Micro-Nano Technology for Genomics and Proteomics BioMEMs - Ozkan
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FIGURE 7.18. Photolithography for patterning of surface free energies [613]. i = photomask placed on top of glass surface coated with photolabile self-assembled monolayer, ii = UV light (filter cube with band pass of 360 to 370 nm) in the presence of aqueous NaOH pH 11.7 yielding hydrophobic (A) and hydrophilic (B) functionalized regions.
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ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI |
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FIGURE 7.19. Generation of patterned surfaces. A = hydrophobic surface; B = hydrophilic surface; i = deposition of photoresist, irradiation and generation of exposed glass surface areas; ii = (tridecafluoro-1,1,2,2- tetrahydro)trichlorosilane in anhydrous toluene; iii = 3-aminopropyltrimethoxysilane in anhydrous toluene; iv = succinic anhydride.
PEPTIDE ARRAYS IN PROTEOMICS AND DRUG DISCOVERY |
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FIGURE 7.20. Generation of surface tension arrays [70]. A = hydrophobic surface; B = hydrophilic surface; i = 0.4% solution of 3-aminopropyldimethylethoxysilane in anhydrous toluene, 72 h; ii = coating with positive photoresist (2.5 µm) and exposure to near UV through a chromium-on-quartz mask followed by removal of the exposed photoresist and oxygen plasma treatment; iii = 0.25% solution of (tridecafluoro-1,1,2,2-tetrahydro)trichlorosilane in anhydrous toluene, 10 min, removal of residual photoresist.
removal of all the permanent protecting groups gives appropriate target peptides tethered to the surface via the C-terminal carboxylic acid function. Incomplete coupling/deprotection reactions during the peptide assembly result in a target peptide contaminated by a variety of deletion and truncation sequences.
7.2.3.1. Stepwise Synthesis on Coherent Surfaces In principle, in situ synthesis has a number of advantages compared to immobilization of pre-synthesized peptides. Normally, yields of peptide synthesis on surfaces are high and consistent over the entire support surface from one array region to another. It also permits combinatorial strategies for constructing large arrays of peptides in a few coupling steps. Several approaches have been used to
186 |
ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI |
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FIGURE 7.21. Generation of surface tension arrays [61]. A = hydrophobic surface; B = hydrophilic surface, X = oxygen; i = 3-(1,1-dihydroperfluoroctyloxy)propyltriethoxysilane; ii = CO2-laser for ablation of fluorosiloxane and exposure of glass surface; iii = glycidyloxypropyltriethoxysilane.
allow spatially addressed peptide synthesis on coherent surfaces and membranes for in situ fabrication of arrays.
Fodor and coworkers described a method for synthesizing large numbers of peptides bound to a planar, solid support by combining the techniques of solid phase peptide
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FIGURE 7.22. Principle of peptide synthesis on coherent surfaces. i = acylation of surface bound amino functions using appropriate side chain (permanent protecting group) and N-terminal protected (temporary protecting group [PG]), carboxyl-activated amino acid derivatives; ii = removal of PG; iii = repeated cycles of acylation and deprotection reactions followed by removal of all permanent protecting groups, yielding the surface bound target peptide sequence contaminated with impurities resulting from incomplete acylation or deprotection reactions (deletion and truncation sequences).
synthesis, photolabile protection, and photolithography, known as light-directed peptide synthesis (LDPS) or Very Large Scale Immobilized Polymer Synthesis [84, 151, 168, 324].
Here, a fraction of sites on a planar support carrying photo-detachable protecting groups, such as nitroveratryl-oxy-carbonyl (Figure 7.23), is exposed to light through a photolithographic mask. The fraction of sites thus deprotected is acylated with a specific
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FIGURE 7.23. Chemical principle enabeling photolithografic patterning of aminosilylated glass surfaces. i = N-(nitroveratryl-oxy-carbonyl)-6-amino hexanoic acid N-hydroxysuccinimide ester; ii = UV-light (365 nm).
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FIGURE 7.24. Chemical principle of photolithografic oligocarbamate synthesis [84]. i = UV-light (365 nm), ii = repetitive cycles of nitrophenylcarbamate treatment and UV-light mediated deprotection of amino groups.
amino acid derivative or building block, itself carrying a photo-detachable protecting group (Figure 7.24). The photo-deprotection is repeated with the mask in a different position, or with a different mask, and a second amino acid derivative or building block is attached either to the functionalized support and/or to the first amino acid residue. After several cycles of acylation and deprotection, with careful attention to the pattern of masking, an array of peptides is built up on the planar support. The final peptide microarray is completely deprotected and exposed to the ligand of interest. Additionally, this technology could be successfully used for the generation of arrays of peptidomimetics such as oligocarbamates (Figure 7.24) [84]. However, this technology has some serious disadvantages. Light-directed peptide synthesis involves a novel set of chemistries, which have to be optimized otherwise the final quality of the surface-bound peptides will cause false positive (if an impurity is active) and/or false negative results (if the target peptide sequence was not synthesized). An interesting alternative to circumvent this limitation is the use of photo-generated acids in combination with Boc-chemistry [171, 265, 309,
PEPTIDE ARRAYS IN PROTEOMICS AND DRUG DISCOVERY |
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406]. Nevertheless, the use of photolithografic masks combined with solid phase peptide synthesis is relatively labor intensive. Related techniques eliminating the mentioned disadvantages associated with photolithografic masks use LED-arrays [174], laser scanning by mirror-arrays [514] or a computer controlled micro-mirror projector [171, 309]. This latter method involves deprotecting part of a surface coated with an amino acid carrying a photo-detachable protecting group by a laser printer beam such as a HeCd laser. Spatial control of the laser beam is accomplished by reflecting the beam from a spinning mirror and employing a shutter to exclude the beam if desired. This programmable laser-activated parallel peptide synthesis permits automated synthesis of immobilized peptide libraries, avoiding the physical scale limitations of photolithography, but does not circumvent the disadvantage of using amino acids with photo-detachable protecting groups. Moreover, there are 20 separate deprotection steps necessary for each position to be varied within a peptide sequence. For example, the synthesis of all possible penta-peptides using the 20 natural amino acids (3.2 million peptides) requires 100 separate deprotection, washing and acylation steps.
A further approach involves physically locating the activated monomer by contacting a confined area of the functionalized surface with monomer solution using masks or physical barriers as demonstrated for the synthesis of oligonucleotides [351]. This allows the synthesis of complex microarrays comprising many different but related sequences within a few coupling steps by combinatorial methods. Flooding the activated monomers through intersecting micro-channels yielded arrays of all sequences of a chosen length [352, 521] but generating circular or diamond shaped reaction chambers by sealing an appropriate shaped mask against a functionalized surface such as glass or aminated polypropylene mounted on glass yielded so-called scanning or tiling-path arrays ([129]; reviewed in [522]).
A very elegant form of spatially addressed compound deposition makes use of modified color laser printers. The cartridges are filled with a solvent/amino acid derivative mixture (high melting point of the solvent yields toner-like powder) resulting in an activated amino acid solution during the laser induced melting process [428].
The use of ink-jet or bubble-jet technologies for the drop-on-demand liquid handling during stepwise oligonucleotide synthesis on glass microscope slides [46, 222, 541] or generation of combinatorial libraries on functionally graded ceramics [368, 369] is described. Analogous ink-jet delivery of activated amino acids to appropriate functionalized surfaces, such as membranes, microscope slides or spinning surfaces in a CD-format [4] for automated synthesis of peptides, have been developed by a number of companies, but is not yet commercially available.
A very simple but extremely robust method for the highly parallel synthesis of peptides on planar surfaces is the SPOT synthesis concept developed by R. Frank [153, 155] and commercialized by the company Jerini AG (www.jerini.com). This method is very flexible and economic relative to other techniques and was recently developed from a semi-automatic procedure [157] to a fully automated system (www.jerini.com). The basic principle involves the spatially addressed deposition of defined volumes of activated amino acid derivatives (or oligopeptides) directly onto a planar surface such as functionalized cellulose (Figures 7.25–7.27), aminated polypropylene or aminopropylsilylated glass slides. The areas contacted by the droplets represent individual micro-reactors allowing the formation of a covalent bond between the amino acid derivative and the surface function. The
190 ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI
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FIGURE 7.25. Amino derivatization of cellulose. i = Fmoc-glycine (n = 1) or Fmoc-6-aminohexane carboxylic acid (n = 5) /TBTU/DIPEA; ii = 20% piperidine in DMF; iii = N-phthaloyl-oxiranyl-methylamine, iv = hydrazine treatment.
resulting spot size is defined by the dispensed volume as well as the physical properties of the surface used. This SPOT synthesis has been reviewed extensively [158, 160–162, 273, 274, 443, 453, 454, 592]. Recent developments such as the introduction of novel polymeric surfaces [591], new linker and cleavage strategies [16, 320, 573] as well as automation (allowing the fully automated synthesis of up to 25,000 peptides within one run) have increased the value of this technique and led to the extension of SPOT synthesis to other molecule classes such as protein domains [546–548], peptide nucleic acids [356, 583], peptomers [16, 617], peptoids [16, 591, 202] and small heterocyclic compounds [488, 490]; see Section 7.5.5). Normally, peptides or peptidomimetics on arrays prepared by SPOT synthesis are assembled in a stepwise manner. For longer peptides this is a laborious and time-consuming procedure. Toepert and coworkers synthesized an oligopeptide array of several thousand triple-substituted variants of the human YAP-WW domain by a combination of classical stepwise SPOT synthesis and native chemical ligation [546–548]. This is one example of how to extend the concept of peptide arrays to arrays of chemically synthesized protein domains. Additionally, SPOT synthesis allows the use of defined amino acid mixtures for acylation reactions leading to the array of libraries concept. The problem of equal amounts of each library member could be circumvented by using either kinetically adjusted mixtures or sub-stoichiometric amounts of amino acid derivatives [269].
It is generally difficult to assess the quality of the peptides made on and bound to a planar surface. The amount of peptide is small for most materials used (Table 7.1). However, analysis of peptides synthesized step by step on cleavable linkers suggests a relatively
PEPTIDE ARRAYS IN PROTEOMICS AND DRUG DISCOVERY |
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FIGURE 7.26. Amino derivatization of cellulose. i = 2-bromomethyl-oxirane; ii = diamino-alkane treatment; iii = 4,7,10-trioxa-1,13-tridecanediamine, iv = 4-[4-(1-aminoethyl)-2-methoxy-5-nitro-phenoxy]-butyric acid /TBTU/DIPEA.
high quality (Wenschuh et al., 2000). Non-destructive measurements can be made by IRspectroscopy, ellipsometry or interferometry [183].
In contrast, pre-synthesized peptides can be assessed before they are attached onto the surface, allowing for quality control. When large numbers of peptide arrays with the same sequences are needed, deposition of pre-synthesized peptides is more economical than in situ synthesis. Deposition is also the method of choice for long peptide sequences, which normally have to be purified to obtain high quality products (see Section 7.2.3.3.).
7.2.3.2. Non-Selective Immobilization of Peptides Non-specific immobilization has the advantage that no specific modification of the peptides is necessary. Additionally, the immobilized peptides within one spot are displayed as a mixture of differently attached molecules, reducing the probability of wrongly presented molecules. On the other hand, non-selective immobilization could prevent effective interaction with the screening probe
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FIGURE 7.27. Linker structure used for the automated SPOT-synthesis of PNA oligomers allowing the parallel synthesis and subsequent removal of individual sequences by treatment with either acid or protease trypsin [583]. i = Fmoc-6-aminohexanioc acid/DIC/HOAt/NMP, 20% piperidine/DMF, Fmoc- 6-aminohexanioc acid/DIC/HOAt/NMP, 20% piperidine/DMF; ii = Fmoc-Glu(OtBu)-OH/ DIC/HOAt/NMP, 20% piperidine/DMF, Fmoc-Lys(Boc)-OH/DIC/HOAt/NMP, 20% piperidine/DMF; iii = Fmoc-{4-[amino-(2,4- dimethoxyphenyl)-methyl]-phenoxy}-acetic acid /DIC/HOAt/NMP, 20% piperidine/DMF.
if the functional group of the peptide used for the immobilization chemistry also represents a key residue.
Several non-selective chemistries have already been described for peptide array applications. In most cases the amino functions of the peptides (free N-terminus or lysine side chains) are targeted by surfaces carrying acid halides, active esters, isocyanates, isothiocyanates, activated double bonds, aldehydes or epoxides. Surface coatings with cyanuric chloride as described for immobilization of synthetic polynucleotides (Lee et al., 2002)