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作者： Tongbin Li Zhixiang Zuo Qi Zhu Ailing Hong Xiaochuan Zhou Xiaolian Gao

peptide microarrays (peptide arrays) have increasingly become an important research tool for studying protein detection, profiling, and protein–protein interactions, and they have the potential to foster high-throughput protein analysis as DNA arrays did for genomics research a decade ago. Recently, technologies have emerged that allow flexible synthesis of high-density peptide arrays based on specific application needs (e.g., phosphopeptide microarrays). To fully unleash the power of this promising research tool, significant efforts are required to develop computational and informatics resources that facilitate the experimental design and data analysis for a wide range of peptide array-based applications. The design of peptide arrays is inherently more complex than that of DNA arrays. We herein introduce µPepArray Pro, a Web-based general-purpose peptide array design program. µPepArray Pro features strong content design capabilities and maximized user control. The program suits the needs of a diversity of design tasks, works with a variety of peptide array configurations, and is highly expandable: new functionalities can be developed and added to µPepArray Pro with relative ease. less

关键词： peptide microarray peptide Array Protein Analysis Genomics Amino Acid Substitution peptide Synthesis microarray Data analysis Swiss-Prot array design µPepArray Pro

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In Situ Chemical Modification of peptide microarrays: Characterization by Desorption/Ionization on Silicon Nanowires

In Situ Chemical Modification of Peptide Microarrays: Charac...

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作者： Gaëlle Piret Hervé Drobecq Rabah Boukherroub Oleg Melnyk

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. less

关键词： Mass Spectrometry LDI Mass Spectrometry microarray peptide microarray Etching MALDI Mycobacterium tuberculosis Methylation MALDI-TOF Matrix-free desorption/ionization on silicon nanowires Heparin-binding hemagglutinin

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High-Density peptide microarrays for Reliable Identification of Phosphorylation Sites and Upstream Kinases

High-Density Peptide Microarrays for Reliable Identification...

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作者： Alexandra Thiele Johannes Zerweck Matthias Weiwad Gunter Fischer Mike Schutkowski

The human genome encodes about 25,000 genes. This number seems to be very small compared to the multitude of different protein functions in highly regulated pathways that are responsible for complex biochemical mechanisms like growth, metabolism, signal transduction and reproduction. Obviously, there are mechanisms creating additional protein diversity. The most important mechanism is post-translational modification (PTM) changing protein surfaces by phosphorylation, sulfation, acetylation, methylation and sumoylation resulting in an about 100-fold higher complexity (1, 2). This chapter presents a very efficient way to detect potential phosphorylation sites in proteins using overlapping peptide scans immobilized on glass slides. Results from 35 different human kinases using peptide microarrays displaying overlapping peptide scans through either all human cyclophilins or all human FK506-binding proteins are shown. Additionally, detection of phosphorylation sites in a proteome-wide manner is demonstrated using peptide microarrays displaying cytomegalovirus proteome in the form of more than 17,000 overlapping peptides. less

关键词： peptide microarray Autoradiography Phosphorimaging Kinase Assay microarray kinase FK506-binding protein Pin1 phosphorylation site overlapping peptides peptidyl-prolyl-cis/trans-isomerase cyclophilin

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High Density peptide microarrays for Proteome-Wide Fingerprinting of Kinase Activities in Cell Lysates

High Density Peptide Microarrays for Proteome-Wide Fingerpri...

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作者： Alexandra Thiele Matthias Weiwad Johannes Zerweck Gunter Fischer Mike Schutkowski

Protein function is highly regulated in pathways that are responsible for complex biochemical mechanisms such as growth, metabolism, and signal transduction. One of the most important mechanisms is posttranslational modification (PTM) changing protein surfaces by phosphorylation, sulfation, acetylation, methylation, glycosylation, and sumoylation resulting in a more than 100-fold higher complexity (Geiss-Friedlander and Melchior, Nat Rev Mol Cell Biol 8, 947–956, 2007; Hunter, Mol Cell 28, 730–738, 2007). This chapter presents a very efficient way to detect potential phosphorylation sites in protein families using overlapping peptides covering the complete primary structures (peptide scans) immobilized on glass slides. Results of kinase activity fingerprinting of cell lysates using peptide microarrays displaying peptide scans through all human peptidyl-prolyl-cis/trans-isomerases are shown. less

关键词： peptide microarray Cell And Tissue Culture microarray CM-2 [Human ESC] Tyrosine kinase Protein kinase Cell lysate Phosphorylation sites Pin1 Overlapping peptides Cyclophilin Serine/threonine kinase Peptidyl-prolyl-cis/trans-isomerase FKBP Parvulin Fluorescent readout Phosphate-specific dye

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Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry

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ANALYTICAL BIOCHEMISTRY 2013年第1期442卷 62-67页

作者： McKee, Christopher J. Hines, Harry B. Ulrich, Robert G. US Army Med Res Inst Infect Dis Frederick MD 21702 USA

microarrays of peptide and recombinant protein libraries are routinely used for high-throughput studies of protein-protein interactions and enzymatic activities. Imaging mass spectrometry (IMS) is currently applied as a method to localize analytes on thin tissue sections and other surfaces. Here, we have applied IMS as a label-free means to analyze protein-peptide interactions in a microarray-based phosphatase assay. This IMS strategy visualizes the entire microarray in one composite image by collecting a predefined raster of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry spectra over the surface of the chip. Examining the bacterial tyrosine phosphatase YopH, we used IMS as a label-free means to visualize enzyme binding and activity with a microarrayed phosphopeptide library printed on chips coated with either gold or indium-tin oxide. Furthermore, we demonstrate that microarray-based IMS can be coupled with surface plasmon resonance imaging to add kinetic analyses to measured binding interactions. The method described here is within the capabilities of many modern MALDI-TOF instruments and has general utility for the label-free analysis of microarray assays. Published by Elsevier Inc.

关键词： MALDI Imaging mass spectrometry peptide microarray YopH Phosphatase assay SPRi

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Time dependent effect of cold ischemia on the phosphoproteome and protein kinase activity in fresh-frozen colorectal cancer tissue obtained from patients

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CLINICAL PROTEOMICS 2021年第1期18卷 1-12页

作者： Buffart, Tineke E. van den Oord, Rosanne A. H. M. van den Berg, Adrienne Hilhorst, Riet Bastiaensen, Niek Pruijt, Hans F. M. van den Brule, Adriaan Nooijen, Peet Labots, Mariette de Goeij-de Haas, Richard R. Dekker, Henk Piersma, Sander R. Pham, Thang V. van der Leij, Theo de Wijn, Rik Ruijtenbeek, Rob Jimenez, Connie R. Verheul, Henk M. W. Vrije Univ Amsterdam Med Ctr Amsterdam UMC Dept Med Oncol Amsterdam Netherlands Netherlands Canc Inst Dept Gastrointestinal Oncol Amsterdam Netherlands Jeroen Bosch Hosp Dept Med Oncol Shertogenbosch Netherlands PamGene Int BV Shertogenbosch Netherlands Jeroen Bosch Hosp Lab Mol Diagnost Shertogenbosch Netherlands Jeroen Bosch Hosp Dept Pathol Shertogenbosch Netherlands GenMab BV Utrecht Netherlands Radboud Univ Nijmegen Med Ctr Dept Med Oncol Geert Grootepl 10 NL-6525 GA Nijmegen Netherlands

Background Based on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics. Methods Biopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT. Results Unsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p < 0.01 and fold change > 2) altered signal intensity in time. In 4 out of 5 patients tumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p < 0.01). Only two phosphopeptides were significantly changed in all time points, including one

关键词： Protein kinase Mass spectrometry Phosphoproteomics peptide microarray Cold ischemia Cancer

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Recognition of Methylated peptides by Drosophila melanogaster Polycomb Chromodomain

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JOURNAL OF PROTEOME RESEARCH 2013年第3期12卷 1467-1477页

作者： Stein, Richard S. L. Li, Nan He, Wei Komives, Elizabeth Wang, Wei Univ Calif San Diego Dept Chem & Biochem La Jolla CA 92093 USA Univ Calif San Diego Dept Cellular & Mol Med La Jolla CA 92093 USA

Lysine methylation is one of the important post-translational modifications (PTMs) that regulate protein functions Up to now, proteomic identification of this PTM remains a challenge due to the lack of effective enrichment methods in mass spectrometry experiments To address this challenge, we present here a systematic approach to predicting peptides in which lysine residues may be methylated to mediate protein protein interactions. We used the chromodomai of the polycomb protein in Drosophila melanogaster as a model system to illustrate the success of this approach. We started with molecular dynamics simulations and free energy analyses on the histone peptides complexed with the polycomb chromodomain to understand how the binding specificity is achieved. We next conducted virtual mutagenesis to quantify each domain and peptide residue's contribution to the domain-peptide recognition, based on which scoring scheme was developed to evaluate the possibility of any lysine-containing peptides to be methylated and recognized by the chromodomain. A peptide microarray experiment on a panel of conserved histone peptides showed a satisfactory prediction accuracy of the scoring scheme. Next, we implemented a bioinformatics pipeline that integrates multiple lines of evidence including conservation, subcellular localization, and mass spectrometry data to scan the fly proteome for a systematic identification of possible methyllysine-containing peptides. These putative chromodomain-binding peptides suggest unknown functions of the important regulator protein polycomb and provide a list of candidate methylation events for follow-up investigations.

关键词： molecular dynamics MM-GBSA mutation matrix peptide microarray methylated lysine

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The immunosignature of canine lymphoma: characterization and diagnostic application

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BMC CANCER 2014年第1期14卷 1-11页

作者： Johnston, Stephen Albert Thamm, Douglas H. Legutki, Joseph Barten Colorado State Univ Flint Anim Canc Ctr Ft Collins CO 80523 USA Arizona State Univ Biodesign Inst Ctr Innovat Med Tempe AZ 85287 USA

Background: Cancer diagnosis in both dogs and humans is complicated by the lack of a non-invasive diagnostic test. To meet this clinical need, we apply the recently developed immunosignature assay to spontaneous canine lymphoma as clinical proof-of-concept. Here we evaluate the immunosignature as a diagnostic for spontaneous canine lymphoma at both at initial diagnosis and evaluating the disease free interval following treatment. Methods: Sera from dogs with confirmed lymphoma (B cell n = 38, T cell n = 11) and clinically normal dogs (n = 39) were analyzed. Serum antibody responses were characterized by analyzing the binding pattern, or immunosignature, of serum antibodies on a non-natural sequence peptide microarray. peptides were selected and tested for the ability to distinguish healthy dogs from those with lymphoma and to distinguish lymphoma subtypes based on immunophenotype. The immunosignature of dogs with lymphoma were evaluated for individual signatures. Changes in the immunosignatures were evaluated following treatment and eventual relapse. Results: Despite being a clonal disease, both an individual immunosignature and a generalized lymphoma immunosignature were observed in each dog. The general lymphoma immunosignature identified in the initial set of dogs (n = 32) was able to predict disease status in an independent set of dogs (n = 42, 97% accuracy). A separate immunosignature was able to distinguish the lymphoma based on immunophenotype (n = 25, 88% accuracy). The individual immunosignature was capable of confirming remission three months following diagnosis. Immunosignature at diagnosis was able to predict which dogs with B cell lymphoma would relapse in less than 120 days (n = 33, 97% accuracy). Conclusion: We conclude that the immunosignature can serve as a multilevel diagnostic for canine, and potentially human, lymphoma.

关键词： Cancer Dog Diagnostic Antibody response peptide microarray

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Making the Most of In Vitro Tests to Diagnose Food Allergy

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JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY-IN PRACTICE 2017年第2期5卷 237-248页

作者： Santos, Alexandra F. Brough, Helen A. Kings Coll London Dept Paediat Allergy Div Asthma Allergy & Lung Biol London England Guys & St Thomas Hosp Childrens Allergy Serv London England

Various in vitro tests assess different aspects of the underlying immune mechanism of IgE-mediated food allergy. Some can be used for diagnostic purposes;specific IgE to allergen extracts is widely available;specific IgE to allergen components is used in most specialist centers, and the basophil activation test is becoming increasingly used clinically. IgE to allergen peptides, T-cell assays, allergen-specific/total IgE ratios, and allergenspecific IgG4/IgE ratios are currently reserved for research. Different factors can modulate the likelihood of IgE-mediated food allergy of a given allergy test result, namely, the patients' age, ethnicity, previous allergic reaction to the identified food, concomitant atopic conditions, and geographical location, and need to be taken into account when interpreting the allergy test results in the clinic. The importance of the specific food, the clinical resources available, and patient preferences are additional aspects that need to be considered when deciding whether an oral food challenge is required to reach an accurate diagnosis of IgE-mediated food allergy. (C) 2017 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology.

关键词： In vitro tests Diagnosis Food allergy Specific IgE Basophil activation test Component-resolved diagnosis IgG4/IgE ratio Specific/total IgE ratio peptide microarray T-cell assay

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Feasibility of an early Alzheimer's disease immunosignature diagnostic test

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JOURNAL OF NEUROIMMUNOLOGY 2013年第1-2期254卷 154-160页

作者： Restrepo, Lucas Stafford, Phillip Johnston, Stephen Albert Arizona State Univ Biodesign Inst Ctr Innovat Med Tempe AZ 85287 USA Arizona State Univ Mol & Cell Biol Program Tempe AZ 85287 USA Arizona State Univ Sch Life Sci Tempe AZ 85287 USA

A practical diagnostic test is needed for early Alzheimer's disease (AD) detection. Immunosignaturing, a technology that employs antibody binding to a random-sequence peptide microarray, generates profiles that distinguish transgenic mice engineered with familial AD mutations (APPswe/PSEN1-dE9) from non-transgenic littermates. It can also detect an AD-like signature in humans. Here, we assess the changes in the immunosignature at different time points of the disease in mice and humans. We also evaluate the accuracy of the late-stage signature as a test to discriminate between young mice with familial AD mutations from non-transgenic littermates. Plasma samples from AD patients were assayed 3-12 months apart, while APPswe/PSEN1-dE9 and non-transgenic controls supplied plasma at monthly intervals until they reached 15 months of age. microarrays with 10,000 random-sequence peptides were used to compare antibody binding patterns. These patterns gradually changed over the life-span of mice. Strong, characteristic signatures were observed in transgenic mice at early, mid and late stages, but these profiles had minimal overlap. The signature of young transgenic mice had an error rate of 18% at classifying plasma samples from late-stage transgenic mice. Conversely, the late-stage transgenic mice signature discriminated between young transgenic mice and littermates with an error rate of 21%. Less distinctive profiles were recognizable throughout the transgenic mice lifespan, being detectable as early as 2 months. The human signature had minimal change on short-term follow-up. Our results call for a reappraisal of the way incipient AD is studied, as biomarkers seen in late-stages of the disease may not be relevant in earlier stages. (C) 2012 Elsevier B.V. All rights reserved.

关键词： Alzheimer's disease Immunosignaturing peptide microarray Dementia diagnosis

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