In Situ Chemical Modification of Peptide Microarrays: Characterization by Desorption/Ionization on Silicon Nanowires
Peptide microarrays are useful devices for the high throughput study of biomolecular or peptide–cell interactions. Whereas the synthesis of unmodified peptide libraries is an easy task and can be performed at reasonable cost, the synthesis of libraries of
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Introduction In situ modification of unmodified peptide microspots and the preparation of daughter microarrays from a parent unmodified peptide microarray can potentially be applied to various posttranslational modifications. In the accompanying chapter, we have in situ methylated the side-chain of Lys residues by reductive methylation. The chemical process used permitted the formation of mono lysine Lys(Me)2 and dimethyl lysine Lys(Me)2 residues. However, other Lys posttranslational modifications such as acetylation (1), formylation (2), or methylene imine formation (3) can potentially Mahesh Uttamchandani and Shao Q. Yao (eds.), Small Molecule Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 669, DOI 10.1007/978-1-60761-845-4_10, © Springer Science+Business Media, LLC 2010
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be studied using the in situ modification method. In this context, the development of an analytical method allowing the characterization of the chemical modifications taking place in the peptide microspot is of prime importance for optimizing the in situ chemical modification or for controlling the level of modification. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a soft ionization technique, developed in the late 1980s, for mass analysis of analytes. MALDI allows the analysis of a wide variety of compounds, including polymers, peptides, and proteins (4). However, due to a competitive desorption of parasitic ions from the matrix, it is difficult to detect low molecular weight compounds (99&, Sigma-Aldrich) silver nitrate 0.1 N (AgNO3). 7. VLSI-grade nitric acid, 65% (HNO3). 8. VLSI-grade hydrochloric acid, 37% (HCl). 9. Acetone.
Peptide Microarray MS Characterization
127
Intensity (%)
SiNWs layer
~200 µm
HBHA
silicon wafer
CH2O NaCNBH3 NiCl2
3.8E+4
1192.64
1191.2
unmodified
1242.4
1293.6
1344.8
0 1396.0
In situ methylation
ions
Laser Me
100 90 80 70 60 50 40 30 20 10 0 1140.0
Me
Intensity (%) 100 90 80 70 60 50 40 30 20 10 0 1140.0
7840.0
1191.2
1242.4
1293.6
1344.8
0 1396.0
Fig. 1. Application of the DIOSiNWs-MS technique for characterizing the in situ methylation of peptide microspots. HBHA peptide H-LPKKAAPAKKAA-NH2 was printed on a silicon wafer substrate covered with a SiNWs layer, and in situ methylated with formaldehyde/NaCNBH3/NiCl2. Analysis of peptide microspots by LDI-MS permitted to characterize the in situ chemical modification of HBHA peptide.
10. Isopropyl alcohol (iPrOH). 11. Dichloromethane (CH2Cl2). 12. Octyldimethylchlorosilane (ODMCS). 13. UV-O Cleaner, Jelight Company Inc., 4 mW/cm2 at 220 nm. 14. Plas-Labs nitrogen glove box. 15. Goniometer system (DIGIDROP, GBX, France) for measuring the contact angles. 2.2. Printing of Peptide Probes
1. Peptide probes. 2. 0.01 M Sodium phosphate buffer pH 7.2 containing 150 mM NaCl (PBS). 3. V-bottomed 384-well microtiter plates (ABgene, Surrey, UK, AB-1058). 4. Noncontact piezoelectric microarrayer (BCA1, Perkin Elmer, MA, USA).
2.3. In Situ Methylation
1. Prepare soluti
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