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This paper presents an enzyme building block for the assembly of megamolecules. The system is based on the inhibition of the human‐derived cellular retinoic acid binding protein II (CRABP2) domain. We synthesized a synthetic retinoid bearing an arylfluorosulfate group, which uses sulfur fluoride exchange click chemistry to covalently inhibit CRABP2. We conjugated both the inhibitor and a fluorescein tag to an oligo(ethylene glycol) backbone and measured a second‐order rate constant for the protein inhibition reaction of approximately 3,600 M−1s−1. We used this new enzyme‐inhibitor pair to assemble multi‐protein structures in one‐pot reactions using three orthogonal assembly chemistries to demonstrate exact control over the placement of protein domains within a single, homogeneous molecule. This work enables a new dimension of control over specificity, orientation, and stoichiometry of protein domains within atomically precise nanostructures. [ABSTRACT FROM AUTHOR]

Abstract The interactions of adherent cells with their insoluble extracellular matrices are complex and challenging to study in the laboratory. Approaches from interface science have been important to preparing models of the biological matrix wherein discrete ligands are immobilized and interact with cellular receptors. A recent theme has been to develop dynamic substrates, where the activities of immobilized ligands can be modulated in real-time during cell culture. This short opinion reviews the strategies to manipulate ligand activity, highlights recent work that has advanced the field and discusses the applications that have been enabled. This work suggests that dynamic substrates will continue to find important uses in basic and applied biointerfaces. Graphical abstract Image 1 Highlights • Cell-surface receptors bind to protein matrix ligands to adhere and receive signals. • In tissue, the interactions are regulated dynamically to control cell behavior. • Bio-interface science seeks to design dynamic substrates that mimic biological matrix. • Strategies based on optical, electrical, mechanical and chemical cues are described. • Dynamic substrates enable studies in biology, cell-based sensing, and diagnostics. [ABSTRACT FROM AUTHOR]

This paper reports a kinetic characterization of the interfacial reaction of N-methylpyrrolidine with a self-assembled monolayer presenting an iodoalkyl group. SAMDI (self-assembled monolayers for matrix-assisted laser desorption/ionization) mass spectrometry was used to determine the extent of reaction for monolayers that were treated with a range of concentrations of the nucleophile for a range of times. These data revealed a second-order rate constant for the reaction that was approximately 100-fold greater than that for the analogous solution-phase reaction. However, addition of sodium iodide to the reaction mixture resulted in a 7-fold decrease in the reaction rate. Addition of bromide and chloride salts also gave slower rate constants for the reaction, but only at 100- and 1000-fold higher concentrations than was observed with iodide, respectively. The corresponding solution-phase reactions, by contrast, had rate constants that were unaffected by the concentration of halide salts. This work provides a well-characterized example illustrating the extent to which the kinetics and properties of an interfacial reaction can depart substantially from their better-understood solution-phase counterparts. [ABSTRACT FROM AUTHOR]

Cell-cell interactions are vital for embryonic organ development and normal function of differentiated cells and tissues. In this study we have developed a self-assembled monolayer-based co-culture system to study tooth morphogenesis. Specifically, we designed a 2-D microenvironment present in the dental tissue by creating a well-structured, laterally organized epithelial and mesenchymal cell co-culture system by patterning the cell-attachment substrate. Chemical modifications were used to develop tunable surface patterns to facilitate epithelial-mesenchymal interactions mimicking the developing tooth. Such a design promoted interactions between monolayer's of the 2 cell types and provided signaling cues that resulted in cellular differentiation and mineralized matrix formation. Gene expression analysis showed that these co-cultures mimicked in-vivo conditions than monolayer cultures of a single cell type. [ABSTRACT FROM AUTHOR]

Summary: This report demonstrates that the mechanical stability of focal adhesions exhibits a biphasic and sensitive pH dependence. These studies used isolated focal adhesions, which retain many of the properties of the intracellular structures, including protein composition and force-dependent reinforcement by cytosolic proteins. The focal adhesion structures are least stable to applied force at a pH of 6.4, and significantly more stable at slightly higher and lower pH values. This trend is consistent with previous work that characterized the pH dependence of cell migration and may therefore be relevant to controlling the invasiveness of metastatic cancer cells. This approach is significant because it allows biochemical studies of large protein complexes previously studied only in cell culture, and therefore offers new opportunities for performing mechanistic studies of a range of factors that contribute to focal adhesion stability. [ABSTRACT FROM AUTHOR]

Summary: Fibrinogen (Fbg) mediates platelet aggregation by binding the αIIbβ3 integrin receptor, but the interaction of the receptor with peptide motifs of Fbg remains unresolved. This paper describes the use of self-assembled monolayers (SAMs) to study the adhesion of αIIbβ3-transfected CHO cells to the GRGDS and HHLGGAKQAGDV motifs within Fbg. Cells adhered to and spread on monolayers presenting either peptide. Cell adhesion could be inhibited by either soluble peptide, demonstrating that the peptides bind competitively to the integrin. A peptide array was used to show that AGD was the minimal binding sequence in HHLGGAKQAGDV and that the receptor recognizes ligands of the form GXGDSC, where X is a hydrophobic or basic residue. This work revises our understanding of the αIIbβ3 specificity and also suggests a new class of antithrombotic agents. [Copyright &y& Elsevier]

This paper introduces a flexible assay for characterizing the activities of the histone deacetylase enzymes. The approach combines mass spectrometry with self-assembled monolayers that present acetylated peptides and enables a label-free and one-Step assay of this biochemical activity. The assay was used to characterize the activity of HDAC8 toward peptides taken from the N-terminal tail of the H4 histone and reveals that a distal region of the peptide substrate interacts with the deacetylase at an exosite and contributes to the activity of the substrate. Specifically, a peptide corresponding to residues 8-19 of H4 and having lysine 12 acetylated is an active substrate, but removal of the KRHR (residues 16-19) sequence abolishes activity. Mutation of glycine 11 to arginine in the peptide lacking the KRHR sequence restores activity, demonstrating that both local and distal sequences act synergistically to regulate the activity of the HDAC. Assays with peptides bearing multiply acetylated residues, but in which each acetyl group is isotopically labeled, permit studies of the processive deacetylation of peptides. Peptide substrates having an extended sequence that includes K20 were used to demonstrate that methylation of this residue directly affects HDAC8 activity at K12. This work provides a mechanistic basis for the regulation of HDAC activities by distal sequences and may contribute to studies aimed at evaluating the role of the histone code in regulating gene expression. [ABSTRACT FROM AUTHOR]

This paper reports a method that combines self-assembled monolayers with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to perform immunoassays on clinical samples. The immunosensors are prepared by immobilizing His-tagged protein G (or A) to a monolayer presenting the Ni2+ chelates, followed by immobilization of IgG antibodies with specificity for the intended analyte. The SAMDI mass spectrometry technique confirms the presence of the two proteins on the immunosensor and additionally provides a label-free analysis of antigens that bind to the sensor. This paper reports examples of detecting several proteins from human serum, including multianalyte assays that resolve each analyte according to their mass-to-charge ratio in the SAMDI spectra. An example is described wherein SAMDI is used to identify a proteolytic fragment of cystatin C in cerebral spinal fluids from patients diagnosed with multiple sclerosis. The SAMDI-TOF immunoassay, which combines well-defined surface chemistries for the selective and reproducible localization of analytes with mass spectrometry for label-free detection of analytes, may offer an alternative methodology to address many of the issues associated with standardized clinical diagnostics. [ABSTRACT FROM AUTHOR]

Methods for transducing the cellular activities of mammalian cells into measurable electronic signals are important in many biotechnical applications, including biosensors, cell arrays, and other cell-based devices. This manuscript describes an approach for functionally integrating cellular activities and electrical processes in an underlying substrate. The cells are engineered with a cell-surface chimeric receptor that presents the nonmammalian enzyme cutinase. Action of this cell-surface cutinase on enzyme substrate self-assembled monolayers switches a nonelectroactive hydroxyphenyl ester to an electroactive hydroquinone, providing an electrical activity that can be identified with cyclic voltammetry. In this way, cell-surface enzymatic activity is transduced into electronic signals. The development of strategies to directly interface the activities of cells with materials will be important to enabling a broad class of hybrid microsystems that combine living and nonliving components. [ABSTRACT FROM AUTHOR]

This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDOS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin α5 or anti-integrin β antibody. This work demonstrates unambiguously that PHSRN alone can support the attachment of cells and that the ROD and PHSRN bind competitively to the integrin receptors. [ABSTRACT FROM AUTHOR]

This paper describes the use of a substituent effects study to understand the mechanistic basis for an interfacial Diels -Alder reaction that does not proceed with standard second-order kinetics. Cyclopentadiene (Cp) undergoes a Diels-Alder reaction with a chemisorbed mercaptobenzoquinone to yield an immobilized Diels-Alder adduct. The pseudo-first-order rate constants are not linearly related to the concentration of diene, but they reach a limiting value with increasing concentrations of diene. The results of a substituent effects study support a mechanism wherein the electrochemical oxidation of hydroquinone produces two states of quinone. The first form, Q*, either reacts with Cp or isomerizes to 0, a form that is significantly less reactive with the diene. The interfacial reaction reaches a maximum rate when the concentration of diene is sufficiently high so that Q* undergoes complete Olels-Alder reaction and does not isomerize to Q. This work provides an example of the use of physical organic chemistry to understand an interfacial reaction. [ABSTRACT FROM AUTHOR]

Peptide arrays have attracted wide interest as tools for discovering biochemical interactions. Because many protein binding and enzyme activities are directed towards peptides, the preparation of arrays having hundreds to thousands of immobilized peptides offers an unprecedented opportunity for identifying interactions of proteins. This short opinion reviews recent progress in the preparation of peptide arrays and their use in the characterization of biomolecular interactions and the discovery of new reagents for biological researches. This body of work establishes the feasibility of this technology and suggests that it will find much wider use in research groups. [Copyright &y& Elsevier]

The adhesion of mammalian cells is mediated by the binding of cell-surface integrin receptors to peptide ligands from the extracellular matrix and the clustering of these receptors into focal adhesion complexes. This paper examines the effect of one mechanistic variable, ligand affinity, on the assembly of focal adhesions (FAs) in order to gain mechanistic insight into this process. This study uses self- assembled monolayers of alkanethiolates on gold as a substrate to present either a linear or cyclic Arg- Gly-Asp peptide at identical densities. Inhibition assays showed that the immobilized cyclic RGD is a higher affinity ligand than linear RGD. 3T3 Swiss fibroblasts attached to substrates presenting the cyclic peptide at twice the rate they attached to substrates presenting the linear peptide. Quantitation of focal adhesions revealed that cells on cyclic RGD had twice the number of FAs as did cells on linear RGD and that these focal adhesions were on average smaller. These findings show that affinity affects the assembly of integrins into focal adhesions and support a model based on competing rates of nucleation and growth of FAs to explain the change in distribution of FAs with ligand affinity. This study is important because it provides a model system that is well-suited for biophysical studies of integrin-mediated cell adhesion and reveals insight into one mechanism utilized by cells to perceive environmental changes. [ABSTRACT FROM AUTHOR]

Recent research has enhanced the development of substrates that serve as models of extracellular matrix and their use in studies of cell adhesion and migration. Advances include the development of methods to prepare substrates having ligands immobilized in controlled densities and patterns, and recent work that is developing dynamic substrates which can modulate, in real-time, the activities of ligands. These technologies are providing new opportunities for studies of cell–extracellular-matrix interactions. [Copyright &y& Elsevier]

Focuses on the technical challenges in the development of a protein-chip technology that matches the availability and performance of gene chips. Essential quality of protein chips to researchers in biology and to commercial programs in drug discovery and diagnostics; Survey of opportunities for immobilizing large numbers of proteins using detection methods.

This paper reports a chemical strategy for preparing carbohydrate arrays and utilizes these arrays for the characterization of carbohydrate-protein interactions. Carbohydrate chips were prepared by the Diels-Alder-mediated immobilization of carbohydrate-cyclopentadiene conjugates to self-assembled monolayers that present benzoquinone and penta(ethylene glycol) groups. Surface plasmon resonance spectroscopy showed that lectins bound specifically to immobilized carbohydrates and that the glycol groups prevented nonspecific protein adsorption. Carbohydrate arrays presenting ten monosaccharides were then evaluated by profiling the binding specificities of several lectins. These arrays were also used to determine the inhibitory concentrations of soluble carbohydrates for lectins and to characterize the substrate specificity of β-1,4-galactosyltransferase. Finally, a strategy for preparing arrays with carbohydrates generated on solid phase is shown. This surface engineering strategy will permit the preparation and evaluation of carbohydrate arrays that present diverse and complex structures. [Copyright &y& Elsevier]

Focuses on the relationship between the rate for an interfacial Diels-Alder reactions and the steric environment around the reacting molecules. Accessibility of quinone toward alkanethiols; Interface reaction with an enthalpy of activation; Lack of convenient analytical techniques.

Focuses on a study which outlined several strategies that are based on electrical, photochemical, thermal and mechanical forces for designing interfaces that are active and can signal changes in the behaviors of attached cells. Methods of surface modification; Comparison of transduction strategies; Applications for dynamic substrates.

Phosphorylation controls important cellular signals and its dysregulation leads to disease. While most phospho-regulation studies are focused on kinases, phosphatases are comparatively overlooked. Combining peptide arrays with SAMDI mass spectrometry, we show that tyrosine phosphatase activity is restricted by basic amino acids adjacent to phosphotyrosines. We validate this model using two β-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K). These mutants introduce a basic residue next to Y654, an established phosphorylation site where modification shifts β-catenin from cell–cell adhesions and towards its essential nuclear role as Wnt-signaling effector. We show that T653-basic mutant β-catenins are less efficiently dephosphorylated by phosphatases, leading to sustained Y654 phosphorylation and elevated Wnt signals, similar to those observed for Y654E phospho-mimic mutant mice. This model rationalizes how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6000+ potentially relevant cancer mutations. [ABSTRACT FROM AUTHOR]

Looks at factors which influence the efficiency of the solid-phase synthesis of peptide-substituted alkanethiols in the preparation of substrates which support cell adhesion. Role of ligands in the interactions of mammalian cells; Strategies required to protect alkanethiol; Details on the preparation of peptides.

Studies the sequence specificity of the natural product distamycin. Synthesis of pyridine-2-carboxamidonetropsin and 1-methylimidazole-2-carboxamidonetropsin; Equilibrium association constants for three peptides at four different binding sites; Use of a high-resolution footprinting reagent for accommodating closely-spaced binding sites; Methods for the alteration of relative specificity.

Describes a method based on microcontact printing and micromolding in capillaries to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. Components of this method; Self-assembled monolayers; Restriction of protein adsorption and cell attachment to the grooves in this substrate.

Examines the enhanced sequence specific recognition in the minor groove of DNA by covalent peptide dimers. Designed peptide binding to the minor groove of double-helical DNA; Enhancement of the binding affinity; Result of DNase I footprint titration experiments.

Discusses the distamycin A and 1-methylimidazole-2-carboxamine-netropsin peptides' simultaneous biding of the five-base-pair sequence 5'-TGTTA-3'. Importance of both molecules for binding the five-base-pair sequence; Binding of peptides as a side-by-side antiparallel heterodimer in the minor groove of double-helical DNA.

Explores the ability of capillary electrophoresis to rationalize charges and electrophoretic mobilities of insulin and its acylated derivative as a function of pH. Characterization of insulin; Acid constant values for two n-terminal ammonium groups; Values of the total charge of insulin; Concentration dependence of eletrophoretic mobility.

Reports on a method by which human and bovine capillary endothelial cells were switched from growth to apoptosis. Methods used; Results; Cell shape found to govern whether individual cells grow or die; Local geometric control of cell growth and viability; Mechanism for developmental regulation within the tissue microenvironment.

Studies a tripeptide containing alternating imidazole and pyrrole carboxamides and with specific binding abilities to six-base pair 5'-(A,T)GCGC(A,T)-3' sites in the minor groove of DNA. Binding patterns of peptides; Antiparallel orientation in minor groove; Recognition of a specific guanine amino group in the GCGC core through a hydrogen bond.

Provides information on a study describing the use of surface plasmon spectroscopy, to investigate the effect of surface wettability on the nonspecific adsorption of proteins, and detergents to self-assembled monolayers (SAMs) of alkanethiolates on gold. Methodology used to conduct the study; Information on surface plasmon resonance spectroscopy; Results of the study.

Describes the eight-step synthesis of the tripeptide ImPImP which contains alternating imidazole and pyrrole carboxamides, and the affinity cleaving analog ImPImP-EDTA-Fe (ImPImPE-Fe(II)). 2:1 peptide-DNA complexes; Synthetic methodology for ImPImP and ImPImPE; Footprinting; Affinity cleaving; Implications for the design of minor groove binding ligands.

Reports on a second generation peptide motif which offers a more general synthetic methodology for the preparation of a wide variety of covalent peptide dimers. Synthesis of hexapeptides; Binding affinities; Implications for design of minor-groove-binding molecules; Preparation of compounds.

The development of peptide and protein microarrays has created enormous opportunities in biomedical research. Current chip-based assays are well suited for identifying candidate protein or enzyme activities but still require conventional solution phase experiments to validate hits. Here, three surface-engineering strategies for microarray design are described and are illustrated in the development of a peptide chip for the quantitative analysis of kinase activity on solid support. These strategies promise to widen the application of microarrays by permitting the evaluation of hits in a chip-based format. [Copyright &y& Elsevier]

Accurate cancer detection and diagnosis is of utmost importance for reliable drug-response prediction. Successful cancer characterization relies on both genetic analysis and histological scans from tumor biopsies. It is known that the cytoskeleton is significantly altered in cancer, as cellular structure dynamically remodels to promote proliferation, migration, and metastasis. We exploited these structural differences with supervised feature extraction methods to introduce an algorithm that could distinguish cancer from non-cancer cells presented in high-resolution, single cell images. In this paper, we successfully identified the features with the most discriminatory power to successfully predict cell type with as few as 100 cells per cell line. This trait overcomes a key barrier of machine learning methodologies: insufficient data. Furthermore, normalizing cell shape via microcontact printing on self-assembled monolayers enabled better discrimination of cell lines with difficult-to-distinguish phenotypes. Classification accuracy remained robust as we tested dissimilar cell lines across various tissue origins, which supports the generalizability of our algorithm. [ABSTRACT FROM AUTHOR]

Modern organic reaction discovery and development relies on the rapid assessment of large arrays of hypothesis-driven experiments. The time-intensive nature of reaction analysis presents the greatest practical barrier for the execution of this iterative process that underpins the development of new bioactive agents. Toward addressing this critical bottleneck, we report herein a high-throughput analysis (HTA) method of reaction mixtures by photocapture on a 384-spot diazirine-terminated self-assembled monolayer, and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) analysis. This analytical platform has been applied to the identification of a single-electron-promoted reductive coupling of acyl azolium species. [ABSTRACT FROM AUTHOR]

Phosphorylation is an important post‐translational modification on proteins involved in many cellular processes; however, understanding of the regulation and mechanisms of global phosphorylation remains limited. Herein, we utilize self‐assembled monolayers on gold for matrix‐assisted laser desorption/ionization mass spectrometry (SAMDI‐MS) with three phosphorylated peptide arrays to profile global phosphatase activity in cell lysates derived from five mammalian cell lines. Our results reveal significant differences in the activities of protein phosphatases on phospho‐ serine, threonine, and tyrosine substrates and suggest that phosphatases play a much larger role in the regulation of global phosphorylation on proteins than previously understood. [ABSTRACT FROM AUTHOR]

The article discusses synthetic biology as the authors demonstrates throughput of the assay by characterizing the cell-free synthesis of HMG CoA, a key intermediate in the biosynthesis of isoprenoids, collecting over 10,000 individual spectra to map more than 800 unique reaction conditions. Topics include metabolic engineering, use of SAMDI mass spectrometry, and biosynthesis of isoprenoids.

The article focuses on the response of stem cells that are cultured in the laboratory differentiate to the mechanical properties of the substrate its form. It says that stem cells provide the possibility of repairing diseased or damaged tissues, such as mesenchymal stem cells (MSCs). It states that MSCs cultured on a nanostructured substrate preserve their multipotency for at least eight weeks. It adds that a study shows that cells that were spread differentiated into bone cells.

Recent advances in the synthesis of carbohydrates and in microarrays for profiling biochemical activities have set the stage for rapid advances in understanding the biological roles for oligosaccharides. An early example by Seeberger and colleagues illustrates the use of these tools in HIV infection . [Copyright &y& Elsevier]

Cell adhesion to the extracellular matrix (ECM) is integral to the survival and function of most cells. Cells use a wide range of receptors, which function by binding ligands in the substratum and transducing signals through the intracellular domain, to regulate functions as diverse as tissue maintenance, immune response, and development. Studies that address the roles for individual receptors are important for understanding these physiological processes but are often complicated by overlapping ligand specificities between receptors. The authors says that a strategy for "rewiring" the interactions between a cell and surface to create unique ligand-receptor bonds between a cell and ECM, but at the same time preserving the biological role of the receptor. This strategy is demonstrated by rewiring a cell-surface integrin receptor for adhesion to a synthetic ligand.

Focuses on the impact of spatial display of cellular ligands and receptors on cell adhesion and communication. Determination of the cell membrane receptors; Localization of mammalian cells; Impact of electrical potential on ligand.

Explores the Diels-Alder reaction for the selective immobilization of protein to electroactive self-assembled monolayers or monomolecular films. Reaction of self-assembled monolayers incorporating a quinone group with cyclopentadiene; Kinetic behavior for the Diels-Alder reaction of cyclopentadiene with quinone attached to a monolayer.

This paper describes the synthesis of giant cyclic molecules having diameters of 10-20 nm. The molecules are prepared through the reactions of a fusion protein building block with small molecule linkers that are terminated in irreversible inhibitors of enzyme domains present in the fusion. This building block has N-terminal cutinase and C-terminal SnapTag domains that react irreversibly with p-nitrophenyl phosphonate (pNPP) and benzylguanine (BG) groups, respectively. We use a bis-BG and a BG-pNPP linker to join these fusion proteins into linear structures that can then react with a bis-pNPP linker that joins the ends into a cyclic product. The last step can occur intramolecularly, to give the macrocycle, or intermolecularly with another equivalent of linker, to give a linear product. Because these are coupled first- and second-order processes, an analysis of product yields from reactions performed at a range of linker concentrations gives rate constants for cyclization. We determined these to be 9.7 x 10[sup -3] s[sup -1], 2.3 x 10[sup -3] s[sup -1], and 8.1 x 10[sup -4] s[sup -1] for the dimer, tetramer, and hexamer, respectively. This work demonstrates an efficient route to cyclic macromolecules having nanoscale dimensions and provides new scaffolds that can be generated using the megamolecule approach. [ABSTRACT FROM AUTHOR]

Emerging peptide array technologies are able to profile molecular activities within cell lysates. However, the structural diversity of peptides leads to inherent differences in peptide signal-to-noise ratios (S/N). These complex effects can lead to potentially unrepresentative signal intensities and can bias subsequent analyses. Within mass spectrometry-based peptide technologies, the relation between a peptide's amino acid sequence and S/N remains largely nonquantitative. To address this challenge, we present a method to quantify and analyze mass spectrometry S/N of two peptide arrays, and we use this analysis to portray quality of data and to design future arrays for SAMDI mass spectrometry. Our study demonstrates that S/N varies significantly across peptides within peptide arrays, and variation in S/N is attributable to differences of single amino acids. We apply supervised machine learning to predict peptide S/N based on amino acid sequence, and identify specific physical properties of the amino acids that govern variation of this metric. We find low peptide-S/N concordance between arrays, demonstrating that different arrays require individual characterization and that global peptide-S/N relationships are difficult to identify. However, with proper peptide sampling, this study illustrates how machine learning can accurately predict the S/N of a peptide in an array, allowing for the efficient design of arrays through selection of high S/N peptides. [ABSTRACT FROM AUTHOR]

Presents a successful second generation molecule, a covelant peptide heterodimer, which specifically binds the designated sequence 5'-TGTTA-3' of the double helical DNA. Structure of the double helical DNA; Previous models of the covalent peptide heterodimer.

Describes the use of microcontact printing for patterning self-assembled monolayers of alkylsiloxanes on the surface of silicon dioxide. Generating patterned self-assembled monolayers (SAM); Comparing properties of SAMs of octadecylsiloxane.

Cell-based assays are finding wider use in evaluating compounds in primary screens for drug development, yet it is still challenging to measure enzymatic activities as an end point in a cell-based assay. This paper reports a strategy that combines state-of-the-art cantilever free polymer pen lithography (PPL) with self-assembled monolayer laser desorption-ionization (SAMDI) mass spectrometry to guide cell localization and measure cellular enzymatic activities. Experiments are conducted with a 384 spot array, in which each spot is composed of ∼400 nanoarrays and each array has a 10 × 10 arrangement of 750 nm features that present extracellular matrix (ECM) proteins surrounded by an immobilized phosphopeptide. Cells attach to the individual nanoarrays, where they can be cultured and treated with small molecules, after which the media is removed and the cells are lysed. Phosphatase enzymes in the proximal lysate can then act on the immobilized phosphopeptide substrate to convert it to the dephosphorylated form. After the lysate is removed, the array is analyzed by SAMDI mass spectrometry to identify the extent of dephosphorylation and, therefore, the amount of enzyme activity in the cell. This novel approach of using nanopatterning to mediate cell adhesion and SAMDI to record enzyme activities in the proximal lysate will enable a broad range of cellular assays for applications in drug discovery and research not possible with conventional strategies. [ABSTRACT FROM AUTHOR]

Raman spectroscopic-based biosensing strategies are often complicated by low signal and the presence of multiple chemical species. While surface-enhanced Raman spectroscopy (SERS) nanostructured platforms are able to deliver high quality signals by focusing the electromagnetic field into a tight plasmonic hot-spot, it is not a generally applicable strategy as it often depends on the specific adsorption of the analyte of interest onto the SERS platform. This paper describes a strategy to address this challenge by using surface potential as a physical binding agent in the context of microneedle sensors. We show that the potential-dependent adsorption of different chemical species allows scrutinization of the contributions of different chemical species to the final spectrum, and that the ability to cyclically adsorb and desorb molecules from the surface enables efficient application of multivariate analysis methods. We demonstrate how the strategy can be used to mitigate potentially confounding phenomena, such as surface reactions, competitive adsorption and the presence of molecules with similar structures. In addition, this decomposition helps evaluate criteria to maximize the signal of one molecule with respect to others, offering new opportunities to enhance the measurement of analytes in the presence of interferants. [ABSTRACT FROM AUTHOR]

This work uses global gene expression analysis to compare the extent to which model substrates presenting peptide adhesion motifs mimic the use of conventional extracellular matrix protein coated substrates for cell culture. We compared the transcriptional activities of genes in cells that were cultured on matrix-coated substrates with those cultured on self-assembled monolayers presenting either a linear or cyclic RGD peptide. Cells adherent to cyclic RGD were most similar to those cultured on native ECM, while cells cultured on monolayers presenting the linear RGD peptide had transcriptional activities that were more similar to cells cultured on the uncoated substrates. This study suggests that biomaterials presenting the cyclic RGD peptide are substantially better mimics of extracellular matrix than are uncoated materials or materials presenting the common linear RGD peptide. [ABSTRACT FROM AUTHOR]