Strategy for surveying the proteome using affinity proteomics and mass spectrometry.
Antibody-based microarrays is a rapidly evolving technology that has gone from the first proof-of-concept studies to more demanding proteome profiling applications, during the last years. Miniaturized microarrays can be printed with large number of antibodies harbouring predetermined specificities, capable of targeting high- as well as low-abundant analytes in complex, nonfractionated proteomes. Consequently, the resolution of such proteome profiling efforts correlate directly to the number of antibodies included, which today is a key limiting factor. To overcome this bottleneck and to be able to perform in-depth global proteome surveys, we propose to interface affinity proteomics with MS-based read-out, as outlined in this technical perspective. Briefly, we have defined a range of peptide motifs, each motif being present in 5-100 different proteins. In this manner, 100 antibodies, binding 100 different motifs commonly distributed among different proteins, would potentially target a protein cluster of 10(4) individual molecules, i.e. around 50% of the nonredundant human proteome. Notably, these motif-specific antibodies would be directly applicable to any proteome in a specie independent manner and not biased towards abundant proteins or certain protein classes. The biological sample is digested, exposed to these immobilized antibodies, whereby motif-containing peptides are specifically captured, enriched and subsequently detected and identified using MS.
Wingren
Christer
C
Department of Immunotechnology, Lund University, Lund, Sweden.
James
Peter
P
Borrebaeck
Carl A K
CA
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*
D020543:Proteome