RESUMO
Pseudomonas aeruginosa is a human opportunistic pathogen responsible for lung infections in cystic fibrosis patients. The emergence of resistant strains and its ability to form a biofilm seem to give a selective advantage to the bacterium and thus new therapeutic approaches are needed. To infect the lung, the bacterium uses several virulence factors, like LecA lectins. These proteins are involved in bacterial adhesion due to their specific interaction with carbohydrates of the host epithelial cells. The tetrameric LecA lectin specifically binds galactose residues. A new therapeutic approach is based on the development of highly affine synthetic glycoclusters able to selectively link with LecA to interfere with the natural carbohydrate-LecA interaction. In this study, we combined atomic force microscopy imaging and molecular dynamics simulations to visualize and understand the arrangements formed by LecA and five different glycoclusters. Our glycoclusters are small scaffolds characterized by a core and four branches, which terminate in a galactose residue. Depending on the nature of the core and the branches, the glycocluster-lectin interaction can be modulated and the affinity increased. We show that glycocluster-LecA arrangements highly depend on the glycocluster architecture: the core influences the rigidity of the geometry and the directionality of the branches, whereas the nature of the branch determines the compactness of the structure and the ease of binding.
Assuntos
Carboidratos/química , Lectinas/química , Microscopia de Força Atômica/métodos , Nanoestruturas/química , Aderência Bacteriana/efeitos dos fármacos , Simulação por Computador , Células Epiteliais/efeitos dos fármacos , Humanos , Modelos Moleculares , Método de Monte Carlo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Multimerização Proteica , Pseudomonas aeruginosa , TermodinâmicaRESUMO
The Gram negative bacterium Pseudomonas aeruginosa (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. It binds to l-fucose, d-mannose and glycan exposing terminal fucose or mannose. Using a competitive assay on microarray, 156 oligosaccharides and polysaccharides issued from fermentation or from the biomass were screened toward their affinity to LecB. Next, the five best ligands (Lewisa, Lewisb, Lewisx, siayl-Lewisx and 3-fucosyllactose) were derivatized with a propargyl aglycon allowing the synthesis of 25 trivalent, 25 tetravalent and 5 monovalent constructions thanks to copper catalyzed azide alkyne cycloaddition. The 55 clusters were immobilized by DNA Directed immobilization leading to the fabrication of a glycocluster microarray. Their binding to LecB was studied. Multivalency improved the binding to LecB. The binding structure relationship of the clusters is mainly influenced by the carbohydrate residues. Molecular simulations indicated that the simultaneous contact of both binding sites of monomer A and D seems to be energetically possible.
Assuntos
Lectinas/química , Oligossacarídeos/química , Pseudomonas aeruginosa/química , Sítios de Ligação , Lectinas/metabolismo , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação ProteicaRESUMO
Anti-infectious strategies against pathogen infections can be achieved through antiadhesive strategies by using multivalent ligands of bacterial virulence factors. LecA and LecB are lectins of Pseudomonas aeruginosa implicated in biofilm formation. A series of 27 LecA-targeting glycoclusters have been synthesized. Nine aromatic galactose aglycons were investigated with three different linker arms that connect the central mannopyranoside core. A low-nanomolar (Kd =19â nm, microarray) ligand with a tyrosine-based linker arm could be identified in a structure-activity relationship study. Molecular modeling of the glycoclusters bound to the lectin tetramer was also used to rationalize the binding properties observed.
Assuntos
Adesinas Bacterianas/química , Galactose/química , Lectinas/química , Pseudomonas aeruginosa/química , Adesinas Bacterianas/metabolismo , Galactose/metabolismo , Lectinas/metabolismo , Ligantes , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Heat shock proteins (HSPs) are over-expressed in a wide range of human cancers. It results in the stimulation of the immune system and consequently in elevated concentration of anti-HSP autoantibodies. Elevated anti-HSP autoantibodies were found in breast cancer patients, and they are associated with tumor metastasis. Therefore, screening these autoantibodies could be of diagnostic and prognostic values. Protein microarrays have already demonstrated their great potential as a diagnostic tool. However, protein diversity requires optimization of the microarray fabrication to achieve high sensitivity and specificity. In this study, seven HSPs were immobilized on six different surface chemistries. After evaluation and optimization with purified antibodies of the six surface chemistries, two surfaces were selected to detect anti-HSP autoantibodies in breast cancer sera. Multiplex detection of anti-HSP autoantibodies allowed discrimination of breast cancer patients (50) from healthy controls (26) with a sensitivity of 86% and a specificity of 100%.
Assuntos
Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Proteínas de Choque Térmico/imunologia , Análise Serial de Proteínas/instrumentação , Análise Serial de Proteínas/métodos , Antígenos de Neoplasias/sangue , Autoanticorpos/imunologia , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/imunologia , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoensaio/métodos , Estadiamento de Neoplasias , Estudos ProspectivosRESUMO
Pseudomonas aeruginosa (PA) is an opportunistic bacterium involved in 10-30% of nosocomial diseases. It causes severe lung injury to cystic fibrosis patients, often leading to patient death. PA strains are multidrug resistant, thus making the design of new therapeutics a challenge for public health. One promising therapeutic option is to design glycoclusters that target the virulence factor of PA. LecA is a galactose-specific lectin that might be involved in adhesion and biofilm formation by PA. The DNA-directed immobilization (DDI) microarray is a powerful tool for screening and understanding of structure-activity relationships between glycoclusters and lectins. High-throughput and multiplexed analysis of lectin-glycocluster interactions on a DDI microarray allows measurement of IC50 and dissociation constant (Kd ) values with minute amounts of material. In order to study the robustness of the DDI microarray in determination of IC50 and Kd values, the impact of glycocluster surface density was investigated. The data obtained show that measured IC50 values were influenced by glycocluster surface density: as the density of glycoclusters increases, the measured IC50 values increase too. In contrast, the measured Kd values were not affected by glycocluster surface density, provided that the experimental conditions allow interaction between glycocluster and lectin at single-molecule level (no surface cluster effect).
Assuntos
Adesinas Bacterianas/metabolismo , Glicoproteínas/metabolismo , Análise em Microsséries , Pseudomonas aeruginosa/metabolismo , Adesinas Bacterianas/química , Aderência Bacteriana , Biofilmes , Transferência Ressonante de Energia de Fluorescência , Glicoproteínas/química , Concentração Inibidora 50 , Cinética , Microscopia de Força Atômica , Ligação Proteica , Pseudomonas aeruginosa/genética , Fatores de VirulênciaRESUMO
Pseudomonas aeruginosa (PA) and Burkholderia ambifaria (BA) are two opportunistic Gram negative bacteria and major infectious agents involved in lung infection of cystic fibrosis patients. Both bacteria can develop resistance to conventional antibiotherapies. An alternative strategy consists of targeting virulence factors in particular lectins with high affinity ligands such as multivalent glycoclusters. LecA (PA-IL) and LecB (PA-IIL) are two tetravalent lectins from PA that recognise galactose and fucose respectively. BambL lectin from BA is trimeric with 2 binding sites per monomer and is also specific for fucose. These three lectins are potential therapeutic targets in an anti-adhesive anti-bacterial approach. Herein, we report the synthesis of 18 oligonucleotide pentofuranose-centered or mannitol-centered glycoclusters leading to tri-, penta- or decavalent clusters with different topologies. The linker arm length between the core and the carbohydrate epitope was also varied leading to 9 galactoclusters targeting LecA and 9 fucoclusters targeting both LecB and BambL. Their dissociation constants (Kd) were determined using a DNA-based carbohydrate microarray technology. The trivalent xylo-centered galactocluster and the ribo-centered fucocluster exhibited the best affinity for LecA and LecB respectively while the mannitol-centered decafucocluster displayed the best affinity to BambL. These data demonstrated that the topology and nature of linkers were the predominant factors for achieving high affinity rather than valency.
Assuntos
Adesinas Bacterianas/metabolismo , Burkholderia/metabolismo , Glicoconjugados/química , Glicoconjugados/farmacologia , Lectinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Sítios de Ligação , Burkholderia/efeitos dos fármacos , Infecções por Burkholderia/tratamento farmacológico , Infecções por Burkholderia/microbiologia , Descoberta de Drogas , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Ligação Proteica , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
Pseudomonas aeruginosa (PA) is a major public health care issue due to its ability to develop antibiotic resistance mainly through adhesion and biofilm formation. Therefore, targeting the bacterial molecular arsenal involved in its adhesion and the formation of its biofilm appears as a promising tool against this pathogen. The galactose-binding LecA (or PA-IL) has been described as one of the PA virulence factors involved in these processes. Herein, the affinity of three tetravalent mannose-centered galactoclusters toward LecA was evaluated with five different bioanalytical methods: HIA, ELLA, SPR, ITC and DNA-based glycoarray. Inhibitory potential towards biofilms was then assessed for the two glycoclusters with highest affinity towards LecA (Kd values of 157 and 194 nM from ITC measurements). An inhibition of biofilm formation of 40% was found for these galactoclusters at 10 µM concentration. Applications of these macromolecules in anti-bacterial therapy are therefore possible through an anti-adhesive strategy.
Assuntos
Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Galactose/química , Galactose/farmacologia , Manose/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Testes de Sensibilidade MicrobianaRESUMO
Pseudomonas aeruginosa (PA) is a major public health issue due to its impact on nosocomial infections as well as its impact on cystic fibrosis patient mortality. One of the main concerns is its ability to develop antibiotic resistance. Therefore, inhibition of PA virulence has been proposed as an alternative strategy to tackle PA based infections. LecA (or PA-IL), a galactose binding lectin from PA, is involved in its virulence. Herein, we aimed at designing high affinity synthetic ligands toward LecA for its inhibition and at understanding the key parameters governing the binding of multivalent galactosylated clusters. Twenty-five glycoclusters were synthesized and their bindings were studied on a carbohydrate microarray. Monosaccharide centered clusters and linear comb-like clusters were synthesized with different linkers separating the core and the galactosyl residues. Their length, flexibility, and aromaticity were varied. Our results showed that the binding profile of LecA to galactosylated clusters was dependent on both the core and the linker and also that the optimal linker was different for each core. Nevertheless, an aryl group in the linker structure drastically improved the binding to LecA. Our results also suggest that optimal distances are preferred between the core and the aromatic group and the core and the galactose.
Assuntos
Adesinas Bacterianas/química , DNA/química , Galactose/química , Pseudomonas aeruginosa/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
COOH-terminated self-assembled monolayers (SAMs) are widely used in biosensor technology to bind different amine-containing biomolecules. A covalent amide bond, however, can be achieved only if the carboxylic acids are activated. This activation process usually consists of forming an N-hydroxysuccinimidyl ester (NHS-ester) by consecutively reacting carboxylic acids with a carbodiimide and NHS. Though many papers report using this method,1-8 the experimental conditions vary greatly between them and chemical characterization at this stage is often omitted. Evidence of an efficient activation is therefore rarely shown. Furthermore, recent publications9-11 have highlighted the complexity of this process, with the possible formation of different byproducts. In this paper, we have conducted a study on NHS activation under different conditions with chemical characterization by polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and time-of-flight secondary ion mass spectroscopy (ToF-SIMS). Our results indicate that the nature of the solvent and carbodiimide and the reactant concentrations play crucial roles in activation kinetics and efficiency.
Assuntos
Carbodi-Imidas/química , Ácidos Carboxílicos/química , Técnicas Biossensoriais , Solventes/química , Espectrometria de Massa de Íon Secundário , Propriedades de SuperfícieRESUMO
A library of 24 new mannose-centered tetragalactoclusters with four different linkers (di- and triethyleneglycol with phosphodiester or phosphorothioate linkages) and six different aromatic aglycons (O-phenyl, S-phenyl, O-benzyl, S-benzyl, O-biphenyl and O-naphthyl) was synthesized. Their interactions with LecA were evaluated on a DNA Directed Immobilization (DDI) based glycocluster array allowing the determination of their IC50 against lactose and the evaluation of their dissociation constant (Kd). Finally, the docking simulations confirm the experimental results and demonstrated that the better affinity of O-biphenyl- and O-naphthyl-galactoside is due to a double interaction between the aromatic ring and the histidine 50 and proline 51 of LecA.
Assuntos
Adesinas Bacterianas/metabolismo , Azidas/química , Compostos de Bifenilo/química , Galactose/química , Galactosídeos/química , Modelos Moleculares , Naftóis/química , Galactosídeos/síntese químicaRESUMO
Antibody microarrays are powerful and high-throughput tools for screening and identifying tumor markers from small sample volumes of only a few microliters. Optimization of surface chemistry and spotting conditions are crucial parameters to enhance antibodies' immobilization efficiency and to maintain their biological activity. Here, we report the implementation of an antibody microarray for the detection of tumor markers involved in colorectal cancer. Three-dimensional microstructured glass slides were functionalized with three different aminated molecules ((3-aminopropyl)dimethylethoxysilane (APDMES), Jeffamine, and chitosan) varying in their chain length, their amine density, and their hydrophilic/hydrophobic balance. The physicochemical properties of the resulting surfaces were characterized. Antibody immobilization efficiency through physical interaction was studied as a function of surface properties as well as a function of the immobilization conditions. The results show that surface energy, steric hindrance, and pH of spotting buffer have great effects on protein immobilization. Under optimal conditions, biological activities of four immobilized antitumor marker antibodies were evaluated in multiplex immunoassay for the detection of the corresponding tumor markers. Results indicated that the chitosan functionalized surface displayed the highest binding capacity and allowed to retain maximal biological activity of the four tested antibody/antigen systems. Thus, we successfully demonstrated the application of amino-based surface modification for antibody microarrays to efficiently detect tumor markers.
Assuntos
Aminas/química , Anticorpos/química , Biomarcadores Tumorais/análise , Quitosana/química , Neoplasias Colorretais/diagnóstico , Propilaminas/química , Silanos/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Análise Serial de Proteínas , Propriedades de SuperfícieRESUMO
Pseudomonas aeruginosa (PA) is a Gram negative opportunistic pathogen and is the major pathogen encounter in the cystic fibrosis (CF) lung airways. It often leads to chronic respiratory infection despite aggressive antibiotic therapy due to the emergence of resistant strains and to the formation of biofilm. The lectin PA-IIL (LecB) is a fucose-specific lectin from PA suspected to be involved in host recognition/adhesion and in biofilm formation. Thus, it can be foreseen as a potential therapeutic target. Herein, 16 fucosylated glycoclusters with antenna-like, linear, or crown-like spatial arrangements were synthesized using a combination of DNA solid-phase synthesis and alkyne azide 1,3-dipolar cycloaddition (CuAAC). Their binding properties toward PA-IIL were then evaluated based on DNA directed immobilization (DDI) carbohydrate microarray. Our results suggested that the antenna-like scaffold was preferred to linear or crown-like glycoclusters. Among the crown-like carbohydrate centered fucosylated glycoclusters, mannose-based core was better than glucose- and galactose-based ones. The influence of the linker arm was also evaluated, and long linkers between fucoses and the core led to a slight better binding than the short ones.
Assuntos
Proteínas de Bactérias/metabolismo , Materiais Biomiméticos/química , DNA de Cadeia Simples/química , Fucose/química , Lectinas/metabolismo , Análise em Microsséries , Pseudomonas aeruginosa , Materiais Biomiméticos/metabolismo , Carbocianinas/química , Reação de Cicloadição , Cicloexanos/química , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Ligação Proteica , Conformação ProteicaRESUMO
Homo- and heterofunctionalized glycoclusters with galactose and/or fucose residues targeting both PA-IL and PA-IIL lectins of Pseudomonas aeruginosa were synthesized using "Click" chemistry and DNA chemistry. Their binding to lectins (separately or in a mixture) was studied using a DNA Directed Immobilization carbohydrate microarray. Homoglycoclusters bind selectively to their lectin while the heteroglycocluster binds simultaneously both lectins with a slight lower affinity.
Assuntos
Fucose/química , Galactose/química , Lectinas/química , Pseudomonas aeruginosa/química , Química Click , Fucose/síntese química , Galactose/síntese química , Lectinas/síntese química , Estrutura MolecularRESUMO
Influenza neuraminidases hydrolyze the ketosidic linkage between N-acetylneuraminic acid and its adjacent galactose residue in sialosides. This enzyme is a tetrameric protein that plays a critical role in the release of progeny virions. Several methods have been described for the determination of neuraminidase activity, usually based on colorimetric, fluorescent, or chemiluminescent detection. However, only a few of these tests allow discrimination of the sialyl-linkage specificity (i.e., α2-3- versus α2-6-linked sialyllactosides) of the neuraminidase. Herein we report a glycoarray-based assay and a MALDI-TOF study for assessing the activity and specificity of two influenza neuraminidases on whole viruses. The human A(H3N2) and avian A(H5N2) neuraminidase activities were investigated. The results from both approaches demonstrated that α2-3 sialyllactoside was a better substrate than α2-6 sialyllactoside for both viruses and that H5N2 virus had a lower hydrolytic activity than H3N2.
Assuntos
Vírus da Influenza A Subtipo H3N2/enzimologia , Vírus da Influenza A Subtipo H5N2/enzimologia , Neuraminidase/metabolismo , Animais , Aves , Humanos , Influenza Aviária/virologia , Influenza Humana/virologia , Análise em Microsséries/métodos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Oligonucleotide glycoconjugates with a mannose or galactose core bearing four galactose residues introduced by phosphoramidite chemistry and copper catalyzed azide alkyne 1,3-dipolar cycloaddition (click chemistry) have been synthesized. A first click reaction allowed the introduction on a solid support of a mannose core on which four pentynyl linkers were introduced using a phosphoramidite derivative. After the elongation of the oligonucleotide, a second click reaction performed either on solid support or in solution allowed the introduction of four galactose azide derivatives. Repeating the phosphoramidite and click chemistries afforded an oligonucleotide glycoconjugate dendrimer bearing 16 galactoses on its periphery.
Assuntos
Galactose/química , Oligonucleotídeos/síntese química , Compostos Organofosforados/química , Alcinos/química , Azidas/química , Catálise , Cobre/química , Ciclização , Manose/química , Estrutura Molecular , Oligonucleotídeos/química , EstereoisomerismoRESUMO
Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. To implement functional protein microarrays, appropriate surface chemistry must be designed so that both the protein structure and the biological activity can be retained. In the current study, two surface chemistries for protein microarrays and immunofluorescent assays were developed. Glass slides were functionalized with N-hydroxysuccinimide (NHS) ester via a monofunctional silane or maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays was then evaluated for the detection of anti-histone autoantibodies present in the sera of patients suffering from a systemic autoimmune disease, namely systemic lupus erythematosus (SLE), and the results were compared with those of the classical enzyme-linked immunosorbent assay (ELISA) and Western blot. The detection limit of our MAMVE copolymer microarrays was 50-fold lower than that of the classical ELISA. Furthermore, 100-fold less volume of biological samples was required with these miniaturized immunoassays.
Assuntos
Imunoensaio/métodos , Análise Serial de Proteínas/métodos , Autoanticorpos/sangue , Western Blotting/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Histonas/química , Histonas/imunologia , Histonas/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Proteínas Imobilizadas/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Miniaturização , Copolímero de Pirano/química , Silanos/químicaRESUMO
Sugar-coated chips: Glycoside clusters are valuable tools for carbohydrate-lectin recognition studies. However, the spatial arrangement of the sugar residues is a key issue in the design of high-affinity glycoclusters. Here the affinities of linear and antenna- and calixarene-based galactoside clusters towards two lectins derived from Pseudomonas aeruginosa and Ricinus communis were compared by means of glycoarrays.Interactions between proteins and carbohydrates are involved in a large number of crucial biological events. Many efforts have been devoted to the design and synthesis of unnatural saccharides displaying high affinities towards targeted lectins. Among others, glycoside clusters have proven to be valuable tools for these recognition studies. However, the spatial arrangements of the sugar residues are a key issue in the design of high-affinity glycoclusters. Here, the affinities of linear and antenna- and calixarene-based galactoside clusters against two lectins, derived from Pseudomonas aeruginosa and Ricinus communis, have been compared by means of glycoarrays.
Assuntos
Adesinas Bacterianas/química , DNA/química , Glicosídeos/química , Lectinas/química , Lectinas de Plantas/química , Triazóis/química , Calixarenos/química , Corantes Fluorescentes/química , Galactosídeos/química , Glicosídeos/síntese química , Análise em MicrossériesRESUMO
Thanks to their specificity and stability in the sera, autoantibodies (AAbs) against tumor-associated antigens (TAAs) are very attractive biomarkers for the development of less invasive serological tests for the diagnosis and prognosis of cancer. Heat shock proteins (HSP) belong to TAAs and they are over-expressed in various human cancers. Elevated HSP can stimulate the immune system to produce anti-HSP antibodies. So far, AAbs against HSP have been identified in the circulation of various cancer patients. Here we will review current literature on the use of anti-HSP antibodies for cancer diagnosis and prognosis. The challenges as well as future directions of AAbs identification in oncology are also discussed.
Assuntos
Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Proteínas de Choque Térmico/imunologia , Neoplasias/diagnóstico , Neoplasias/imunologia , Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Neoplasias/sangueRESUMO
Site-directed mutagenesis has greatly helped researchers both to understand the precise role of specific residues in coding sequences and to generate variants of proteins that have acquired new characteristics. Today's demands for more complete functional cartographies of proteins and advances in selection and screening technologies require that site-directed mutagenesis be adapted for high-throughput applications. We describe here the first generation of a library of single and multiple site-directed mutants using a mixture of oligonucleotides synthesized on DNA chips. We have used the human interleukin 15 (IL15) gene as a model, of which 37 codons were simultaneously targeted for substitution by any of eight possible codons. Ninety-six clones were sequenced, exhibiting a broad spectrum of targeted substitutions over the whole gene length with no unwanted mutations. Libraries produced using such pools of oligonucleotides open new perspectives to direct the evolution of proteins in vitro, by enabling the simple, rapid, and cost-effective generation of large tailor-made genetic diversities from any gene.
Assuntos
Biblioteca Gênica , Interleucina-15/genética , Mutagênese Sítio-Dirigida/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligonucleotídeos/genéticaRESUMO
Single-step orthogonal chemical functionalization procedures have been developed with patterned gold on silica surfaces. Different combinations of a silane and a thiol were simultaneously deposited on a gold/silica heterogeneous substrate. The orthogonality of the functionalization (i.e., selective grafting of the thiol on the gold areas and the silane on the silica) was demonstrated by X-ray photoelectron spectroscopy (XPS) as well as time-of-flight secondary ion mass spectrometry (ToF-SIMS) mapping. The orthogonal functionalization was used to immobilize proteins onto gold nanostructures on a silica substrate, as demonstrated by atomic force microscopy (AFM). These results are especially promising in the development of future biosensors where the selective anchoring of target molecules onto nanostructured transducers (e.g., nanoplasmonic biosensors) is a major challenge.