Number of Detected Proteins as the Function of the Sensitivity of Proteomic Technology in Human Liver Cells

Abstract

Aims: The main goal of the Russian part of C-HPP is to detect and functionally annotate missing proteins (PE2-PE4) encoded by human chromosome 18. To achieve this goal it is necessary to use the most sensitive methods of analysis. Background: However, identifying such proteins in a complex biological mixture using mass spectrometry (MS)-based methods is difficult due to the insufficient sensitivity of proteomic analysis methods. A possible solution to the problem is the pre-fractionation of a complex biological sample at the sample preparation stage Objective: The aims of this study are to measure the detection limit of SRM SIS analysis using a standard set of UPS1 proteins and find a way to enhance the sensitivity of the analysis and to detect proteins encoded by the human chromosome 18 in liver tissue samples and compare the data with transcriptomic analysis of the same samples. Methods: Mass spectrometry, data-dependent acquisition, selected reaction monitoring, high-performance liquid chromatography, data-dependent acquisition in combination with pre-fractionation by alkaline reversed-phase chromatography, selected reaction monitoring in combination with pre-fractionation by alkaline reversed-phase chromatography methods were used in this study. Results: The results revealed that 100% of UPS1 proteins in a mixture could only be identified at a concentration of at least 10-9 М. The decrease in concentration leads to protein losses associated with technology sensitivity, and no UPS1 protein is detected at a concentration of 10-13 М. Therefore, the two-dimensional fractionation of samples was applied to improve sensitivity. The human liver tissue was examined by selected reaction monitoring and shotgun methods of MS analysis using one-dimensional and two-dimensional fractionation to identify the proteins encoded by human chromosome 18. A total of 134 proteins were identified. The overlap between proteomic and transcriptomic data in human liver tissue was ~50%. Conclusion: The sample concentration technique is well suited for a standard UPS1 system that is not contaminated with a complex biological sample. However, it is not suitable for use with a complex biological protein mixture. Thus, it is necessary to develop more sophisticated fractionation systems for the detection of all low-copy proteins. This weak convergence is due to the low sensitivity of proteomic technology compared to transcriptomic approaches. Also, total mRNA was used to perform RNA-seq analysis, but not all detected mRNA molecules could be translated into proteins. This introduces additional uncertainty in the data; in the future, we plan to study only translated mRNA molecules-the translatome. Data is available via ProteomeXchange with identifier PXD026997.

Some structural features of the peptide profile of myelin basic protein-hydrolyzing antibodies in schizophrenic patients

“…Based on the values of the relative molar content of riBAQ proteins, it can be seen that the purity of immunoglobulins has reached more than 92%, and the rest of the proteins are impurities from plasma proteins, that were identified due to the high sensitivity of mass spectrometry. Shotgun MS is capable to detect proteins at a concentration of 10 −9 M–10 −12 M ( Vavilov et al, 2022 ). Standard Coomassie staining, used in the work, allows visualization from 100 ng of protein in a SDS-PAGE spot ( Weiss, Weiland & Görg, 2009 ), which exceeds the detection limits of mass spectrometry by 2–5 orders of magnitude in terms of protein amount.…”