This study has been done by all the authors listed below, who share the authorship. The abstract has been submitted by the coordinator of the EuPA Standardization Initiative.
Single cell analysis at the proteome level has become feasible as new technologies for sample preparation and mass spectrometry analysis ensure efficient protein extraction and processing, as well as analytical sensitivity in the attomolar range. However, the implementation of high sensitivity proteomics (HSP) protocols remains a challenge. In this context, the EuPA standardization initiative launched the Proteomics Multicentric Experiment (PME13) with the main objectives of benchmarking HSP analysis protocols, providing tools to facilitate HSP implementation and assessing laboratory performance. The reference sample used in the study consisted of a serial dilution of E. coli protein extract from 100 to 1 ng, provided to participants in intact and trypsin digested formats. A pilot analysis was conducted to optimise the analytical pipeline and to minimize sources of uncertainty. After analysis, DDA and DIA datasets were generated and submitted for centralized processing along with metadata specific to each participant. Data were collected via NextCloud, processed using MaxQuant v2.0.2.0 and integrated, visualized and analyzed using R. Searches were mainly performed using default parameters, agreement between runs within the same concentration level and sample, and 1% FDR at peptide and protein level. Thirty-six laboratories from eight different countries requested PME13 samples, almost half of which (17 out of 36) had not participated in previous PMEs. We received DDA results from 28 out of 36 laboratories (78%) and DIA results from 16 out of 23 laboratories (70%). Overall, our data indicate that the reference sample provides a reliable basis for benchmarking, establishing protocols and assessing performance. The influence of analytical variables and the correlation with the physico-chemical properties of the analyzed peptides are presented and discussed.