure and organization of the matrisome require maintenance as it adapts to tissue growth needs. Matrisome proteins become glycosylated in the secretory pathway, may be proteolytically processed and cross-linked. The resulting physical and biochemical characteristics reflect the organized networks that depend on numerous molecular interactions that arise from PTMs.
      To date, the ability to apply established proteomics methods that depend on database searching to such highly modified and heterogeneous proteins remains far from adequate (
      • Aebersold R.
      • Agar J.N.
      • Amster I.J.
      • Baker M.S.
      • Bertozzi C.R.
      • Boja E.S.
      • Costello C.E.
      • Cravatt B.F.
      • Fenselau C.
      • Garcia B.A.
      • Ge Y.
      • Gunawardena J.
      • Hendrickson R.C.
      • Hergenrother P.J.
      • Huber C.G.
      • Ivanov A.R.
      • Jensen O.N.
      • Jewett M.C.
      • Kelleher N.L.
      • Kiessling L.L.
      • Krogan N.J.
      • Larsen M.R.
      • Loo J.A.
      • Ogorzalek Loo R.R.
      • Lundberg E.
      • MacCoss M.J.
      • Mallick P.
      • Mootha V.K.
      • Mrksich M.
      • Muir T.W.
      • Patrie S.M.
      • Pesavento J.J.
      • Pitteri S.J.
      • Rodriguez H.
      • Saghatelian A.
      • Sandoval W.
      • Schluter H.
      • Sechi S.
      • Slavoff S.A.
      • Smith L.M.
      • Snyder M.P.
      • Thomas P.M.
      • Uhlen M.
      • Van Eyk J.E.
      • Vidal M.
      • Walt D.R.
      • White F.M.
      • Williams E.R.
      • Wohlschlager T.
      • Wysocki V.H.
      • Yates N.A.
      • Young N.L.
      • Zhang B.
      How many human proteoforms are there?.
      ). Although we can detect many matrisome proteins using proteomics, the low sequence coverage leaves many structural elements of functional interest unidentified. In addition, the heterogeneity of glycosylation at each of the many glycosylation sites of matrisome proteins results in astronomically large numbers of possible proteoforms, if taken as multiples of the variants at each site. Although the existence of such large numbers of proteoforms seems unlikely, the number of functional proteoforms that exist in a given biological context remains largely undefined.
      In this review, we summarize methods for proteomics, glycomics and glyoproteomics of matrisome molecules. The goal is to characterize matrisome molecular structure in the greatest detail possible using wide-angle omics experiments. The high extent of matrisome protein glycosylation and other post-translational modifications requires special consideration of sample workup and proteomics database searching. We summarize matrisome physiology with emphasis on brain diseases. We summarize experimental approaches for matrisome workup and mass spectrometric analysis.

      Extracellular Matrix Physiology and Pathophysiology

      Dysregulation of the cellular microenvironment occurs in cancers (
      • Bissell M.J.
      • Hines W.C.
      Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression.
      ,
      • Mouw J.K.
      • Ou G.
      • Weaver V.M.
      Extracellular matrix assembly: a multiscale deconstruction.
      ,
      • Werb Z.
      • Lu P.
      The Role of Stroma in Tumor Development.
      ), neurodevelopmental and neuropsychiatric diseases (
      • Pantazopoulos H.
      • Berretta S.
      In sickness and in health: perineuronal nets and synaptic plasticity in psychiatric disorders.
      ,
      • Sorg B.A.
      • Berretta S.
      • Blacktop J.M.
      • Fawcett J.W.
      • Kitagawa H.
      • Kwok J.C.
      • Miquel M.
      Casting a wide net: role of perineuronal nets in neural plasticity.
      ). Known as the matrisome, networks of extracellular matrix and cell surface molecules control the availability of growth factors to cellular receptors and the mechanical-physical properties of the cell microenvironment. Currently, the limited understanding of the regulation of matrisome glycosylation hinders understanding of the roles of glycosylation-dependent matrisome networks in the basic mechanisms necessary for the targeted intervention of many diseases.
      The extracellular environment (
      • Chautard E.
      • Fatoux-Ardore M.
      • Ballut L.
      • Thierry-Mieg N.
      • Ricard-Blum S.
      MatrixDB, the extracellular matrix interaction database.
      ,
      • Chautard E.
      • Ballut L.
      • Thierry-Mieg N.
      • Ricard-Blum S.
      MatrixDB, a database focused on extracellular protein-protein and protein-carbohydrate interactions.
      ,
      • Hynes R.O.
      • Naba A.
      Overview of the matrisome–an inventory of extracellular matrix constituents and functions.
      ) consists of glycoproteins, proteoglycans, collagens, and their interacting partners. Matrisome protein functions are elaborated by biosynthetic enzymes of the secretory pathway that generate mature molecules with spatially and temporally regulated glycosylation. Thus, glycoproteins have context-specific structures and biological functions that remain largely undefined because of the lack of effective methods for quantifying changes to site-specific protein glycosylation. This means that it is necessary to achieve complete matrisome protein coverage in order to determine the changes to these molecules that occur during disease mechanisms.
      Progress in developing treatments that target interactions among cells and their extracellular microenvironments, including dysregulated cell growth, morphogenesis, and host-pathogen interactions, is limited by the ability to quantify the extent to which changes in matrisome networks resulting from altered glycoprotein glycosylation determine disease mechanisms. Many matrisome proteins contain lectin domains that recognize glycan epitopes. Thus, the glycosylation of matrisome molecules determines their binding interactions with other matrisome molecules and with soluble growth factors. The result is organized assemblies of matrisome molecules that compose the spatially and temporally regulated microenvironments through which cells receive signals in physiology and pathophysiology.
      The core matrisome consists of 195 glycoproteins, 44 collagens and 35 proteoglycans (
      • Hynes R.O.
      • Naba A.
      Overview of the matrisome–an inventory of extracellular matrix constituents and functions.
      ,
      • Naba A.
      • Clauser K.R.
      • Hoersch S.
      • Liu H.
      • Carr S.A.
      • Hynes R.O.
      The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices.
      ,

      http://matrisomeproject.mit.edu.

      ), many of which are glycosylated. Extensive proteomics studies have cataloged the abundances of matrisome molecules in different tissues (
      • Naba A.
      • Clauser K.R.
      • Hoersch S.
      • Liu H.
      • Carr S.A.
      • Hynes R.O.
      The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices.
      ); however, such studies have not defined the glycosylation states of matrisome molecules necessary to define networks of interactions with lectin-containing binding partners. Many matrisome molecules have several points of glycosylation, each with microheterogeneity, each representing a functional epitope that needs to be defined in order to characterize biological functions (
      • Cummings R.D.
      The repertoire of glycan determinants in the human glycome.
      ).
      Glycan branching regulates glycoprotein dynamics and the residency of cell surface cytokine receptors (
      • Ryczko M.C.
      • Pawling J.
      • Chen R.
      • Abdel Rahman A.M.
      • Yau K.
      • Copeland J.K.
      • Zhang C.
      • Surendra A.
      • Guttman D.S.
      • Figeys D.
      • Dennis J.W.
      Metabolic reprogramming by hexosamine biosynthetic and golgi N-glycan branching pathways.
      ). Interactions among cell surface and extracellular glycoproteins with lectins including galectins, C-type lectins, and siglecs, drive the clustering of cell surface molecules into networks that define tissue microenvironments. These organized assemblies of extracellular molecules (the matrisome) adapt cellular microenvironments to phenotypic needs. Although glycosylated proteins represent potential therapeutic targets, their macro- and micro-heterogeneities pose a significant challenge to exploitation. Because their functions depend on glycosylation and other PTMs, it is necessary to produce detailed proteolytic maps of matrisome proteins and follow the changes that occur during aging and disease development.

      The Dynamic Brain Matrisome

      The brain extracellular space has been referred to as the final frontier in neuroscience (
      • Nicholson C.
      • Hrab[caron]etová S.
      Brain extracellular space: the final frontier of neuroscience.
      ). Many of the matrisome molecules common to systemic organs are not found in brain (
      • Bonneh-Barkay D.
      • Wiley C.A.
      Brain extracellular matrix in neurodegeneration.
      ). Matrisome functions depends on networks of interaction among glycosylated proteins and glycan-binding lectins. As illustrated in Fig. 1 for the brain, networks of cell surface and extracellular glycoproteins and proteoglycans bind many families of growth factors and growth factor receptors (
      • Dityatev A.
      • Wehrle-Haller B.
      • Pitkanen A.
      Preface. Brain extracellular matrix in health and disease.
      ,
      • Yamaguchi Y.
      Lecticans: organizers of the brain extracellular matrix.
      ,
      • Bandtlow C.E.
      • Zimmermann D.R.
      Proteoglycans in the Developing Brain: New Conceptual Insights for Old Proteins.
      ). They modulate receptor tyrosine kinase signaling pathways at the heart of mechanisms including tissue stiffness and growth factor transport.
      Figure thumbnail gr1
      Fig. 1Brain matrisome types include blood-brain barrier, interstitial matrix, and perineuronal nets. These structures are composed of matrisome molecules including hyaluronan, collagens, glycoproteins, and proteoglycans. Matrisome structure is spatially and temporally regulated, dynamic, and becomes altered during the pathogenesis of neuropsychiatric and neurodegenerative diseases.
      In the brain, the extracellular space occupies ∼20% of brain volume (
      • Nicholson C.
      • Hrab[caron]etová S.
      Brain extracellular space: the final frontier of neuroscience.
      ,
      • Sykova E.
      • Nicholson C.
      Diffusion in brain extracellular space.
      ). As shown in Fig. 1, matrisome in the central nervous system includes the interstitial matrix, basement membranes, and perineuronal nets (PNNs), the fine structures of which vary spatially and temporally (
      • Dauth S.
      • Grevesse T.
      • Pantazopoulos H.
      • Campbell P.H.
      • Maoz B.M.
      • Berretta S.
      • Parker K.K.
      Extracellular matrix protein expression is brain region dependent.
      ,
      • Dityatev A.
      • Schachner M.
      Extracellular matrix molecules and synaptic plasticity.
      ). The matrisome provides the environment necessary for cell homeostasis, repair, regeneration, and neural plasticity in a brain region-specific manner (
      • Dityatev A.
      • Seidenbecher C.I.
      • Schachner M.
      Compartmentalization from the outside: the extracellular matrix and functional microdomains in the brain.
      ,
      • Kwok J.C.
      • Dick G.
      • Wang D.
      • Fawcett J.W.
      Extracellular matrix and perineuronal nets in CNS repair.
      ,
      • Rowlands D.
      • Lensjo K.K.
      • Dinh T.
      • Yang S.
      • Andrews M.R.
      • Hafting T.
      • Fyhn M.
      • Fawcett J.W.
      • Dick G.
      Aggrecan directs extracellular matrix mediated neuronal plasticity.
      ).
      The basement membranes that line cerebral blood vessels consist of collagen IV, laminins and heparan sulfate proteoglycans (HSPGs) perlecan and agrin (
      • Randles M.J.
      • Humphries M.J.
      • Lennon R.
      Proteomic definitions of basement membrane composition in health and disease.
      ,
      • Randles M.
      • Lennon R.
      Applying proteomics to investigate extracellular matrix in health and disease.
      ). In the brain, neural interstitial matrix separates cells and consists of networks of chondroitin sulfate proteoglycans (CSPGs), tenascins, hyaluronan, and link proteins. The PNN consist of many of the same CSPGs, tenascins, hyaluronan, and link proteins condensed that surround some neuronal cell bodies and dendrites.
      Dysregulation of matrisome molecular networks characterize pathophysiologies of cancer (
      • Stowell S.R.
      • Ju T.
      • Cummings R.D.
      Protein glycosylation in cancer.
      ,
      • Ju T.
      • Aryal R.P.
      • Kudelka M.R.
      • Wang Y.
      • Cummings R.D.
      The Cosmc connection to the Tn antigen in cancer.
      ), autoimmune (
      • Stuchlova Horynova M.
      • Raska M.
      • Clausen H.
      • Novak J.
      Aberrant O-glycosylation and anti-glycan antibodies in an autoimmune disease IgA nephropathy and breast adenocarcinoma.
      ,
      • Benkhoucha M.
      • Molnarfi N.
      • Santiago-Raber M.L.
      • Weber M.S.
      • Merkler D.
      • Collin M.
      • Lalive P.H.
      IgG glycan hydrolysis by EndoS inhibits experimental autoimmune encephalomyelitis.
      ,
      • Green R.S.
      • Stone E.L.
      • Tenno M.
      • Lehtonen E.
      • Farquhar M.G.
      • Marth J.D.
      Mammalian N-glycan branching protects against innate immune self-recognition and inflammation in autoimmune disease pathogenesis.
      ), cardiovascular (
      • McGarrah R.W.
      • Kelly J.P.
      • Craig D.M.
      • Haynes C.
      • Jessee R.C.
      • Huffman K.M.
      • Kraus W.E.
      • Shah S.H.
      A novel protein glycan-derived inflammation biomarker independently predicts cardiovascular disease and modifies the association of HDL subclasses with mortality.
      ,
      • Akinkuolie A.O.
      • Buring J.E.
      • Ridker P.M.
      • Mora S.
      A novel protein glycan biomarker and future cardiovascular disease events.
      ,
      • Menni C.
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      • Lauc G.
      • Valdes A.M.
      Glycosylation profile of immunoglobulin G is cross-sectionally associated with cardiovascular disease risk score and subclinical atherosclerosis in two independent cohorts.
      ), and neuropsychiatric diseases (
      • Pantazopoulos H.
      • Berretta S.
      In sickness and in health: perineuronal nets and synaptic plasticity in psychiatric disorders.
      ,
      • Sorg B.A.
      • Berretta S.
      • Blacktop J.M.
      • Fawcett J.W.
      • Kitagawa H.
      • Kwok J.C.
      • Miquel M.
      Casting a wide net: role of perineuronal nets in neural plasticity.
      ,
      • Dauth S.
      • Grevesse T.
      • Pantazopoulos H.
      • Campbell P.H.
      • Maoz B.M.
      • Berretta S.
      • Parker K.K.
      Extracellular matrix protein expression is brain region dependent.
      ). In the brain, region-specific regulation of matrisome molecule glycosylation controls the neuronal microenvironment and becomes dysregulated in neuropsychiatric diseases (
      • Pantazopoulos H.
      • Berretta S.
      In sickness and in health: perineuronal nets and synaptic plasticity in psychiatric disorders.
      ,
      • Sorg B.A.
      • Berretta S.
      • Blacktop J.M.
      • Fawcett J.W.
      • Kitagawa H.
      • Kwok J.C.
      • Miquel M.
      Casting a wide net: role of perineuronal nets in neural plasticity.
      ,
      • Dauth S.
      • Grevesse T.
      • Pantazopoulos H.
      • Campbell P.H.
      • Maoz B.M.
      • Berretta S.
      • Parker K.K.
      Extracellular matrix protein expression is brain region dependent.
      ,
      • Foscarin S.
      • Raha-Chowdhury R.
      • Fawcett J.W.
      • Kwok J.C.F.
      Brain ageing changes proteoglycan sulfation, rendering perineuronal nets more inhibitory.
      ). For example, in neurodegeneration, proteoglycans bind to and play roles in the aggregation of proteins including Aβ, tau, prion protein, and α-synuclein (
      • Papy-Garcia D.
      • Christophe M.
      • Huynh M.B.
      • Fernando S.
      • Ludmilla S.
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      Glycosaminoglycans, protein aggregation and neurodegeneration.
      ,
      • Lehri-Boufala S.
      • Ouidja M.O.
      • Barbier-Chassefiere V.
      • Henault E.
      • Raisman-Vozari R.
      • Garrigue-Antar L.
      • Papy-Garcia D.
      • Morin C.
      New roles of glycosaminoglycans in alpha-synuclein aggregation in a cellular model of Parkinson disease.
      ). In Alzheimer's disease, proteoglycans participate in amyloid plaque formation, leading to disease pathology from altered proteolytic processing and accumulation of toxic aggregates (
      • Bergamaschini L.
      • Rossi E.
      • Vergani C.
      • De Simoni M.G.
      Alzheimer's disease: another target for heparin therapy.
      ,
      • van Horssen J.
      • Wesseling P.
      • van den Heuvel L.P.
      • de Waal R.M.
      • Verbeek M.M.
      Heparan sulphate proteoglycans in Alzheimer's disease and amyloid-related disorders.
      ).
      Perineuronal nets are lattices of matrisome molecules that surround the cell body and dendrites of neurons. They are thought to serve as a reservoir for cations and provide the connectional architecture that controls synaptic plasticity. Deficits in PNN structure appear to contribute to dysfunction in cortical circuitry in schizophrenia (
      • Berretta S.
      Extracellular matrix abnormalities in schizophrenia.
      ). The number of PNN in visual cortex increases during postnatal development, paralleling the critical period for synaptic plasticity and playing an important role in critical period closure. Significantly, the adult inability to repair spinal cord injury can be restored by the treatment of the injury site with chondroitinase enzymes (
      • Laabs T.
      • Carulli D.
      • Geller H.M.
      • Fawcett J.W.
      Chondroitin sulfate proteoglycans in neural development and regeneration.
      ,
      • Bradbury E.J.
      • Moon L.D.
      • Popat R.J.
      • King V.R.
      • Bennett G.S.
      • Patel P.N.
      • Fawcett J.W.
      • McMahon S.B.
      Chondroitinase ABC promotes functional recovery after spinal cord injury.
      ) with the implication that CSPGs maintain the extracellular environment in adult neural tissue that limits neural plasticity (
      • Kwok J.C.
      • Dick G.
      • Wang D.
      • Fawcett J.W.
      Extracellular matrix and perineuronal nets in CNS repair.
      ,
      • Carulli D.
      • Kwok J.C.
      • Pizzorusso T.
      Perineuronal nets and CNS plasticity and repair.
      ,
      • Berretta S.
      • Pantazopoulos H.
      • Markota M.
      • Brown C.
      • Batzianouli E.T.
      Losing the sugar coating: Potential impact of perineuronal net abnormalities on interneurons in schizophrenia.
      ,
      • Carulli D.
      • Pizzorusso T.
      • Kwok J.C.
      • Putignano E.
      • Poli A.
      • Forostyak S.
      • Andrews M.R.
      • Deepa S.S.
      • Glant T.T.
      • Fawcett J.W.
      Animals lacking link protein have attenuated perineuronal nets and persistent plasticity.
      ). PNN structure differs spatially and temporally in the brain in association with injury, repair, development, aging, learning, memory, neuropsychiatric diseases, neurodegeneration, and in response to drug abuse (
      • Sorg B.A.
      • Berretta S.
      • Blacktop J.M.
      • Fawcett J.W.
      • Kitagawa H.
      • Kwok J.C.
      • Miquel M.
      Casting a wide net: role of perineuronal nets in neural plasticity.
      ,
      • Horii-Hayashi N.
      • Sasagawa T.
      • Matsunaga W.
      • Nishi M.
      Development and structural variety of the chondroitin sulfate proteoglycans-contained extracellular matrix in the mouse brain.
      ,
      • Miyata S.
      • Nadanaka S.
      • Igarashi M.
      • Kitagawa H.
      Structural variation of chondroitin sulfate chains contributes to the molecular heterogeneity of perineuronal nets.
      ).

      Variation in Matrisome Molecular Structure Among Brain Regions, With Development, Aging, and Pathologies

      Traditional antibody-based techniques including immunohistochemistry show spatial and temporal regulation of matrisome molecule expression in the brain (
      • van Kuppevelt T.H.
      • Dennissen M.A.
      • van Venrooij W.J.
      • Hoet R.M.
      • Veerkamp J.H.
      Generation and application of type-specific anti-heparan sulfate antibodies using phage display technology. Further evidence for heparan sulfate heterogeneity in the kidney.
      ,
      • Thompson S.M.
      • Fernig D.G.
      • Jesudason E.C.
      • Losty P.D.
      • van de Westerlo E.M.
      • van Kuppevelt T.H.
      • Turnbull J.E.
      Heparan sulfate phage display antibodies identify distinct epitopes with complex binding characteristics: insights into protein binding specificities.
      ,
      • Rickelt S.
      • Hynes R.O.
      Antibodies and methods for immunohistochemistry of extracellular matrix proteins.
      ). Although antibody binding indicates the levels of individual epitopes, the antibody specificity and underlying structure are assumed. Matrisome molecule glycosylation can also be stained using lectins, leaving the underlying matrisome site-specific glycosylation structure undefined (
      • Sorg B.A.
      • Berretta S.
      • Blacktop J.M.
      • Fawcett J.W.
      • Kitagawa H.
      • Kwok J.C.
      • Miquel M.
      Casting a wide net: role of perineuronal nets in neural plasticity.
      ,
      • Berretta S.
      Extracellular matrix abnormalities in schizophrenia.
      ,
      • Pantazopoulos H.
      • Boyer-Boiteau A.
      • Holbrook E.H.
      • Jang W.
      • Hahn C.G.
      • Arnold S.E.
      • Berretta S.
      Proteoglycan abnormalities in olfactory epithelium tissue from subjects diagnosed with schizophrenia.
      ).
      Because antibodies bind to discrete structural epitopes on highly complex matrisome molecules, the changes in structure unrelated to such epitopes are not defined by antibody-based techniques. This is illustrated for the aggrecan proteoglycan in Fig. 2. Aggrecan contains three globular domains and an extended region modified with more than 100 CS chains. The C-terminal G3 domain has EGF-like repeats, a complement regulatory module, and a C-type lectin module (
      • Iozzo R.V.
      • Schaefer L.
      Proteoglycan form and function: A comprehensive nomenclature of proteoglycans.
      ) and interacts with tenascins. The globular domains are N-glycosylated, and the extended domains also carry keratan sulfate, mucin-type O-glycans, and O-mannose glycans (
      • Pacharra S.
      • Hanisch F.G.
      • Muhlenhoff M.
      • Faissner A.
      • Rauch U.
      • Breloy I.
      The lecticans of mammalian brain perineural net are O-mannosylated.
      ). Although this provides a parts list for aggrecan, we have little information on how the variation of glycosylation modulates the functions of aggrecan in a weight-bearing tissue such as cartilage versus brain. Further, although it is clear that CS structure varies among brain regions, there is little information on the fine structures of the resulting matrisome molecular networks. The same reasoning applies to the other brain matrisome network glycoproteins, proteoglycans, and collagens.
      Figure thumbnail gr2
      Fig. 2Model for the structure of aggrecan, showing glycosylation with CS, HS, N-glycans, mucin-type O-GalNAc glycans, and O-Man glycans. The G1 and G2 domains contain link modules with homology to HAPLN proteins. The G3 domain has two epidermal growth factor (EGF)-like repeats; a C-type lectin domain, and a complement regulatory protein domain (CRP).
      It is clear, however, that matrisome glycosylation changes during development and with disease states. This can be seen from the alteration of CS sulfation during development in many tissues, including cartilage (
      • Sorrell J.M.
      • Caterson B.
      Detection of age-related changes in the distributions of keratan sulfates and chondroitin sulfates in developing chick limbs: an immunocytochemical study.
      ) and brain (
      • Miyata S.
      • Kitagawa H.
      Chondroitin sulfate and neuronal disorders.
      ,
      • Miyata S.
      • Kitagawa H.
      Formation and remodeling of the brain extracellular matrix in neural plasticity: Roles of chondroitin sulfate and hyaluronan.
      ). Further, staining of brain tissue with Wisteria floribunda agglutinin (WFA), a lectin that binds GalNAc residues, has been used to identify brain region pathologies associated with schizophrenia (
      • Pantazopoulos H.
      • Murray E.A.
      • Berretta S.
      Total number, distribution, and phenotype of cells expressing chondroitin sulfate proteoglycans in the normal human amygdala.
      ,
      • Ajmo J.M.
      • Eakin A.K.
      • Hamel M.G.
      • Gottschall P.E.
      Discordant localization of WFA reactivity and brevican/ADAMTS-derived fragment in rodent brain.
      ,
      • Costa C.
      • Tortosa R.
      • Domenech A.
      • Vidal E.
      • Pumarola M.
      • Bassols A.
      Mapping of aggrecan, hyaluronic acid, heparan sulphate proteoglycans and aquaporin 4 in the central nervous system of the mouse.
      ,
      • Hartig W.
      • Brauer K.
      • Bigl V.
      • Bruckner G.
      Chondroitin sulfate proteoglycan-immunoreactivity of lectin-labeled perineuronal nets around parvalbumin-containing neurons.
      ). The extreme complexities of matrisome proteins concerning glycosylation, cross-linking and other PTMs, drives the need for many more validated antibody reagents than are available to date (
      • Rickelt S.
      • Hynes R.O.
      Antibodies and methods for immunohistochemistry of extracellular matrix proteins.
      ). Such lectin and/or antibody staining studies do not define the glycosite changes that underlie dysregulated interactions with lectin-containing binding partners that give rise to pathologies.

      Matrisome Proteomics

      Matrisome Sample Preparation Methods

      As summarized in Table I and reviewed in detail (
      • Krasny L.
      • Paul A.
      • Wai P.
      • Howard B.A.
      • Natrajan R.C.
      • Huang P.H.
      Comparative proteomic assessment of matrisome enrichment methodologies.
      ), methods for enrichment of matrisome proteins for proteomics studies include tissue decellularization and extraction of matrisome components from tissue homogenates. Proteomics researchers often use decellularization to remove cellular components prior to solubilization of matrisome molecules (
      • Mallis P.
      • Gontika I.
      • Poulogiannopoulos T.
      • Zoidakis J.
      • Vlahou A.
      • Michalopoulos E.
      • Chatzistamatiou T.
      • Papassavas A.
      • Stavropoulos-Giokas C.
      Evaluation of decellularization in umbilical cord artery.
      ,
      • Maghsoudlou P.
      • Georgiades F.
      • Smith H.
      • Milan A.
      • Shangaris P.
      • Urbani L.
      • Loukogeorgakis S.P.
      • Lombardi B.
      • Mazza G.
      • Hagen C.
      • Sebire N.J.
      • Turmaine M.
      • Eaton S.
      • Olivo A.
      • Godovac-Zimmermann J.
      • Pinzani M.
      • Gissen P.
      • De Coppi P.
      Optimization of liver decellularization maintains extracellular matrix micro-architecture and composition predisposing to effective cell seeding.
      ,
      • Lindsey M.L.
      • Hall M.E.
      • Harmancey R.
      • Ma Y.
      Adapting extracellular matrix proteomics for clinical studies on cardiac remodeling post-myocardial infarction.
      ,
      • Johnson T.D.
      • Hill R.C.
      • Dzieciatkowska M.
      • Nigam V.
      • Behfar A.
      • Christman K.L.
      • Hansen K.C.
      Quantification of decellularized human myocardial matrix: A comparison of six patients.
      ,
      • Hsueh M.F.
      • Khabut A.
      • Kjellstrom S.
      • Onnerfjord P.
      • Kraus V.B.
      Elucidating the molecular composition of cartilage by proteomics.
      ,
      • Mayorca-Guiliani A.E.
      • Madsen C.D.
      • Cox T.R.
      • Horton E.R.
      • Venning F.A.
      • Erler J.T.
      ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix.
      ,
      • Barrett A.S.
      • Wither M.J.
      • Hill R.C.
      • Dzieciatkowska M.
      • D'Alessandro A.
      • Reisz J.A.
      • Hansen K.C.
      Hydroxylamine chemical digestion for insoluble extracellular matrix characterization.
      ,
      • de Castro Bras L.E.
      • Ramirez T.A.
      • DeLeon-Pennell K.Y.
      • Chiao Y.A.
      • Ma Y.
      • Dai Q.
      • Halade G.V.
      • Hakala K.
      • Weintraub S.T.
      • Lindsey M.L.
      Texas 3-step decellularization protocol: looking at the cardiac extracellular matrix.
      ,
      • Gaetani R.
      • Aouad S.
      • Demaddalena L.L.
      • Straessle H.
      • Dzieciatkowska M.
      • Wortham M.
      • Bender H.R.
      • Nguyen-Ngoc K.V.
      • Schmid-Schoenbein G.W.
      • George S.C.
      • Hughes C.C.W.
      • Sander M.
      • Hansen K.C.
      • Christman K.L.
      Evaluation of different decellularization protocols on the generation of pancreas-derived hydrogels.
      ). The use of a chaotrope solubilizes some matrisome components, leaving an insoluble pellet rich in fibrillar collagens (
      • Goddard E.T.
      • Hill R.C.
      • Barrett A.
      • Betts C.
      • Guo Q.
      • Maller O.
      • Borges V.F.
      • Hansen K.C.
      • Schedin P.
      Quantitative extracellular matrix proteomics to study mammary and liver tissue microenvironments.
      ). The yield of such matrisome components improves with chemical digestion with cyanogen bromide (
      • Hill R.C.
      • Wither M.J.
      • Nemkov T.
      • Barrett A.
      • D'Alessandro A.
      • Dzieciatkowska M.
      • Hansen K.C.
      Preserved proteins from extinct bison latifrons identified by tandem mass spectrometry; hydroxylysine glycosides are a common feature of ancient collagen.
      ). Alternatively, hydroxylamine cleavage at Asn-Gly sites has also been used to solubilize matrisome from insoluble pellets prior to tryptic digestion and proteomics (
      • Barrett A.S.
      • Wither M.J.
      • Hill R.C.
      • Dzieciatkowska M.
      • D'Alessandro A.
      • Reisz J.A.
      • Hansen K.C.
      Hydroxylamine chemical digestion for insoluble extracellular matrix characterization.
      ,
      • Hill R.C.
      • Wither M.J.
      • Nemkov T.
      • Barrett A.
      • D'Alessandro A.
      • Dzieciatkowska M.
      • Hansen K.C.
      Preserved proteins from extinct bison latifrons identified by tandem mass spectrometry; hydroxylysine glycosides are a common feature of ancient collagen.
      ). For matrisome protein quantification among tissue sample cohorts, Hansen et al. have developed targeted proteomics (
      • Dempsey S.G.
      • Miller C.H.
      • Hill R.C.
      • Hansen K.C.
      • May B.C.H.
      Functional insights from the proteomic inventory of ovine forestomach matrix.
      ,
      • Banerjee A.
      • Silliman C.C.
      • Moore E.E.
      • Dzieciatkowska M.
      • Kelher M.
      • Sauaia A.
      • Jones K.
      • Chapman M.P.
      • Gonzalez E.
      • Moore H.B.
      • D'Alessandro A.
      • Peltz E.
      • Huebner B.E.
      • Einerson P.
      • Chandler J.
      • Ghasabayan A.
      • Hansen K.
      Systemic hyperfibrinolysis after trauma: a pilot study of targeted proteomic analysis of superposed mechanisms in patient plasma.
      ,
      • Calle E.A.
      • Hill R.C.
      • Leiby K.L.
      • Le A.V.
      • Gard A.L.
      • Madri J.A.
      • Hansen K.C.
      • Niklason L.E.
      Targeted proteomics effectively quantifies differences between native lung and detergent-decellularized lung extracellular matrices.
      ).
      Table IAn overview of proteomics approaches for matrisome Proteins
      Enrichment method prior to LC-MS/MS analysisTissue typeNumber of matrisome proteins identifiedReferenceLimitations
      High pH reversed phase fractionationHuman knee articular cartilage196 (out of total 653 protein IDs)Önnerfjord et al.(
      • Folkesson E.
      • Turkiewicz A.
      • Englund M.
      • Onnerfjord P.
      Differential protein expression in human knee articular cartilage and medial meniscus using two different proteomic methods: a pilot analysis.
      )
      Additional fractionation step
      Commercial compartmental protein extraction kit and urea solublizationMurine normal and melanoma tumor tissues200Naba et al. (
      • Naba A.
      • Clauser K.R.
      • Hoersch S.
      • Liu H.
      • Carr S.A.
      • Hynes R.O.
      The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices.
      )
      Semi-quantitative estimates
      Decellularization, solubilization using a three-stage extraction (salt, detergent, guanidine HCl)Atherosclerotic plaque tissue∼200Mayr et al. (
      • Langley S.R.
      • Willeit K.
      • Didangelos A.
      • Matic L.P.
      • Skroblin P.
      • Barallobre-Barreiro J.
      • Lengquist M.
      • Rungger G.
      • Kapustin A.
      • Kedenko L.
      • Molenaar C.
      • Lu R.
      • Barwari T.
      • Suna G.
      • Yin X.
      • Iglseder B.
      • Paulweber B.
      • Willeit P.
      • Shalhoub J.
      • Pasterkamp G.
      • Davies A.H.
      • Monaco C.
      • Hedin U.
      • Shanahan C.M.
      • Willeit J.
      • Kiechl S.
      • Mayr M.
      Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques.
      )
      False positives and limited list of proteins
      Decellularization, solubilization using a three-stage extraction (salt, detergent, guanidine HCl)Pig coronary arteries151Mayr et al. (
      • Suna G.
      • Wojakowski W.
      • Lynch M.
      • Barallobre-Barreiro J.
      • Yin X.
      • Mayr U.
      • Baig F.
      • Lu R.
      • Fava M.
      • Hayward R.
      • Molenaar C.
      • White S.J.
      • Roleder T.
      • Milewski K.P.
      • Gasior P.
      • Buszman P.P.
      • Buszman P.
      • Jahangiri M.
      • Shanahan C.M.
      • Hill J.
      • Mayr M.
      Extracellular matrix proteomics reveals interplay of aggrecan and aggrecanases in vascular remodeling of stented coronary arteries.
      )
      Limited availability of porcine antibodies
      ERLIC off-line fractionationRat brain17Berretta S et al. (
      • Dauth S.
      • Grevesse T.
      • Pantazopoulos H.
      • Campbell P.H.
      • Maoz B.M.
      • Berretta S.
      • Parker K.K.
      Extracellular matrix protein expression is brain region dependent.
      )
      Additional fractionation step
      On-slide tissue digestion (no enrichment)Rat brain (Striatum and Substantia-nigra)15Zaia J et al. (
      • Raghunathan R.
      • Polinski N.K.
      • Klein J.A.
      • Hogan J.D.
      • Shao C.
      • Khatri K.
      • Leon D.
      • McComb M.E.
      • Manfredsson F.P.
      • Sortwell C.E.
      • Zaia J.
      Glycomic and proteomic changes in aging brain nigrostriatal pathway.
      )
      Low protein coverage

      Proteomics Data Acquisition Methods for the Analysis of Matrisome Proteins

      Present discovery proteomics methods suffice to identify matrisome proteins based on the presence of minimally modified peptides using database searching. Such peptides have been used in targeted proteomics assays for quantification of matrisome molecules based on inferred core protein abundances (
      • Dempsey S.G.
      • Miller C.H.
      • Hill R.C.
      • Hansen K.C.
      • May B.C.H.
      Functional insights from the proteomic inventory of ovine forestomach matrix.
      ,
      • Banerjee A.
      • Silliman C.C.
      • Moore E.E.
      • Dzieciatkowska M.
      • Kelher M.
      • Sauaia A.
      • Jones K.
      • Chapman M.P.
      • Gonzalez E.
      • Moore H.B.
      • D'Alessandro A.
      • Peltz E.
      • Huebner B.E.
      • Einerson P.
      • Chandler J.
      • Ghasabayan A.
      • Hansen K.
      Systemic hyperfibrinolysis after trauma: a pilot study of targeted proteomic analysis of superposed mechanisms in patient plasma.
      ,
      • Calle E.A.
      • Hill R.C.
      • Leiby K.L.
      • Le A.V.
      • Gard A.L.
      • Madri J.A.
      • Hansen K.C.
      • Niklason L.E.
      Targeted proteomics effectively quantifies differences between native lung and detergent-decellularized lung extracellular matrices.
      ). Data-independent analysis (DIA) has the advantage that all precursor ions are subjected to collisional dissociation. Using the sequential window acquisition of all theoretical fragment ion spectra (SWATH)-MS DIA method (
      • Gillet L.C.
      • Navarro P.
      • Tate S.
      • Rost H.
      • Selevsek N.
      • Reiter L.
      • Bonner R.
      • Aebersold R.
      Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis.
      ), fragment ion spectra for all precursors are acquired within the specified m/z range and retention time window. Interpretation of such datasets in which tandem mass spectra show product ions from co-eluting peptides requires the use of spectral libraries (
      • Rosenberger G.
      • Bludau I.
      • Schmitt U.
      • Heusel M.
      • Hunter C.L.
      • Liu Y.
      • MacCoss M.J.
      • MacLean B.X.
      • Nesvizhskii A.I.
      • Pedrioli P.G.A.
      • Reiter L.
      • Rost H.L.
      • Tate S.
      • Ting Y.S.
      • Collins B.C.
      • Aebersold R.
      Statistical control of peptide and protein error rates in large-scale targeted data-independent acquisition analyses.
      ). Huang et al. developed a spectral library of 201 matrisome proteins and compared the performance of SWATH versus data-dependent acquisition (DDA) for analysis of unfractionated tissue extracts (
      • Krasny L.
      • Bland P.
      • Kogata N.
      • Wai P.
      • Howard B.A.
      • Natrajan R.C.
      • Huang P.H.
      SWATH mass spectrometry as a tool for quantitative profiling of the matrisome.
      ). They reported a 15–20% improvement in peptide reproducibility and a 54% increase in several matrisome proteins identified relative to DDA. Önnerfjord et al. used high pH reversed-phase fractionation of tryptic digests as a workflow for cartilage proteomics (
      • Folkesson E.
      • Turkiewicz A.
      • Englund M.
      • Onnerfjord P.
      Differential protein expression in human knee articular cartilage and medial meniscus using two different proteomic methods: a pilot analysis.
      ). Because of the additional fractionation step, they reported 653 proteins identified. They used DDA data to build spectral libraries for interpretation of a subset of identified proteins using DIA. They showed that DIA produced a more precise measurement of peptide abundances than DDA.
      Naba et al. used a commercial Cytosol/Nucleus/Membrane/Cytoskeleton compartmental protein extraction kit to enrich intracellular and matrisome proteins in separate fractions from tissue (
      • Naba A.
      • Clauser K.R.
      • Hoersch S.
      • Liu H.
      • Carr S.A.
      • Hynes R.O.
      The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices.
      ,
      • Naba A.
      • Clauser K.R.
      • Lamar J.M.
      • Carr S.A.
      • Hynes R.O.
      Extracellular matrix signatures of human mammary carcinoma identify novel metastasis promoters.
      ,
      • Naba A.
      • Clauser K.R.
      • Whittaker C.A.
      • Carr S.A.
      • Tanabe K.K.
      • Hynes R.O.
      Extracellular matrix signatures of human primary metastatic colon cancers and their metastases to liver.
      ,
      • Naba A.
      • Clauser K.R.
      • Hynes R.O.
      Enrichment of extracellular matrix proteins from tissues and digestion into peptides for mass spectrometry analysis.
      ). The tryptic digests of the matrisome -enriched pellets were solubilized using urea prior to LC-tandem MS (
      • Naba A.
      • Pearce O.M.T.
      • Del Rosario A.
      • Ma D.
      • Ding H.
      • Rajeeve V.
      • Cutillas P.R.
      • Balkwill F.R.
      • Hynes R.O.
      Characterization of the extracellular matrix of normal and diseased tissues using proteomics.
      ). This approach resulted in the identification of ∼250 matrisome proteins from tissue (
      • Naba A.
      • Clauser K.R.
      • Lamar J.M.
      • Carr S.A.
      • Hynes R.O.
      Extracellular matrix signatures of human mammary carcinoma identify novel metastasis promoters.
      ,
      • Naba A.
      • Clauser K.R.
      • Whittaker C.A.
      • Carr S.A.
      • Tanabe K.K.
      • Hynes R.O.
      Extracellular matrix signatures of human primary metastatic colon cancers and their metastases to liver.
      ,
      • Gocheva V.
      • Naba A.
      • Bhutkar A.
      • Guardia T.
      • Miller K.M.
      • Li C.M.
      • Dayton T.L.
      • Sanchez-Rivera F.J.
      • Kim-Kiselak C.
      • Jailkhani N.
      • Winslow M.M.
      • Del Rosario A.
      • Hynes R.O.
      • Jacks T.
      Quantitative proteomics identify Tenascin-C as a promoter of lung cancer progression and contributor to a signature prognostic of patient survival.
      ,
      • Naba A.
      • Clauser K.R.
      • Mani D.R.
      • Carr S.A.
      • Hynes R.O.
      Quantitative proteomic profiling of the extracellular matrix of pancreatic islets during the angiogenic switch and insulinoma progression.
      ,
      • Hu Z.
      • Gu H.
      • Hu J.
      • Hu S.
      • Wang X.
      • Liu X.
      • Jiao X.
      • Liu X.
      Quantitative proteomics identify an association between extracellular matrix degradation and immunopathology of genotype VII Newcastle disease virus in the spleen in chickens.
      ).
      Mayr et al. extracted matrisome proteins from vascular tissue using decellularization, solubilization using a three-stage extraction (salt, detergent, guanidine HCl) and MS-based quantification (
      • Didangelos A.
      • Yin X.
      • Mandal K.
      • Baumert M.
      • Jahangiri M.
      • Mayr M.
      Proteomics characterization of extracellular space components in the human aorta.
      ,
      • Didangelos A.
      • Yin X.
      • Mandal K.
      • Saje A.
      • Smith A.
      • Xu Q.
      • Jahangiri M.
      • Mayr M.
      Extracellular matrix composition and remodeling in human abdominal aortic aneurysms: a proteomics approach.
      ,
      • Schiller H.B.
      • Fernandez I.E.
      • Burgstaller G.
      • Schaab C.
      • Scheltema R.A.
      • Schwarzmayr T.
      • Strom T.M.
      • Eickelberg O.
      • Mann M.
      Time- and compartment-resolved proteome profiling of the extracellular niche in lung injury and repair.
      ,
      • Barallobre-Barreiro J.
      • Lynch M.
      • Yin X.
      • Mayr M.
      Systems biology-opportunities and challenges: the application of proteomics to study the cardiovascular extracellular matrix.
      ,
      • Lynch M.
      • Barallobre-Barreiro J.
      • Jahangiri M.
      • Mayr M.
      Vascular proteomics in metabolic and cardiovascular diseases.
      ). In studies of human venous tissue, they reported the identification of ∼150 matrisome proteins. They identified a proteomics 4-biomarker signature for atherosclerotic plaques from a comparison of vascular matrisome in human carotid artery specimens (
      • Langley S.R.
      • Willeit K.
      • Didangelos A.
      • Matic L.P.
      • Skroblin P.
      • Barallobre-Barreiro J.
      • Lengquist M.
      • Rungger G.
      • Kapustin A.
      • Kedenko L.
      • Molenaar C.
      • Lu R.
      • Barwari T.
      • Suna G.
      • Yin X.
      • Iglseder B.
      • Paulweber B.
      • Willeit P.
      • Shalhoub J.
      • Pasterkamp G.
      • Davies A.H.
      • Monaco C.
      • Hedin U.
      • Shanahan C.M.
      • Willeit J.
      • Kiechl S.
      • Mayr M.
      Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques.
      ). In this work, they report 110 matrisome -associated proteins from guanidine HCl extraction and 87 from the salt fractions with an overlap of 51. They also performed matrisome proteomics studies of restenosis and thrombosis following coronary stent implantation in pigs, for which they report the identification of 151 matrisome proteins (
      • Suna G.
      • Wojakowski W.
      • Lynch M.
      • Barallobre-Barreiro J.
      • Yin X.
      • Mayr U.
      • Baig F.
      • Lu R.
      • Fava M.
      • Hayward R.
      • Molenaar C.
      • White S.J.
      • Roleder T.
      • Milewski K.
      • Gasior P.
      • Buszman P.P.
      • Buszman P.E.
      • Jahangiri M.
      • Shanahan C.
      • Hill J.M.
      • Mayr M.
      Extracellular matrix proteomics reveals interplay of aggrecan and aggrecanases in vascular remodeling of stented coronary arteries.
      ,
      • Fava M.
      • Barallobre-Barreiro J.
      • Mayr U.
      • Lu R.
      • Didangelos A.
      • Baig F.
      • Lynch M.
      • Catibog N.
      • Joshi A.
      • Barwari T.
      • Yin X.
      • Jahangiri M.
      • Mayr M.
      Role of ADAMTS-5 in aortic dilatation and extracellular matrix remodeling.
      ).
      Berretta et al. demonstrated, using a combination of immunohistochemistry and proteomics, that matrisome molecule expression is brain region-dependent (
      • Dauth S.
      • Grevesse T.
      • Pantazopoulos H.
      • Campbell P.H.
      • Maoz B.M.
      • Berretta S.
      • Parker K.K.
      Extracellular matrix protein expression is brain region dependent.
      ). For this work, fresh rat brains were dissected, and regions were snap frozen. Tryptic peptides were fractionated using ERLIC, and the resulting fractions analyzed using LC-MS. The fold change abundances of a set of 17 matrisome molecules, including tenascins, hyalectans, link proteins, and others were reported for a set of five rat brain regions.

      MALDI Imaging of Matrisome Proteins

      As described in detail in recent reviews (
      • Casadonte R.
      • Caprioli R.M.
      Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry.
      ,
      • Ryan D.J.
      • Spraggins J.M.
      • Caprioli R.M.
      Protein identification strategies in MALDI imaging mass spectrometry: a brief review.
      ,
      • Caprioli R.M.
      Imaging mass spectrometry: a perspective.
      ), MALDI imaging mass spectrometry (IMS) produces 2-dimensional maps of the distributions of ions desorbed from the surfaces of tissue slides. The advantage is that the maps can be produced at ∼25 μm or better resolution and with impressive ion-specific spatial resolution patterns. The disadvantages are that in the absence of a separation step, the dynamic range of protein/peptide detection is limited, and identification of observed proteins or peptides can be cumbersome. Drake et al. have demonstrated the use of MALDI-imaging to visualize proteins and peptides from matrisome-rich tissues, including heart (
      • Angel P.M.
      • Baldwin H.S.
      • Gottlieb Sen D.
      • Su Y.R.
      • Mayer J.E.
      • Bichell D.
      • Drake R.R.
      Advances in MALDI imaging mass spectrometry of proteins in cardiac tissue, including the heart valve.
      ). They demonstrated the use of matrix metalloproteinase enzymes to localize collagen and elastin peptides on the surfaces of the tumor and cardiac tissue slides (
      • Angel P.M.
      • Comte-Walters S.
      • Ball L.E.
      • Talbot K.
      • Mehta A.
      • Brockbank K.G.M.
      • Drake R.R.
      Mapping extracellular matrix proteins in formalin-fixed, paraffin-embedded tissues by MALDI imaging mass spectrometry.
      ). This group also pioneered MALDI-based imaging of glycans at ∼25 μm spatial resolution on tissue slides (
      • Drake R.R.
      • Powers T.W.
      • Jones E.E.
      • Bruner E.
      • Mehta A.S.
      • Angel P.M.
      MALDI mass spectrometry imaging of N-linked glycans in cancer tissues.
      ,
      • Drake R.R.
      • West C.A.
      • Mehta A.S.
      • Angel P.M.
      MALDI mass spectrometry imaging of N-linked glycans in tissues.
      ) for which they derivatized sialic acid residues to prevent dissociation resulting from the MALDI process.

      Matrisome Glycomics and Proteomics from Histological Slides

      We developed a workflow for profiling GAGs, N-glycans, and proteins from tissue slides (
      • Shi X.
      • Shao C.
      • Mao Y.
      • Huang Y.
      • Wu Z.L.
      • Zaia J.
      LC-MS and LC-MS/MS studies of incorporation of 34SO3 into glycosaminoglycan chains by sulfotransferases.
      ,
      • Shao C.
      • Shi X.
      • Phillips J.J.
      • Zaia J.
      Mass spectral profiling of glycosaminoglycans from histological tissue surfaces.
      ). We applied the method to comparative glycomics profiling from invertebrates (
      • Ramachandra R.
      • Namburi R.B.
      • Ortega-Martinez O.
      • Shi X.
      • Zaia J.
      • Dupont S.T.
      • Thorndyke M.C.
      • Lindahl U.
      • Spillmann D.
      Brittlestars contain highly sulfated chondroitin sulfates/dermatan sulfates that promote FGF2 induced cell signaling.
      ,
      • Armistead J.S.
      • Wilson I.B.
      • van Kuppevelt T.H.
      • Dinglasan R.R.
      A role for heparan sulfate proteoglycans in Plasmodium falciparum sporozoite invasion of anopheline mosquito salivary glands.
      ,
      • Dierker T.
      • Shao C.
      • Haitina T.
      • Zaia J.
      • Hinas A.
      • Kjellen L.
      Nematodes join the family of chondroitin sulfate-synthesizing organisms: Identification of an active chondroitin sulfotransferase in Caenorhabditis elegans.
      ), mammalian organ tissues (
      • Shi X.
      • Zaia J.
      Organ-specific heparan sulfate structural phenotypes.
      ,
      • Staples G.O.
      • Shi X.
      • Zaia J.
      Extended NS domains reside at the non-reducing end of heparan sulfate chains.
      ,
      • Leymarie N.
      • McComb M.E.
      • Naimy H.
      • Staples G.O.
      • Zaia J.
      Differential characterization and classification of tissue specific glycosaminoglycans by tandem mass spectrometry and statistical methods.
      ), skeletal muscle (
      • Tran T.H.
      • Shi X.
      • Zaia J.
      • Ai X.
      Heparan sulfate 6-O-endosulfatases (Sulfs) coordinate the Wnt signaling pathways to regulate myoblast fusion during skeletal muscle regeneration.
      ), kidney tissues (
      • Schumacher V.A.
      • Schlotzer-Schrehardt U.
      • Karumanchi S.A.
      • Shi X.
      • Zaia J.
      • Jeruschke S.
      • Zhang D.
      • Pavenstaedt H.
      • Drenckhan A.
      • Amann K.
      • Ng C.
      • Hartwig S.
      • Ng K.H.
      • Ho J.
      • Kreidberg J.A.
      • Taglienti M.
      • Royer-Pokora B.
      • Ai X.
      WT1-dependent sulfatase expression maintains the normal glomerular filtration barrier.
      ,
      • Reine T.M.
      • Kolseth I.B.
      • Meen A.J.
      • Lindahl J.P.
      • Jenssen T.G.
      • Reinholt F.P.
      • Zaia J.
      • Shao C.
      • Hartmann A.
      • Kolset S.O.
      Effects of restoring normoglycemia in type 1 diabetes on inflammatory profile and renal extracellular matrix structure after simultaneous pancreas and kidney transplantation.
      ) leukocytes (
      • Shao C.
      • Shi X.
      • White M.
      • Huang Y.
      • Hartshorn K.
      • Zaia J.
      Comparative glycomics of leukocyte glycosaminoglycans.
      ), stem cell niche (
      • Langsdorf A.
      • Schumacher V.
      • Shi X.
      • Tran T.
      • Zaia J.
      • Jain S.
      • Taglienti M.
      • Kreidberg J.A.
      • Fine A.
      • Ai X.
      Expression regulation and function of heparan sulfate 6-O-endosulfatases in the spermatogonial stem cell niche.
      ), and tumor tissues (
      • Shao C.
      • Shi X.
      • Phillips J.J.
      • Zaia J.
      Mass spectral profiling of glycosaminoglycans from histological tissue surfaces.
      ,
      • Thelin M.
      • Svensson K.J.
      • Shi X.
      • Bagher M.
      • Axelsson J.
      • Isinger-Ekstrand A.
      • van Kuppevelt T.H.
      • Johansson J.
      • Nilbert M.
      • Zaia J.
      • Belting M.
      • Maccarana M.
      • Malmstrom A.
      Dermatan sulfate is involved in the tumorigenic properties of esophagus squamous cell carcinoma.
      ). We have investigated brain aging (
      • Raghunathan R.
      • Polinski N.K.
      • Klein J.A.
      • Hogan J.D.
      • Shao C.
      • Khatri K.
      • Leon D.
      • McComb M.E.
      • Manfredsson F.P.
      • Sortwell C.E.
      • Zaia J.
      Glycomic and proteomic changes in aging brain nigrostriatal pathway.
      ) and neuropathological diseases including glioma (
      • Shao C.
      • Shi X.
      • Phillips J.J.
      • Zaia J.
      Mass spectral profiling of glycosaminoglycans from histological tissue surfaces.
      ). Our method provides a readout of GAG quantities, domain structures, and non-reducing end structures using simple enzyme digestions with minimal need for workup. The final proteomics of tryptic peptides identifies ∼1200 proteins from the 10 nL tissue volume, providing deeper coverage than can be obtained from an MS imaging approach.
      This approach requires small tissue volumes, minimal sample workup, and reduces the effort required per biospecimen for glycomics and proteomics studies. Fresh frozen slides are washed with a series of solvents, thereby denaturing tissue proteins. Formalin-fixed, paraffin embedded tissue slides require dewaxing, re-hydration, and high pH antigen retrieval prior to enzymatic digestion. Although proteins are denatured in both cases, the observed glycomics and proteomics profiles reflect tissue processing biases that remain to be studied in detail. Nonetheless, the analysis of matrisome molecules from tissue slides offers an attractive option to extraction from wet tissue in terms of lower sample quantities and effort required. For example, in a study of aging rat brain from tissue slides with no enrichment, we observed 9–11% of total proteins of extracellular origin, corresponding to 15 matrisome molecules (
      • Raghunathan R.
      • Polinski N.K.
      • Klein J.A.
      • Hogan J.D.
      • Shao C.
      • Khatri K.
      • Leon D.
      • McComb M.E.
      • Manfredsson F.P.
      • Sortwell C.E.
      • Zaia J.
      Glycomic and proteomic changes in aging brain nigrostriatal pathway.
      ).

      Matrisome Glycoproteomics

      Application of a conventional discovery proteomics workflow with database searching identified Matrisome molecules based on the presence of unmodified peptides. Although homogeneous PTMs including phosphorylation, acetylation, methylation, and ubiquitination are amenable to proteomics database searching (
      • Choudhary C.
      • Mann M.
      Decoding signalling networks by mass spectrometry-based proteomics.
      ,
      • Hart G.W.
      • Ball L.E.
      Post-translational modifications: a major focus for the future of proteomics.
      ), glycosylation is heterogeneous as a rule. This multiplies the number of PTM forms of a given matrisome molecule glycopeptide, thereby dividing the precursor ion signals, and multiplying the size of the proteomics search space and the difficulty of assigning the glycopeptide with confidence (
      • Desaire H.
      • Hua D.
      When can glycopeptides be assigned based solely on high-resolution mass spectrometry data?.
      ,
      • Dallas D.C.
      • Martin W.F.
      • Hua S.
      • German J.B.
      Automated glycopeptide analysis–review of current state and future directions.
      ,
      • Mechref Y.
      Use of CID/ETD mass spectrometry to analyze glycopeptides.
      ,
      • Hinneburg H.
      • Stavenhagen K.
      • Schweiger-Hufnagel U.
      • Pengelley S.
      • Jabs W.
      • Seeberger P.H.
      • Silva D.V.
      • Wuhrer M.
      • Kolarich D.
      The art of destruction: optimizing collision energies in quadrupole-time of flight (Q-TOF) instruments for glycopeptide-based glycoproteomics.
      ,
      • Hu H.
      • Khatri K.
      • Klein J.
      • Leymarie N.
      • Zaia J.
      A review of methods for interpretation of glycopeptide tandem mass spectral data.
      ,
      • Leymarie N.
      • Zaia J.
      Effective use of mass spectrometry for glycan and glycopeptide structural analysis.
      ,
      • Thaysen-Andersen M.
      • Packer N.H.
      • Schulz B.L.
      Maturing glycoproteomics technologies provide unique structural insights into the N-glycoproteome and its regulation in health and disease.
      ). As shown in Fig. 3, the presence of complex glycosylation alters the collisional dissociation pattern of peptides significantly. Glycopeptide collisional dissociation tandem mass spectra show low m/z oxonium signature ions that indicate the presence of glycosylation. The spectra also show peptide+saccharide ions, the abundances of which depend on the extent of vibrational excitation of the precursor ions. If relatively low collision energies are used, then product ions resulting from losses of saccharide units are abundant. At higher collision energies, peptide plus from one to a few monosaccharide units are observed. Under such conditions, dissociation of the peptide backbone is often observed albeit at relatively low abundances. Thus, the most confident collisional tandem mass spectra for glycopeptide precursor ions contain all three ion types as shown for example for an aggrecan glycopeptide in Fig. 3 (
      • Klein J.A.
      • Meng L.
      • Zaia J.
      Deep sequencing of complex proteoglycans: a novel strategy for high coverage and site-specific identification of glycosaminoglycan-linked peptides.
      ).
      Figure thumbnail gr3
      Fig. 3Higher energy collisional dissociation tandem mass spectrum of an aggrecan glycopeptide showing the presence of oxonium ions (green), peptide+saccharide ions (golden), and peptide backbone dissociation ions (red or blue) (
      • Klein J.A.
      • Meng L.
      • Zaia J.
      Deep sequencing of complex proteoglycans: a novel strategy for high coverage and site-specific identification of glycosaminoglycan-linked peptides.
      ).

      Lectin Enrichment of Glycopeptides

      Investigators have used WFA and concanavalin A (ConA) lectin enrichment of guanidine HCl extracts to enrich glycoproteins from human cardiac tissue from which they reported identification of 65 glycosylation sites from 35 extracellular proteins (
      • Barallobre-Barreiro J.
      • Gupta S.K.
      • Zoccarato A.
      • Kitazume-Taneike R.
      • Fava M.
      • Yin X.
      • Werner T.
      • Hirt M.N.
      • Zampetaki A.
      • Viviano A.
      • Chong M.
      • Bern M.
      • Kourliouros A.
      • Domenech N.
      • Willeit P.
      • Shah A.M.
      • Jahangiri M.
      • Schaefer L.
      • Fischer J.W.
      • Iozzo R.V.
      • Viner R.
      • Thum T.
      • Heineke J.
      • Kichler A.
      • Otsu K.
      • Mayr M.
      Glycoproteomics reveals decorin peptides with anti-myostatin activity in human atrial fibrillation.
      ,
      • Barallobre-Barreiro J.
      • Baig F.
      • Fava M.
      • Yin X.
      • Mayr M.
      Glycoproteomics of the extracellular matrix: a method for intact glycopeptide analysis using mass spectrometry.
      ). Wheat germ agglutinin (WGA) was used to enrich glycoproteins from mouse brain to identify O-mannosylated peptides from neurofascin 186 (
      • Pacharra S.
      • Hanisch F.G.
      • Breloy I.
      Neurofascin 186 is O-mannosylated within and outside of the mucin domain.
      ) and PNN associated hyalectan proteoglycans (
      • Pacharra S.
      • Hanisch F.G.
      • Muhlenhoff M.
      • Faissner A.
      • Rauch U.
      • Breloy I.
      The lecticans of mammalian brain perineural net are O-mannosylated.
      ). O-Mannosylated peptides have been enriched from tissue extracts digested using trypsin and peptide-N-glycosidase F using ConA lectin chromatography (
      • Vester-Christensen M.B.
      • Halim A.
      • Joshi H.J.
      • Steentoft C.
      • Bennett E.P.
      • Levery S.B.
      • Vakhrushev S.Y.
      • Clausen H.
      Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins.
      ,
      • Bartels M.F.
      • Winterhalter P.R.
      • Yu J.
      • Liu Y.
      • Lommel M.
      • Mohrlen F.
      • Hu H.
      • Feizi T.
      • Westerlind U.
      • Ruppert T.
      • Strahl S.
      Protein O-mannosylation in the murine brain: occurrence of mono-O-mannosyl glycans and identification of new substrates.
      ). A set of 16 O-mannosylated glycoproteins were identified, several belonging to the cadherin superfamily, using this approach.

      Proteoglycan Glycoproteomics

      Enzymatic digestion of GAG chains leaves a glycopeptide with linker saccharide attached to the core protein. Such linker glycopeptides can be identified by the presence of a diagnostic oxonium ion for CS and HS proteoglycans (
      • Gomez Toledo A.
      • Nilsson J.
      • Noborn F.
      • Sihlbom C.
      • Larson G.
      Positive mode LC-MS/MS analysis of chondroitin sulfate modified glycopeptides derived from light and heavy chains of the human inter-alpha-trypsin inhibitor complex.
      ). The linker saccharide glycopeptides detected for CSPGs were modified with sulfate, phosphate, fucose and/or sialic acid (
      • Nilsson J.
      • Noborn F.
      • Gomez Toledo A.
      • Nasir W.
      • Sihlbom C.
      • Larson G.
      Characterization of glycan structures of chondroitin sulfate-glycopeptides facilitated by sodium ion-pairing and positive mode LC-MS/MS.
      ,
      • Noborn F.
      • Gomez Toledo A.
      • Green A.
      • Nasir W.
      • Sihlbom C.
      • Nilsson J.
      • Larson G.
      Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans.
      ,
      • Noborn F.
      • Gomez Toledo A.
      • Sihlbom C.
      • Lengqvist J.
      • Fries E.
      • Kjellen L.
      • Nilsson J.
      • Larson G.
      Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans.
      ). This approach has been used to analyze PGs from biological fluids including urine and cerebrospinal fluid (
      • Noborn F.
      • Gomez Toledo A.
      • Sihlbom C.
      • Lengqvist J.
      • Fries E.
      • Kjellen L.
      • Nilsson J.
      • Larson G.
      Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans.
      ). We used a similar approach for analysis of purified proteoglycans including aggrecan, decorin, brevican and neurocan (
      • Klein J.A.
      • Meng L.
      • Zaia J.
      Deep sequencing of complex proteoglycans: a novel strategy for high coverage and site-specific identification of glycosaminoglycan-linked peptides.
      ). In order to interpret the glycopeptide tandem mass spectra automatically from the LC-tandem MS datasets, we optimized our GlycReSoft software (
      • Maxwell E.
      • Tan Y.
      • Tan Y.
      • Hu H.
      • Benson G.
      • Aizikov K.
      • Conley S.
      • Staples G.O.
      • Slysz G.W.
      • Smith R.D.
      • Zaia J.
      GlycReSoft: A software package for automated recognition of glycans from LC/MS Data.
      ,
      • Klein J.
      • Carvalho L.
      • Zaia J.
      Application of network smoothing to glycan LC-MS profiling.
      ) for interpretation of linker-glycopeptides.
      We have observed glycopeptides abundances too low for confident identification when analyzing proteolytic digests from tissue slides. It, therefore, appears that enrichment steps will be necessary to allow glycoproteomics from the tissue. Although such enrichment remains a challenge from small tissue volumes such as obtained from tissue slides, it seems feasible from wet tissue extracts.

      CONCLUSIONS

      For researchers interested in profiling abundances of matrisome core proteins, the use of decellularization or enrichment methods combined with targeted MS or DIA MS seems appropriate. As in other areas of proteomics, the use of multidimensional separations increases the number of proteins identified at the expense of analysis time and cost. One of these separation dimensions can be designed to enrich glycopeptides, thus increasing the ability to detect matrisome determinants of molecular networks. Such enrichment steps are most readily applied to tissue extracts. The analysis of tissue slides has potential benefits in terms of throughput, cost, and applicability to pathological workflows. The tissue volume, however, is rather low, making use of enrichment steps challenging. On the other hand, tissue slides can be microdissected (
      • Budnik B.
      • Levy E.
      • Harmange G.
      • Slavov N.
      SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation.
      ), increasing the ability to select cell populations of interest for subsequent proteomics. Robotic approaches for manipulation of microdissected tissue have been described (
      • Zhu Y.
      • Piehowski P.D.
      • Zhao R.
      • Chen J.
      • Shen Y.
      • Moore R.J.
      • Shukla A.K.
      • Petyuk V.A.
      • Campbell-Thompson M.
      • Mathews C.E.
      • Smith R.D.
      • Qian W.J.
      • Kelly R.T.
      Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells.
      ,
      • Zhu Y.
      • Dou M.
      • Piehowski P.D.
      • Liang Y.
      • Wang F.
      • Chu R.K.
      • Chrisler W.B.
      • Smith J.N.
      • Schwarz K.C.
      • Shen Y.
      • Shukla A.K.
      • Moore R.J.
      • Smith R.D.
      • Qian W.J.
      • Kelly R.T.
      Spatially resolved proteome mapping of laser capture microdissected tissue with automated sample transfer to nanodroplets.
      ). It may, therefore, be feasible to use glycopeptide enrichment in such robotic workflows to enable the application of glycoproteomics LC-MS methods to microdissected tissue. This will enable profiling of designated matrisome glycosites as a means for assessing changes to extracellular networks during disease mechanisms.

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