Edge spreading lithography and its application to the fabrication of mesoscopic gold and silver rings

A new form of edge lithography, edge spreading lithography (ESL), has been demonstrated and applied to the formation of coinage metal rings. In this process, alkanethiols are delivered from a flat PDMS stamp to the surface of a metal film through a two-dimensional array of spherical silica colloids. The thiols further spread on the metal surface, forming highly ordered SAMs in the form of a ring pattern. Following lift-off of beads, the pattern in the SAMs can be transferred into the metal film through wet chemical etching, with SAMs serving as the resist. The dimensions of the rings can be readily controlled by several parameters such as the beads diameter, the concentration of the thiol solution, and the contact time between the stamp and the silica beads.     

细管电泳-激光诱导荧光检测用于多个胞内蛋白激酶的抑制剂筛选和选择性评价

发展了一种基于毛细管电泳(CE)-激光诱导荧光(LIF)检测的多个细胞内源激酶的抑制剂平行筛选及选择性评价方法。CE高效的分离能力和LIF检测器的高选择性,使得同时测试多个胞内激酶的活性成为可能。共4种细胞系、3种特异性蛋白激酶底物肽、2种选择性蛋白激酶抑制剂和1种非选择性蛋白激酶抑制剂用于方法的建立。特异性底物肽与细胞裂解液混合后孵育,被其相应的激酶选择性地磷酸化,利用CE-LIF分离检测磷酸化产物和底物肽。同时测定一个抑制剂对几种蛋白激酶的抑制活性,用于评价抑制剂的选择性。与传统的单靶标筛选模式相比,这种基于细胞裂解液的多靶标筛选方法能提供更多的信息,更加高效,且细胞裂解液作为一种廉价的激酶来源大大降低了筛选成本。     

A Simple Method for Fabricating Patterned Curvilinear Microstructures in Poly(dimethylsiloxane) by Selective Wetting

The fabrication of patterned microstructures in poly(dimethylsiloxane) (PDMS) is a prerequisite for soft lithography. Herein, curvilinear surface relief microstructures in PDMS are fabricated through a simple three‐stage approach combining microcontact printing (μCP), selective surface wetting/dewetting and replica molding (REM). First, using an original PDMS stamp (first‐generation stamp) with linear relief features, a chemical pattern on gold substrate is generated by μCP using hexadecanethiol (HDT) as an ink. Then, by a dip‐coating process, an ordered polyethylene glycol (PEG) polymer‐dot array forms on the HDT‐patterned gold substrate. Finally, based on a REM process, the PEG‐dot array on gold substrate is used to fabricate a second‐generation PDMS stamp with microcavity array, and the second‐generation PDMS stamp is used to generate third‐generation PDMS stamp with microbump array. These fabricated new‐generation stamps are utilized in μCP and in micromolding in capillaries (MIMIC), allowing the generation of surface micropatterns which cannot be obtained using the original PDMS stamp. The method will be useful in producing new‐generation PDMS stamps, especially for those who want to use soft lithography in their studies but have no access to the microfabrication facilities.     

Microfabrication using elastomeric stamp deformation

Elastomeric stamp deformation has been utilized for the contact printing (CP) of self-assembled monolayers (SAMs) and, more recently, polymers and proteins. Here, we take advantage of this well-studied phenomenon to fabricate a series of new metal thin-film patterns not present on the original stamp. The rounded patterns are of nanoscale thickness, long-range order, and are created from elastomeric stamps with only straight-edged features. The metal was printed onto the surface of an alpha,omega-alkanedithiol self-assembled monolayer (SAM). The new shapes are controlled by a combination of stamp geometry design and the application of external pressure. Previously published rules on stamp deformation for contact printing of SAMs are invalid because the coating is instead a thin-metal film. This method represents a new pathway to micropatterning metal thin films, leading to shapes with higher complexity than the original lithographic masters.     

Letter: correlation between phosphorylation ratios by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis and enzyme kinetics

To identify the correlation between the phosphorylation ratios by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analysis and enzyme kinetics (K(m), V(max), and V(max)/K(m)) is important to understand whether MALDI-TOF MS can be applied for monitoring the properties of peptides that are substrates of protein kinases. The correlation between phosphorylation ratios and enzyme kinetics was examined using peptides for protein kinase C (PKC) and for 60 kDa phosphoprotein, encoded by the cellular sarcoma gene (c-Src). Phosphorylation ratios, analyzed by MALDI-ToF MS, showed higher correlation coefficient (r = > +0.7) for V(max)/K(m) compared with that (r = < -/+0.6) for K(m) or V(max). For ion modes, a higher correlation coefficient between phosphorylation ratios and V(max)/K(m) was identified in the positive mode (r = > +0.7) compared to that in the negative mode (r = < +0.5). These results suggest that MALDI-ToF MS is a useful tool to evaluate V(max)/K(m) of peptides for protein kinases.     

Patterning NHS-terminated SAMs on germanium

Here we report a simple, robust approach to patterning functional SAMs on germanium. The protocol relies on catalytic soft-lithographic pattern transfer from an elastomeric stamp bearing pendant immobilized sulfonic acid moieties to an NHS-functionalized bilayer molecular system comprising a primary ordered alkyl monolayer and a reactive ester secondary overlayer. The catalytic polyurethane-acrylate stamp was used to form micrometer-scale features of chemically distinct SAMs on germanium. The methodology represents the first example of patterned SAMs on germanium, a semiconductor material.     

MALDI‐MS Patterning of Caspase Activities and Its Application in the Assessment of Drug Resistance

Mass spectrometry (MS) has been widely used for enzyme activity assays. Herein, we propose a MALDI‐MS patterning strategy for the convenient visual presentation of multiple enzyme activities with an easy‐to‐prepare chip. The array‐based caspase‐activity patterned chip (Casp‐PC) is fabricated by hydrophobically assembling different phospholipid‐tagged peptide substrates on a modified ITO slide. The advantages of amphipathic phospholipids lead to high‐quality mass spectra for imaging analysis. Upon the respective cleavage of these substrates by different caspases, such as caspase‐1, ‐2, ‐3, and ‐8, to produce a mass shift, the enzyme activities can be directly evaluated by MALDI‐MS patterning by m/z‐dependent imaging of the cleavage products. The ability to identify drug‐sensitive/resistant cancer cells and assess the curative effects of anticancer drugs is demonstrated, indicating the applicability of the method and the designed chip.     

Rapid evaluation and screening of interfacial reactions on self-assembled monolayers

This paper reports 16 chemical reactions for elaborating the structures of self-assembled monolayers (SAMs) of alkanethiolates on gold. This work takes advantage of matrix-assisted laser desorption/ionization and time-of-flight mass spectrometry (MALDI-TOF MS) to rapidly characterize the products and yields of reactions that occur with molecules attached to monolayers. The paper also describes a method for screening reaction conditions, wherein monolayers are treated with an array of reactants and mass spectrometry is used to identify those regions that undergo reactions to give new, and unanticipated, products in high yield. These examples serve to increase the collection of reactions that can be used to elaborate the structures, and therefore the properties, of self-assembled monolayers of alkanethiolates on gold and to introduce label-free methods for screening interfacial reactions.     

An Adaptor Domain‐Mediated Autocatalytic Interfacial Kinase Reaction

This paper describes a model system for studying the autocatalytic phosphorylation of an immobilized substrate by a kinase enzyme. This work uses self‐assembled monolayers (SAMs) of alkanethiolates on gold to present the peptide substrate on a planar surface. Treatment of the monolayer with Abl kinase results in phosphorylation of the substrate. The phosphorylated peptide then serves as a ligand for the SH2 adaptor domain of the kinase and thereby directs the kinase activity to nearby peptide substrates. This directed reaction is intramolecular and proceeds with a faster rate than does the initial, intermolecular reaction, making this an autocatalytic process. The kinetic non‐linearity gives rise to properties that have no counterpart in the corresponding homogeneous phase reaction: in one example, the rate for phosphorylation of a mixture of two peptides is faster than the sum of the rates for phosphorylation of each peptide when presented alone. This work highlights the use of an adaptor domain in modulating the activity of a kinase enzyme for an immobilized substrate and offers a new approach for studying biochemical reactions in spatially inhomogeneous settings.     

Design,characterization, and evaluation of peptide arrays allowing the direct monitoring of MMP activities

Characterization of matrix metalloprotease (MMP) activities is of increasing interest for cancer prognosis or treatment follow-up. Indeed, MMP-1, -2 and -9 are widely involved in the growth of many tumors and progression steps such as angiogenesis, invasion, and metastasis. Fluorogenic peptide MMP substrates were previously synthesized with the aim of detecting MMP activities. One of their drawbacks is their limited solubility in biological media. Grafting them onto a solid support represented a novel way to yield efficient analysis devices whilst at the same time decreasing the quantities of peptides used. Novel peptide arrays were designed in order to detect MMP activities in biological fluids. Silicon plates were used as the solid support for the design of these novel tools. These were functionalized by organic self-assembled monolayers (SAMs) on which fluorogenic peptides were covalently coupled. SAM and peptide grafting on silicon plates were confirmed by epifluorescence, ellipsometry, and FT-IR analysis. Enzymatic assays were monitored by fluorescence spectrometry and showed that immobilized linear peptides were recognized and cleaved by MMPs.     

The Characterization of Self-Assembled Monolayers on Copper Surfaces by Low-Temperature Plasma Mass Spectrometry

We describe direct analysis of self-assembled monolayers (SAMs) on copper surfaces by low temperature plasma (LTP) mass spectroscopy (MS). Two kinds of SAMs formed from n-dodecylmercaptan (NDM) and l-phenyl-5-mercaptotetrazole (PMTA) were prepared on copper by spontaneous chemisorption. With the LTP probe, desorption and ionization of the SAMs was easily achieved, and the ions produced were introduced into MS for analysis. Characteristic fragment ions from NDM SAMs, mainly [M + M - H](+) (M is the NDM molecule) and from PMTA SAMs, mainly [M + H - S](+) (M is the PMTA molecule), were both absent in the MS spectra of neat NDM and PMTA samples. This provided evidence of the formation of SAMs on copper. As a supplementary method, LTP-MS is helpful in obtaining information on the barrier properties of SAMs on copper, such as inhibitor efficiency (IE) and the surface adsorption concentration of corrosive electrolyte (Γ*) surrounding copper. Aiming for an evaluation of the reliability of LTP-MS, a comparative study of our method and the traditional method of cyclic voltammetry (CV) showed a correlation coefficient higher than 0.97. In addition, a rough, simple procedure for imaging of the distribution of the molecules adsorbed on copper surface was presented. The study supplied a rapid and simple method for direct investigation of SAMs on copper.     

β2-微球蛋白连续表位的免疫亲和质谱研究

采用Endoproteinase Glu-C, Lys-C和Trypsin 3种蛋白酶分别水解β2-微球蛋白, 产生一系列肽段, 利用固定在琼脂糖珠上的单克隆抗体与其发生免疫亲和反应. 利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术, 对抗原决定簇肽段-抗体复合物进行系统研究, 结果表明, 与抗体结合部位即连续表位的位点为肽段(59~69)(DWSFYLLYYTE). 该研究方法简便、准确, 可用来对其它抗原连续表位的快速测定.     

Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin

Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin‐IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin‐ IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.     

MALDI-TOF MS detection of dilute, volume-limited peptide samples with physiological salt levels

This paper presents a highly efficient sample preparation technique for matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The purpose of the research is to use a conventional MALDI support to directly and conveniently detect sub-nM levels of peptides from volume-limited samples with physiological salt levels. In this new method, highly uniform matrix-nitrocellulose spots with a 500 microm diameter were conveniently generated by direct contact of a capillary tip to a stainless steel MALDI plate. An array of 50 microspots can be blotted from 1 microL matrix-nitrocellulose solution within 1 min. It was found that the addition of high concentration nitrocellulose to the alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution is critical for the formation of microspots. Samples are deposited on top of those microspots and incubated for 3 min. The CHCA-nitrocellulose surface shows a significant peptide binding capability for sub-nM levels of peptide. Restricting the matrix spot diameter to 500 microm gives an analyte enrichment effect because the peptides are confined to a small solid-phase surface area. Selective peptide binding is seen even with >0.15 M salt levels. Loading small aliquots of samples with multiple applications allows low level peptide detection down to 100 pM. Push-pull perfusates collected from the rat striatum were successfully analyzed with the microspot method.     

Improved peptide sequencing using isotope information inherent in tandem mass spectra

We demonstrate here the use of natural isotopic 'labels' in peptides to aid in the identification of peptides with a de novo algorithm. Using data from ion trap tandem mass spectrometric (MS/MS) analysis of 102 tryptic peptides, we have analyzed multiple series of peaks within LCQ MS/MS spectra that 'spell' peptide sequences. Isotopic peaks from naturally abundant isotopes are particularly prominent even after peak centroiding on y- and b-series ions and lead to increased confidence in the identification of the precursor peptides. Sequence analysis of the MS/MS data is accomplished by finding sequences and subsequences in a hierarchical manner within the spectra.     

Click chemistry by microcontact printing on self-assembled monolayers: a structure-reactivity study by fluorescence microscopy

Microcontact printing (μCP) has developed into a powerful tool to functionalize surfaces with patterned molecular monolayers. μCP can also be used to induce a chemical reaction between a molecular ink and a self-assembled monolayer (SAM) in the nanoscale confinement between stamp and substrate. In this paper, we investigate the Huisgen 1,3-dipolar cycloaddition, the Diels-Alder cycloaddition and the thiol-ene/yne reaction induced by μCP. A range of fluorescent alkyne inks were printed on azide SAMs and fluorescence microscopy was used to monitor the extent of the 1,3-dipolar cycloaddition on a glass substrate. The rate of cycloaddition depends on the reactivity of the alkyne and on the presence of Cu(I). The cycloaddition is accelerated by Cu(I) but it also proceeds readily in the absence of Cu(I). In addition, a range of fluorescent diene inks were printed on alkene SAMs on glass. In this case, fluorescence microscopy was used to monitor the rate of the Diels-Alder cycloaddition as well as its retro-reaction. Finally, fluorescent thiol inks were printed on alkene SAMs on glass, and fluorescent alkenes and alkynes were printed on thiol SAMs. It is shown that reactions by μCP follow structure-reactivity relationships similar to solution reactions. Under optimized conditions all reactions lead to dense microarrays of addition products within minutes of printing time.     

Acoustic deposition with NIMS as a high-throughput enzyme activity assay

Mass spectrometry (MS)-based enzyme assay has been shown to be a useful tool for screening enzymatic activities from environmental samples. Recently, reported approaches for high-specificity multiplexed characterization of enzymatic activities allow for providing detailed information on the range of enzymatic products and monitoring multiple enzymatic reactions. However, the throughput has been limited by the slow liquid-liquid handling and manual analysis. This rapid communication demonstrates the integration of acoustic sample deposition with nanostructure initiator mass spectrometry (NIMS) imaging to provide reproducible measurements of multiple enzymatic reactions at a throughput that is tenfold to 100-fold faster than conventional MS-based enzyme assay. It also provides a simple means for the visualization of multiple reactions and reaction pathways.     

Patterning functional materials using channel diffused plasma-etched self-assembled monolayer templates

A simple and cost-effective methodology for large-area micrometer-scale patterning of a wide range of metallic and oxidic functional materials is presented. Self-assembled monolayers (SAM) of alkyl thiols on Au were micropatterned by channel-diffused oxygen plasma etching, a method in which selected areas of SAM were protected from plasma oxidation via a soft lithographic stamp. The patterned SAMs were used as templates for site-selective electrodeposition, electroless deposition and solution-phase deposition of functional materials such as ZnO, Ni, Ag thin films, and ZnO nanowires. The patterned SAMs and functional materials were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), atomic force microscopy (AFM), and tunneling AFM (TUNA).     

Preparation and functions of self‐assembled monolayers of helix peptides

Self‐assembled monolayers (SAMs) of helix peptides oriented vertically to a gold surface were prepared. Negative surface potentials of a few hundred millivolts were observed for the helix peptide SAMs when they were immobilized on gold through the N terminal of the peptides. However, positive surface potentials were generated in the helix peptide SAMs when the N terminal of the peptides was directed the opposite way. The large dipole moment of the helical peptide was thought to be the major factor for generation of the surface potential. The effect of the dipole moment on the electron transfer through the helix peptide SAMs was investigated. Photocurrent generation by photoexcitation of the N‐ethylcarbazolyl group of the peptide SAMs was accelerated by the dipole moment directed toward the gold substrate. Helical peptides were thus shown to be a suitable medium for electron transfer. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 4826–4831, 2000     

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.