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ABSTRACT: Hepatocyte growth factor/scatter factor (HGF/SF) is the high affinity ligand of MET tyrosine kinase receptor. We report here the total synthesis of a biotinylated analogue of human HGF/SF N domain. Functionally, N domain is part of the HGF/SF high affinity binding site for MET and also the main HGF/SF binding site for heparin. The 97 Aa linear chain featuring a C-terminal biotin group was assembled in high yield using an N-to-C one-pot three segments assembly strategy relying on a sequential Native Chemical Ligation (NCL)/bis(2-sulfanylethyl)amido (SEA) native peptide ligation process. The folded protein displayed the native disulfide bond pattern and showed the ability to bind heparin.
Bioorganic & medicinal chemistry 03/2013; · 2.82 Impact Factor
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Frederik Gwinner,
Adelina E Acosta-Martin,
Ludovic Boytard,
Maggy Chwastyniak,
Olivia Beseme, Hervé Drobecq,
Sophie Duban-Deweer,
Francis Juthier,
Brigitte Jude,
Philippe Amouyel,
Florence Pinet,
Benno Schwikowski
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ABSTRACT: In this study, we developed a novel computational approach based on protein-protein interaction (PPI) networks to identify a list of proteins that might have remained undetected in differential proteomic profiling experiments. We tested our computational approach on two sets of human smooth muscle cell (SMC) protein extracts which were affected differently by DNase I treatment. Differential proteomic analysis by saturation DIGE resulted in the identification of 41 human proteins. The application of our approach to these 41 input proteins consisted of four steps: 1) Compilation of a human PPI network from public databases, 2) Calculation of interaction scores based on functional similarity, 3) Determination of a set of candidate proteins that are needed to efficiently and confidently connect the 41 input proteins, and 4) Ranking of the resulting 25 candidate proteins. Two of the three highest-ranked proteins, beta-arrestin 1 and beta-arrestin 2, were experimentally tested, revealing that their abundance levels in human SMC samples were indeed affected by DNase I treatment. These proteins had not been detected during the experimental proteomic analysis. Our study suggests that our computational approach may represent a simple, universal, and cost-effective means to identify additional proteins that remain elusive for current 2D gel-based proteomic profiling techniques.
Proteomics 02/2013; · 4.43 Impact Factor
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ABSTRACT: Ets-1 is a transcription factor that regulates many genes involved in cancer progression and in tumour invasion. It is a poor prognostic marker for breast, lung, colorectal and ovary carcinomas. Here, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel interaction partner of Ets-1. We show that Ets-1 activates, by direct interaction, the catalytic activity of PARP-1 and is then poly(ADP-ribosyl)ated in a DNA-independent manner. The catalytic inhibition of PARP-1 enhanced Ets-1 transcriptional activity and caused its massive accumulation in cell nuclei. Ets-1 expression was correlated with an increase in DNA damage when PARP-1 was inhibited, leading to cancer cell death. Moreover, PARP-1 inhibitors caused only Ets-1-expressing cells to accumulate DNA damage. These results provide new insight into Ets-1 regulation in cancer cells and its link with DNA repair proteins. Furthermore, our findings suggest that PARP-1 inhibitors would be useful in a new therapeutic strategy that specifically targets Ets-1-expressing tumours.
PLoS ONE 01/2013; 8(2):e55883. · 4.09 Impact Factor
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ABSTRACT: In this paper, we report on the functionalization of silicon nanostructured (NanoSi) surface with an organic layer of nitrilotriacetic acid (NTA) and its subsequent use as an affinity surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) interface for histidine-tagged peptide enrichment and mass spectrometry analysis. The NTA terminal groups are immobilized onto the NanoSi surface via very stable Si-C covalent bonds. The NTA-modified NanoSi (NTA-NanoSi) interface was characterized by contact angle measurements, Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The NTA-NanoSi interface has shown a good selectivity toward His-tagged peptide and permits its enrichment from an artificial mixture of both tagged and untagged peptides and its subsequent mass spectrometry detection with good signal/noise ratio.
The Analyst 10/2012; · 4.23 Impact Factor
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Kunie Ando,
Pierre Dourlen,
Anne-Véronique Sambo,
Alexis Bretteville,
Karim Bélarbi,
Valérie Vingtdeux,
Sabiha Eddarkaoui, Hervé Drobecq,
Antoine Ghestem,
Séverine Bégard, [......],
Claude-Alain Maurage,
Marie-Laure Caillet-Boudin,
Yann Verdier,
Joelle Vinh,
Isabelle Landrieu,
Marie-Christine Galas,
David Blum,
Malika Hamdane,
Nicolas Sergeant,
Luc Buée
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ABSTRACT: A prerequisite to dephosphorylation at Ser-Pro or Thr-Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction of AD brain tissue. Furthermore, in all tau pathology conditions, acetylation of Pin1 may also contribute to the differences observed. In conclusion, Pin1 displays several posttranslational modifications, which are specific in tauopathies and may be useful as biomarker.
Neurobiology of aging 08/2012; · 5.94 Impact Factor
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Kunie Ando,
Pierre Dourlen,
Anne-Véronique Sambo,
Alexis Bretteville,
Karim Bélarbi,
Valérie Vingtdeux,
Sabiha Eddarkaoui, Hervé Drobecq,
Antoine Ghestem,
Séverine Bégard, [......],
Claude-Alain Maurage,
Marie-Laure Caillet-Boudin,
Yann Verdier,
Joelle Vinh,
Isabelle Landrieu,
Marie-Christine Galas,
David Blum,
Malika Hamdane,
Nicolas Sergeant,
Luc Buée
[show abstract]
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ABSTRACT: A prerequisite to dephosphorylation at Ser-Pro or Thr-Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction
Neurobiology of aging. 08/2012;
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ABSTRACT: The paper reports on the use of a titanium oxide (TiO(2)) nanotube layer as a sensitive substrate for surface-assisted laser desorption-ionization mass spectrometry (SALDI-MS) of peptides and small molecules. The nanotube layers were prepared by electrochemical anodization of titanium foil. The optimized TiO(2) nanotubes morphology coupled to a controlled surface chemistry allowed desorption-ionization (D/I) of a peptide mixture (Mix1) with a detection limit of 10 femtomoles for the neurotensin peptide. The performance of the TiO(2) nanotubes for the D/I of small molecules was also tested for the detection of sutent, a small tyrosine kinase inhibitor, and verapamil. A detection limit of 50 fmol was obtained for these molecules, as compared to 500 fmol using classical matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). Both amorphous and anatase TiO(2) layers displayed a comparable performance for D/I of analyte molecules. In a control experiment, we have performed D/I of analyte molecules on a flat TiO(2) layer. The absence of signal emphasizes the role of the nanostructured substrate in the D/I process.
The Analyst 05/2012; 137(13):3058-63. · 4.23 Impact Factor
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ABSTRACT: Bis(2-sulfanylethyl)amido (SEA) side-chain derivatives of aspartic and glutamic acids enable the synthesis of tail-to-side chain cyclic or branched peptides using standard Fmoc-SPPS followed by SEA native peptide ligation.
Organic Letters 04/2012; 14(9):2222-5. · 5.86 Impact Factor
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ABSTRACT: Three in one: Native chemical ligation (NCL) and bis(2-sulfanylethyl)amido (SEA) ligation allow the one-pot assembly of three peptide segments in the N-to-C direction. The SEA group is switched off by intramolecular disulfide bond formation during NCL. Then, a phosphine switches it on to trigger the second SEA ligation step. The K1 domain of the hepatocyte growth factor was synthesized and found to be biologically active.
Angewandte Chemie International Edition 11/2011; 51(1):209-13. · 13.45 Impact Factor
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ABSTRACT: This paper reports on the use of boron-doped diamond nanowires (BDD NWs) as an inorganic substrate for matrix-free laser desorption/ionization mass spectrometry (LDI-MS) analysis of small molecules. The diamond nanowires are prepared by reactive ion etching (RIE) with oxygen plasma of highly boron-doped (the boron level is 10(19) B cm(-3)) or undoped nanocrystalline diamond substrates. The resulting diamond nanowires are coated with a thin silicon oxide layer that confers a superhydrophilic character to the surface. To minimize droplet spreading, the nanowires were chemically functionalized with octadecyltrichlorosilane (OTS) and then UV/ozone treated to reach a final water contact angle of 120°. The sub-bandgap absorption under UV laser irradiation and the heat confinement inside the nanowires allowed desorption/ionization, most likely via a thermal mechanism, and mass spectrometry analysis of small molecules. A detection limit of 200 zeptomole for verapamil was demonstrated.
Nanoscale 11/2011; 4(1):231-8. · 5.91 Impact Factor
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ABSTRACT: The chaperone/protease DegP belongs to the HtrA superfamily and is involved in protein quality control in the periplasm of Gram-negative bacteria. In Escherichia coli, typical substrates are unfolded or misfolded globular proteins that trigger the rearrangement of inactive DegP hexamers into substrate-sequestering 12- or 24-mers 'cages' for refolding or degradation. In Bordetella pertussis, DegP(Bp) facilitates, in addition, the secretion of FHA, a long β-helical adhesin that passes through the periplasm in an extended conformation. We show that DegP(Bp) exists as soluble trimers and as a membrane-associated form. Different substrates interact differently with the distinct forms of DegP(Bp), and membrane-associated DegP(Bp) has high affinity for non-native FHA. Unlike more globular substrates, FHA does not efficiently mediate rearrangement of trimers into proteolytically active, short-lived dodecamers. In contrast to these dodecamers, membrane-associated DegP(Bp) is not committed to substrate degradation, although it is proteolytically competent. In B. pertussis, membrane-associated DegP(Bp) thus represents a specific functional form serving as a holding chaperone for client proteins including FHA. If FHA secretion is impaired, membrane-associated DegP(Bp) participates in its degradation. This form of DegP(Bp) is appropriate to handle substrates unsuitable to be sequestered in cages or non-folded, secretory proteins that must not be degraded.
Molecular Microbiology 06/2011; 80(6):1625-36. · 5.01 Impact Factor
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ABSTRACT: We present for the first time an electrowetting on dielectric (EWOD) microfluidic system coupled to a surface-assisted laser desorption-ionization (SALDI) silicon nanowire-based interface for mass spectrometry (MS) analysis of small biomolecules. Here, the transfer of analytes has been achieved on specific locations on the SALDI interface followed by their subsequent mass spectrometry analysis without the use of an organic matrix. To achieve this purpose, a device comprising a digital microfluidic system and a patterned superhydrophobic/superhydrophilic silicon nanowire interface was developed. The digital microfluidic system serves for the displacement of the droplets containing analytes, via an electrowetting actuation, inside the superhydrophilic patterns. The nanostructured silicon interface acts as an inorganic target for matrix-free laser desorption-ionization mass spectrometry analysis of the dried analytes. The proposed device can be easily used to realize several basic operations of a Lab-on-Chip such as analyte displacement and rinsing prior to MS analysis. We have demonstrated that the analysis of low molecular weight compounds (700 m/z) can be achieved with a very high sensitivity (down to 10 fmol μL(-1)).
Lab on a Chip 03/2011; 11(9):1620-8. · 5.67 Impact Factor
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Louise H Lefrançois,
Céline Pujol,
Christelle C Bodier,
Ana Paula Teixeira-Gomez, Hervé Drobecq,
Marie-Laure Rosso,
Dominique Raze,
André Alves Dias,
Jean-Pierre Hugot,
Ofelia Chacon,
Raul G Barletta,
Camille Locht,
Maria Cristina Vidal Pessolani,
Franck Biet
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ABSTRACT: Mycobacterium avium subsp. paratuberculosis (Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne's disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin-Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn's disease.
Microbes and Infection 02/2011; 13(6):585-94. · 3.10 Impact Factor
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ABSTRACT: Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named "chips from chips", which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen.
Journal of Proteome Research 10/2010; 9(12):6467-78. · 5.11 Impact Factor
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ABSTRACT: Depletion of major blood proteins is one of the most promising approaches to accessing low-abundance biomarkers. This study compared the use of combinatorial peptide ligand library (CPLL) and albumin and immunoglobulins (IgGs) depletion technology for accessing these low-abundance proteins in plasma using 2-DE in an acidic restricted pH range (4-7). Compared with native plasma, both techniques enlarge the visibility of other proteins than albumin and IgG, but there were marked differences in their composition. An increase of the number of spots was detected compared with native plasma (157 spots) with 427 and 557 spots, respectively, detected with albumin and IgG depletion, and CPLL treatment. We selected 70 spots to be identified by MALDI-TOF related to their absence in the 2-D gels from native or albumin and IgG-depleted plasma. The 42 spots identified corresponded to 24 different proteins, with more than half of the proteins which did not belong to the major plasma proteins. CPLL treatment allowed the accessibility to proteolytic fragments obtained from major plasma proteins. We found a large superiority of the CPLL approach over the albumin and IgG depletion process. These findings show the utility of depleting major blood proteins to be able to access low-abundance proteins and the potential of CPLL to select and identify candidate biomarkers in clinical studies.
Electrophoresis 08/2010; 31(16):2697-704. · 3.30 Impact Factor
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Nicolas Lamblin,
Philippe Ratajczak,
David Hot,
Emilie Dubois,
Maggy Chwastyniak,
Olivia Beseme, Hervé Drobecq,
Yves Lemoine,
Mohammad Koussa,
Philippe Amouyel,
Florence Pinet
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ABSTRACT: Abdominal aortic aneurysms (AAA) are defined by an increased aortic diameter and characterized by impairment of the extracellular matrix, macrophages infiltration and decreased density of smooth muscle cells. Our aim is to identify the key molecules involved in the pathogenesis of AAAs. This study investigated transcriptomic and proteomic profiles of macrophages from AAA patients (>50 mm aortic diameter) (n = 24) and peripheral arterial occlusion (PAO) patients without AAA detected (n = 18), who both needed a surgery. An antibody protein microarray, generated by printing antibodies onto membranes against proteins selected from the transcriptomic and proteomic analysis, was performed to validate the proteins differentially expressed specifically in macrophages and plasma from the same patients. We found a restricted number of proteins differentially expressed between AAA and PAO patients: TIMP-3, ADAMTS5, and ADAMTS8 that differ significantly in plasma of AAA patients compared to PAO patients, as found in the macrophages. In contrast to plasma MMP-9, soluble glycoprotein V (sGPV) and plasmin-antiplasmin complex levels, plasma TIMP-3 levels were not correlated to AAA size but interestingly correlated to sGPV, a platelet activation marker. Combining transcriptomic and proteomic is a valid approach to identify diseases causing proteins and potential biomarkers.
Journal of Proteome Research 07/2010; 9(7):3720-9. · 5.11 Impact Factor
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ABSTRACT: The integrated analysis of intracellular trafficking pathways is one of the current challenges in the field of cell biology, and functional proteomics has become a powerful technique for the large-scale identification of proteins or lipids and the elucidation of biological processes in their natural contexts. For this, new dynamic strategies must be devised to trace proteins that follow a specific pathway such that their initial and final destinations can be detected by automated means.
Here, we report a novel vectorial strategy for trafficking pathway analysis. This strategy is based on a chemical modification of plasma membrane proteins with a bSuPeR (biotinylated sulfation site peptide reagent) and metabolic labelling in the Golgi apparatus, such that plasma membrane proteins that traffic via the retrograde route become detectable in complex mixtures. Efficient synthesis schemes are presented for tailor-made chemical tools that are then applied to the step-by-step validation of the strategy, using a known retrograde cargo protein: the STxB (Shiga toxin B-subunit). bSuPeR modification at the plasma membrane does not affect STxB transport to the Golgi apparatus, where the protein is metabolically labelled, allowing its detection in cell lysates.
Our vectorial concept proposes a new chemical approach for traffic-based profiling of proteins that may prove to be applicable to the analysis of diverse endocytic pathways.
Biology of the Cell 06/2010; 102(6):351-9. · 3.60 Impact Factor
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ABSTRACT: Chronic heart failure following myocardial infarction (MI) is characterized by progressive left ventricular remodelling (LVR). Despite significant improvements in MI management, LVR remains a frequent complication. Although several risk factors have been identified, such as infarct size, LVR is difficult to predict in clinical practice.
Using a rat model of MI and phosphoproteomic technology, we discovered that remodelling is associated with decreased levels of myocardial and plasma serine(208)-phosphorylated troponin T (TnT). To confirm the association in human plasma, we developed new specific polyclonal antibodies against human/rat serine(207/208)-phosphorylated TnT and tested plasma obtained in the first week after MI from patients with low, intermediate, and high remodelling a year later. We observed a significant decrease of serine(207)-phosphorylated TnT and of the serine(207)-phosphorylated TnT/total TnT ratio in those with intermediate or high LVR. These differences remained statistically significant when adjusted for other determinants of LVR. In contrast, baseline B-type natriuretic peptide levels were not associated with LVR.
The level of circulating phosphorylated TnT could be a new biomarker of LVR.
European Heart Journal 04/2010; 32(1):115-23. · 10.48 Impact Factor
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ABSTRACT: This paper reports on the use of silicon nanowires (SiNWs), easily prepared in a single step by chemical etching of crystalline silicon in HF/AgNO(3) aqueous solution, as a highly sensitive substrate for laser desorption/ionization mass spectrometry (LDI-MS) analysis. The SiNWs' diameter and length depend on the etchant concentration and dissolution time. Optimized LDI substrate consists of nanowires with an average diameter in the range of 20-100 nm and 2.5 mum in length. The optimized SiNWs' surface morphology coupled to a controlled surface chemistry allowed a significant LDI-MS performance through measurements of a broad range of analytes, including small molecules, peptides, and a bovine serum albumin (BSA) digest. A signal-to-noise ratio of 250 was ascertained for a 10 fmol bradykinin pick, in reflector mode acquisition. Likewise, the sutent, a small tyrosine kinase inhibitor, could be observed down to 10 fmol, as compared to 500 fmol limit detection using the classical matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We have further investigated the optical properties of the nanowires, and our results suggest that they have a small or no effect on the desorption/ionization (D/I) process. On the contrary, the surface morphology and thermal properties of the silicon nanostructures are found to be the essential features contributing to the D/I performance.
Langmuir 01/2010; 26(2):1354-61. · 4.19 Impact Factor
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ABSTRACT: 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 modified peptides remains expensive and time consuming. This bottleneck led us to examine the possibility to produce modified peptide microspots by in situ chemical modification of unmodified peptide microspots. The great advantage would be the preparation of a series of complex microarrays (daughter microarrays) starting from an easy-to-make and cost-effective unmodified peptide microarray (parent microarray). One step toward this goal has been presented in the accompanying chapter dealing with the in situ methylation methodology for studying the specificity of antibodies directed toward methylated epitopes. Here we describe the development of a novel desorption/ionization on silicon nanowires mass spectrometry (DIOSiNWs-MS) technique for characterizing the in situ chemical modification of peptides.
Methods in molecular biology (Clifton, N.J.) 01/2010; 669:125-33.
