Protein splice variant/isoform discrimination and quantitative measurements thereof
View Patent ↗The invention relates to methods, reagents and apparatus for detecting protein isoforms (e.g., those due to alternative splicing, or different disease protein isoforms or degradation products) in a sample, including using combinations of capture agents, each combination being unique to the splicing variant to be detected/measured.
1. A multiplex detection apparatus for detecting plural different protein isoforms in a sample comprising a mixture of proteins, the multiplex detection apparatus comprising: (a) an apparatus or reagents for digesting said mixture of proteins to produce epitope-bearing peptide fragments, such that each epitope-bearing fragment comprises at least portions of the expression products of exon(s) encoding at least a portion of the respective proteins comprising first and second epitope; (b) a set of immobilized capture agents, individual ones of which bind to either first or second epitopes on peptide fragments generated by digestion of proteins in said sample, wherein said first epitopes are different from said second epitopes; and (c) a set of detectably labeled binding agents which bind to respective second epitopes on respective said epitope-bearing peptide fragments, the binding of a capture agent-detectably labeled binding agent pair being unambiguously indicative of the presence of respective isoforms in the sample.
2. The apparatus of claim 1 , wherein said second epitope is produced from an exon different from the exon from which said first epitope is produced.
3. The apparatus of claim 1 , wherein the capture agents are immobilized on a solid surface in an array and the detectably labeled binding agents comprise optically detectable labels.
4. The apparatus of claim 1 , further comprising a protocol specifying directions for digesting said mixture of proteins in said sample to reliably produce said peptide fragments.
5. The apparatus of claim 1 , wherein the capture agents are immobilized on beads.
6. The apparatus of claim 1 , wherein said isoforms are splice variant isoforms.