On October 1 2011 Prot-HiSPRA Consortium started the activities on developing hardware, software, and sample preparation methods for proteomics analysis of minute protein amounts in biological samples. The Consortium set to develop fast, reproducible, and as simple as possible on-line methods for application with biological samples relevant for clinical diagnosis. We have chosen to use spent culturing media utilized in in-vitro fertilization (IVF), analyze the secretome of fertilized oocytes, which are to be re-implanted into women’s uterus during the IVF procedure, and to search for distinct proteins, which could help to better characterize positively and negatively labeled blastocysts.
The idea was based on assumption that growing embryos secrete distinct proteins during their development, and that these proteins can be used to distinguish between embryos with a higher potential for successful pregnancy upon first implantation. Classical proteomics approach for analysis of secreted proteins involves time consuming sample preparation steps and cannot be employed for analysis of samples in situation where clinicians need rapid answers for fast proceedings. Classical proteomics analysis, usually, requires 18-24 hours to deliver the result. However, this time span is too long for the IVF procedure during which the results must be either immediately available, or at latest, within a few hours. In order to provide the analysis of secreted proteins fast, reliable, and reproducible, new analytical approaches for sample preparation, peptide separation, and data analysis are needed. These methods should enable fast depletion of high abundant proteins form spent culturing media; in this case it was human serum albumin, fast protein digestion – minutes instead of hours – and peptide separation with highest possible separation resolution. Peptide and protein identification would be performed by means of mas spectrometry and database search. This goal could be only reached by establishing new methods for sample preparation and developing new hardware for online depletion, online digest, and parallel chromatographic separation of multiple samples simultaneously. At the same time, software solutions for prediction of peptide retention times in HPLC and synchronization of separation and mass spectrometric identification. At the same time, software solutions for prediction of peptide retention times in HPLC and synchronization of separation and mass spectrometric identification were also develped.