Abstract
Antibody-based microarrays are a rapidly evolving affinity-proteomic methodology that recently has shown great promise in clinical applications. The resolution of these proteomic analyses is, however, directly related to the number of data-points, i.e. antibodies, included on the array. Currently, this is a key bottleneck because of limited availability of numerous highly characterized antibodies. Here, we present a conceptually new method, denoted global proteome survey, opening up the possibility to probe any proteome in a species-independent manner while still using a limited set of antibodies. We use context-independent-motif-specific antibodies directed against short amino acid motifs, where each motif is present in up to a few hundred different proteins. First, the digested proteome is exposed to these antibodies, whereby motif-containing peptides are enriched, which then are detected and identified by mass spectrometry. In this study, we profiled extracts from human colon tissue, yeast cells lysate, and mouse liver tissue to demonstrate proof-of-concept.
Highlights
From the ‡Department of Immunotechnology, Lund University, Lund, Sweden, and CREATE Health, BMC D13, Lund, Sweden; §BioInvent International AB, SE-223 70 Lund, Sweden; ¶School of Medicine and Medical Sciences, Conway Institute, University College Dublin, Dublin 4, Ireland
We present proof-of-concept for a conceptually new method opening up the possibility to probe proteomes in a species independent manner, while still using a limited set of antibodies
Despite recent achievements within the field of affinity proteomics [7], two major bottlenecks remain before antibody microarray-based analysis can be performed in a more global-scale and/or in discovery mode
Summary
Proteomic Analysis and Discovery Using Affinity Proteomics and Mass Spectrometry*□S. Niclas Olsson‡ʈ, Christer Wingren‡ʈ, Mikael Mattsson§, Peter James‡, David O’ Connell¶, Fredrik Nilsson§, Dolores J. To advance further and set a novel standard for proteomics, the most attractive features of affinity proteomics could be combined with those of MS-based proteomics This has previously been attempted, where antibodies have initially been used to enrich specific proteins [17,18,19,20] or peptides [21,22,23,24] followed by subsequent MS analysis. The biological sample is digested, exposed to these antibodies, whereby motif-containing peptides are captured, enriched, and subsequently detected and identified (and quantified) using single- or tandem-MS In this manner, 100 of these context independent motif specific (CIMS) antibodies, would theoretically cover almost 50% of the nonredundant human proteome [25], a concept supported by a recent in-silico study of the human proteome [26]
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