Using automated capillary gel electrophoresis to detect multiple proteins from single oocyte and blastocyst

Traditional protein analysis by Western blot often requires a pool of large amount of samples that limits its utilization for human reproductive research. The objective of this study was to develop a highly sensitive and reproducible protocol to analyze metabolic proteins from single oocyte and embryo from humans or animals. Antibodies for metabolic proteins including mitogen-activated protein kinase 1 (MAPK1), signal transducer and activator of transcription 3 (STAT3), AKT serine threonine kinase 1 (AKT1), pyruvate kinase M1/2 (PKM2), pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), their phosphorylated versions (p-protein), and lactate dehydrogenase C (LDHC) were validated using serial dilutions (15ng- 1000ng) of human cumulus cells on the automated capillary gel electrophoresis platform (Jess, ProteinSimple). Multiplex immunoassay was performed with proteins of different band sizes, and the same sample was reconstituted and reused multiple times to maximize the number of proteins to be detected from a single oocyte/blastocyst. Apart from detection of total proteins in each capillary, chemiluminiscent detection of target proteins was automatically performed at exposure times ranging from 4 to 512s. After assay completion, results were generated as electropherograms by Compass software designed for Jess platform. Clear peak at the associated molecular weight (MW) indicated presence of target protein. Using this approach, we examined the expression of multiple proteins from human germinal vesicle (GV) stage oocyte and bovine blastocyst. When validating metabolic protein antibodies in human cumulus cells, we found that all proteins could be detected at the lowest used concentration of 15ng. However, as protein concentration increased, there was also an increase in non-specific peaks and background. Best peaks for all proteins were observed in 50-100ng range. Clear peaks for STAT3, p-AKT, and p-MAPK were observed in one bovine blastocyst. However, when same lysate was reused, only p-PDH peak was observed. Clear peaks of p-PKM2 and p-PDH were observed in one human GV oocyte. Peaks for p-AKT and p-MAPK was observed when this lysate was reused for the first time. Second time reuse revealed detection of LDHC, MAPK1, and PDH. We have shown presence of 11 metabolic proteins in human cumulus cells (15ng to 1000ng). Using Jess platform, we have also detected presence of these metabolic proteins in a single human oocyte and bovine blastocyst by multiplexing as well as reconstituting the same oocyte or blastocyst lysate.