Micro-Nano Technology for Genomics and Proteomics BioMEMs - Ozkan

PEPTIDE ARRAYS IN PROTEOMICS AND DRUG DISCOVERY

173

either the initiation of peptide synthesis or the immobilization of peptides. It was often demonstrated that the surface-bound peptides’ accessibility to the proteins or enzymes used in screening is a critical factor. Insertion of a spacer between the peptide and the surface is an effective way to circumvent this drawback (Figure 7.11B). Generally, all linker molecules introduced to transform a given surface function into a functional group suitable for amino acid or peptide attachment can be considered as spacers. Such spacers can improve the efficiency of peptide/ligand interaction on surfaces as demonstrated with FLAG epitope peptides recognized by the monoclonal anti-FLAG M2 antibody [581]. The signal increased with the length of spacer introduced between the epitope and the surface. Additionally, for protein tyrosine kinase p60c-src it was demonstrated that only incorporation of the long and hydrophilic 1-amino-4,7,10-trioxa-13-tridecanamine succinimic acid building block spacer allowed effective phosphorylation of the glass surface-bound peptides [143]. Moreover, insertion of hydrophilic dextran structures between the surface and the presented peptides (Figures 7.12 and 7.13) was described as necessary for efficient enzyme substrate interaction [337].

The loading of glass surfaces is too low for several biochemical reactions (Tables 7.1, 7.2). Several different approaches for generating dendrimeric spacers/linkers are used to increase the density of immobilized peptides (Figure 7.11C, 7.12–7.16). Alternatively, the effective surface per area can be increased by the use of porous silicon [306, 347, 462]. Examples for one-step generation of dendrimeric structures (Figure 7.11C) are represented by poly-lysine coated glass slides (Table 7.2) or aziridine polymerization onto aminopropylsilylated glass surfaces (Figure 7.16) [85, 257, 388]. An interesting approach for increasing the density of reactive functions is surface modification by adsorption of structured α-helical peptides [332] or proteins (bovine serum albumin, BSA; [338]) yielding amino modified polymers. The BSA amino functions were transformed into active esters by treatment with N,N-disuccinimidyl carbonate (Figure 7.15).

More sophisticated procedures for preparing multiple linker structures by multistep functionalization of glass surfaces have been reported (Figures 7.14, 7.17) [31, 33, 197, 242, 258, 302, 410]). Alternatively, surface loading could be improved by coatings employing three-dimensional layers such as hydrogels (Rubina et al., 2003), semi-wet gels [258], agarose films [5], acrylamide gel pads [367, 430, 565] or gelatine pads [136].

7.2.2. Generation of Micro-Structured Surfaces

Micro-structured or micro-patterned surfaces represent an alternative way to achieve spatially addressed deposition of molecules. This could be useful for generating microreactors [604] or improving array regularity. Two major principles are used for micropatterning: contact printing techniques (see Section 7.2.4.1.) and photolithografic technologies. The use of micro-contact printing for micro-patterning [36, 107, 225, 226, 240, 283, 284, 349, 507] has been reviewed extensively [60, 365, 366, 512, 601, 602] and is therefore not discussed in detail. Dip-Pen nanolithography/scanning probe lithography [51, 67, 249, 256, 302, 385, 519, 577, 580, 598] and microfluidic channel networks [83, 103, 105, 398] were used for nanoand micro-patterned immobilization of biomolecules such

174	ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI

i

O

O

OH

O

O

HN

O	O
ii
H2N	O
O	N
HO	H

iii

O

OH

O

OH

O

OH

OO

O	OH	O
OH		OH
	O	O	O

HOH

N O

O N

H

O

O

iv

OH

O

OH

O

OH

OO

O	OH	O
NH2 HN		OH
	O	O	O

HOH

N O

O N

H

NH2 O

FIGURE 7.12. Transformation of porous polypropylene into hydrophilic matrix suitable for enzyme profiling [337]. i = oxidation using Cr2O3/H2SO4/H2O, 54:54:80 (wt,wt,wt), ii = 5% oxalylchloride in CH2Cl2, 2 h, room temperature followed by treatment with 10% 2,2’-(ethylenedioxy)diethylamine in CH2Cl2, iii = carboxymethylated dextran, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in water, 12 h, room temperature, iv = carbonyl diimidazole, acetonitrile, 30 min, 0.3 M diaminopropane in acetonitrile, 1 h.

TABLE 7.2. Selected suppliers of modified surfaces

surface function	world wide web	dimension [mm]		material
amino	www.aims-scientific-products.de	80	× 120	cellulose
amino		100	× 150	cellulose
amino		235	× 250	cellulose
streptavidin		100	× 150	cellulose
amino		240	× 240	polypropylene
amino		80	× 120	polypropylene
amino		240	× 240	polypropylene
amino		80	× 120	polypropylene
bromide		100	× 150	cellulose
carboxyl		100	× 150	cellulose
Fmoc-ß-Alanine		100	× 150	cellulose
Fmoc-Proline		100	× 150	cellulose
mercapto		100	× 150	cellulose
hydroxyl		240	× 240	polypropylene
hydroxyl		80	× 120	polypropylene
hydroxyl		240	× 240	polypropylene
hydroxyl		80	× 120	polypropylene
epoxy	www.quantifoil.com	75	× 25	glass
aldehyde		75	× 25	glass
epoxy	www.noabdiagnostics.com	75	× 25	glass
aldehyde		75	× 25	glass
NHS ester		75	× 25	glass
aldehyde	www.aat-array.com	75	× 25	glass
amino	www.sigmaaldrich.com	75.5	× 25	glass
poly-L-lysine		75.5	× 25.5	glass
carboxyl	www.nuncbrand.com	76	× 25	heat stable polymer
amino		76	× 25	glass
epoxy		76	× 25	glass
aldehyde		76	× 25	glass
amino	www.eriesci.com	n.a.		glass
poly-lysine		n.a.		glass
epoxy		n.a.		glass

loading

400 nmol/ cm2

400 nmol/ cm2

400 nmol/ cm2

2 nmol/ cm2

80 nmol/ cm2

80 nmol/ cm2

600 nmol/ cm2

600 nmol/ cm2

800 nmol/ cm2

500 nmol/ cm2

800 nmol/ cm2

600 nmol/ cm2

600 nmol/ cm2

150 nmol/ cm2

150 nmol/ cm2

1500 nmol/ cm2

1500 nmol/ cm2 n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

(cont.)

DISCOVERY DRUG AND PROTEOMICS IN ARRAYS PEPTIDE

175

TABLE 7.2. Continued

surface function	world wide web		dimension [mm]	material	loading
amino	www.arrayit.com	76	× 25	glass	8 pmol/cm2
amino		76	× 25	glass	8 pmol/cm2
aldehyde		76	× 25	glass	8 pmol/cm2
poly-L-Lysine		76	× 25	glass	n.a.
acrylic		76	× 25	glass	8 pmol/cm2
carboxyl		76	× 25	glass	8 pmol/cm2
cyanato		76	× 25	glass	8 pmol/cm2
epoxy		76	× 25	glass	8 pmol/cm2
mercapto		76	× 25	glass	8 pmol/cm2
amino	www.xenopore.com	75	× 25 or 22 × 40	glass	16 pmol/cm2
aldehyde		75	× 25 or 22 × 40	glass	16 pmol/cm2
epoxy		75	× 25 or 22 × 40	glass	16 pmol/cm2
maleimide		75	× 25 or 22 × 40	glass	16 pmol/cm2
nitrilotriacetic acid		75	× 25 or 22 × 40	glass	n.a.
streptavidin		75	× 25 or 22 × 40	glass	n.a.
biotin		75	× 25 or 22 × 40	glass	n.a.
mercapto		75	× 25 or 22 × 40	glass	16 pmol/cm2
amino	www.corning.com/lifesciences	75.5 × 25.3		glass	n.a.
substituted antraquinones	www.exiqon.com	75	× 25	injection molded polymer	n.a.
amino	www.greinerbioone.com	75	× 25	glass	n.a.
aldehyde				glass	n.a.
streptavidin			× 25 or 24 × 60	glass	n.a.
epoxy	www.asperbio.com	75		glass	n.a.
amino		75	× 25 or 24 × 60	glass	n.a.
isothiocyanate		75	× 25 or 24 × 60	glass	n.a.
epoxy	www.genescan.com	75.7 × 25.4		glass	16 pmol/mm2
amino	www.perkinelmer.com/	75	× 25	glass coated with modified acrylamide	n.a.
	areas/proteomics/chem2.asp	active surface: 12 × 40 or		polymer
		12	× 12 (two pad)

176

SCHUTKOWSKI MIKE AND MERGENER-SCHNEIDER JENS REINEKE, ULRICH

epoxy	www.mwgbiotech.com	75	× 25	glass	n.a.
mercapto	www.ApogentDiscoveries.com	75	× 25	glass	n.a.
animo	www.csem.ch	75	× 25	glass	n.a.
carboxyl		22	× 20 available for print	metal oxides	4.2 pmol/mm2
maleimide				metal sulfides	3.4 pmol/mm2
mercapto				silicon
				silicon nitride
		75.5 × 25		plastics
amino	www.us.schott.com			glass	n.a.
isothiocyanate	www.picorapid.de	76	× 26	glass	n.a.
amino-reactive	www.ucb-group.com	75	× 25	glass	n.a.
		variable size		cellophane	n.a.
		variable size		polypropylene	n.a.
amino	[e-mail]	120 × 80		highly crosslinked, polyaminated,	15 nmol/mm2
			× 25 (slide)	polyurea coated polypropylene fleece
amino	www.pall.com	75		positive charged nylon membrane bound	n.a.
		60	× 22 (membrane)	to glass
aldehyde	www.pall.com	Ultrabind US450 membrane roll		modified polyethersulfone	n.a.
		3000 × 320
amine reactive	www.accelr8.com	75	× 25	glass or silicon	n.a.
thiol-reactive					n.a.
biotin			× 25		n.a.
NHS ester	www1.amershambiosciences.com	75		glass	50 fmol/mm2

n.a. not available

DISCOVERY DRUG AND PROTEOMICS IN ARRAYS PEPTIDE

177

178	ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI

i X Y

R HN

CF3

ii

				X Y
		S	HN
	O	HN		CF3
	OH
OH	O	O
	OH	O
		OH	O
		NH	OH
	S	OH	O
			OH
		NH		O
				OH
			OH	O
				OH

CF3

N

N

FIGURE 7.13. Photobonding to and functionalization of surfaces such as glass, polystyrene, silicon nitride or polyurethane membranes. i = photochemical coupling of N-substituted m-3-(trifluoromethyl)diaziridin-3- yl-anilines, R = 4-maleimidobutyryl residue [91, 442]; ii = Optodex treatment [71].

as proteins, but no applications for the creation of peptide arrays have been described so far.

Surfaces modified by amino groups protected by photosensitive protection groups can be used to generate microstructures simply by irradiation through a lithographic mask [480, 605]. A very similar approach [613] starts from the derivatization of glass surfaces with a photolabile self-assembled monolayer (Figure 7.18). Irradiation led to release of hydrophobic moieties yielding hydrophilic, carboxy modified areas (B) within a hydrophobic (A) environment.

Alternatively, photoresist coatings can be used in combination with photolithographic masks to create micro-patterns. Following irradiation, areas protected by photoresist coatings will not undergo modification upon treatment with tridecafluoro- 1,1,2,2-tetrahydro)trichlorosilane in anhydrous toluene (Figure 7.19). After removal of the

PEPTIDE ARRAYS IN PROTEOMICS AND DRUG DISCOVERY

179

H2N

H2N

i

O

OO

	N	N
	O
	O	H
	O	H
	O
		N
	N

OO

O

ii

OO

	NH2
H2N	HN	N
	PAMAM	H
	HN	H
H2N		N
	NH2

OO

N	S
C			O	O
S		NH
N	NH	HN			N
H					H
	PAMAM
H					H
	NH	HN			N
N
S		NH	O	O
	S
C		S
N			O	O
		NH
			HO	3	N
iii					H
			HO		H
				3	N
		NH
			O	O
N		S
	S		O	O
C		NH
S
N	NH	HN			N
H	PAMAM				H
H					H
	NH	HN			N
N
S		NH	O	O
C	S
N

FIGURE 7.14. Glass surface modification/activation via starbust dendrimer coating [33]. i = treatment of aminosilylated glass with homobifunctional disuccinimidylglutarate in CH2Cl2 containing 1% diisopropylethylamine, 2 h; ii = 10% PANAM starbust monomer, 12 h; iii = activation and intermolecular crosslinking using homobifunctional 1,4-phenylenediisothiocyanate in CH2Cl2 containing 1% pyridine, 2 h.

protecting layer the newly exposed glass surfaces can be transformed into hydrophilic areas by aminopropylsilylation and acylation. Butler and coworkers produced so-called surface tension arrays [70] for the on-chip synthesis of oligonucleotides using a similar principle (Figure 7.20). The resulting hydrophilic areas (B), surrounded by hydrophobic areas (A) with a very low surface tension, represent micro-reactors holding the reaction solvent in area B due to differences in the wetting characteristics. The perfluorosilane modified areas A have wetting properties similar to Teflon. Alternatively, Brennan used a laser for

180	ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI

H2N

i

OO

NN H

O
	ii
NH2
H2N		O
BSA	N	N
	H	H

H2N

NH2

i

O

O	O		N
N	O	NH	O
O				O
HN
O		BSA	N	N
			H	H

NHN

O O O NH

N

O O

FIGURE 7.15. Multistep functionalization of glass surfaces [338]. i = N,N-disuccinimidyl carbonate, diisopropylamine, dimethylformamide, 3 h, room temperature; ii = PBS pH 7.5, 1% bovine serum albumin (BSA), 12 h, room temperature.

PEPTIDE ARRAYS IN PROTEOMICS AND DRUG DISCOVERY

ii

HO

i

		NH2
		H2N	NH2 HO
	H2N		HO
		H2N
	H2N		NH2
	H2N		HO
			NH2
	H N			H2N
	2		NH HO
	H2N	H2N
			2
	H2N		NH2 HO
	H2N			H N
	H2N	H N	NH	2
				2
	H2N	2
		H2N	HO
	H2N
		H2N
			HO
		H2N
	H2N		NH2
		H2N
	H2N	H2N	HO
	H N	HN	NH2
	2
		NH	NH HO
		2	2
		H2N
		H2N

181

	CH3
H2N	Si
	O
	CH3

iii

NH2

N

CH3

N Si

O

CH3

N

NH2

FIGURE 7.16. Aziridine-polymerization [85, 257, 388] and poly-lysine coating. i = the glass surface is coated by covering it with poly-L-lysine solution and drying the film; the polymer film is bound to the surface by electrostatic interactions between silyl-OH functions and positive charged amino groups in the polymer; ii = ethoxydimethylaminopropylsilane/toluene, iii = aziridine / methylene chloride / acetic acid.

ablation of fluorosiloxanes (Figure 7.21) to create micro-patterned surfaces starting from a uniformly hydrophobic modified glass slide [61]. Furthermore, photolithographic techniques have been reported for the micro-patterning of films ([188, 268, 381, 606]; reviewed in [186, 305]). Micro-mirror mediated patterning of biomolecules has been reported [303, 304, 510].

182			ULRICH REINEKE, JENS SCHNEIDER-MERGENER AND MIKE SCHUTKOWSKI
			O2N	O		O
		i	O	N	ii	nN
					H2N		N
				H		H	H
H2N
	iii		O
			N	iv	H	H		O
			H
					N	N
				H2N	N		N	N
					H		H	H
					v
			O	O	O	O
			N	N	N	N
				N	N
			H			H
				O	O

			vi
		H	H
H2N	O	N	N	O	NH2
		2			2

OO

			O			O
H N	O	N	N	N	N	N
				N	N
2		2 H	H			H
				O	O
		H2N	O	2 N	N	O 2	NH2
				H	H

FIGURE 7.17. Multistep functionalization of aminoalkylsilylated glass surfaces or plasma-aminated polypropylene surfaces [31]. i = 4-nitrophenylchloroformiate, diisopropylamine, CH2Cl2, 2 h; ii = diamine, dimethylformamide, 12 h; iii = acryloylchloride, diisopropylamine, dimethylformamide, 24 h, iv = tetraethylenepentamine, dimethylformamide, 12 h; v = acryloylchloride, diisopropylamine, dimethylformamide, 24 h; vi = 1,4-bis(3-aminopropoxy)-butane, dimethylformamide, 12 h.

7.2.3. Peptide Array Preparation

Peptides are usually synthesized step by step using appropriate side chain (permanent protecting group) and N-terminal protected (temporary protecting group) amino acid derivatives (Figure 7.22). Generally, fluorenyl-oxy-carbonyl- (Fmoc), tert-butyl-oxy-carbonyl- (Boc) and nitroveratryl-oxy-carbonyl (Nvoc) moieties are used as temporary protecting groups (PG) while preparing peptide arrays. Subsequent to amino acylation of surfacebound amino functions, the PG of the surface-bound amino acid derivative is removed by base (Fmoc), acid (Boc) or light (Nvoc) treatment. Repeated amino acylation and deprotection reactions yield a surface-bound target peptide in the side chain protected state. Final