Comparative Secretomics Gives Access to High Confident Secretome Data: Evaluation of Different Methods for the Determination of Bona Fide Secreted Proteins

Abstract

Secretome analysis is broadly applied to understand the interplay between cells and their microenvironment. In particular, the unbiased analysis by mass spectrometry‐based proteomics of conditioned medium has been successfully applied. In this context, several approaches have been developed allowing to distinguish proteins actively secreted by cells from proteins derived from culture medium or proteins released from dying cells. Here, three different methods comparing conditioned medium and lysate by quantitative mass spectrometry‐based proteomics to identify bona fide secreted proteins are evaluated. Evaluation in three different human cell lines reveals that all three methods give access to a similar set of bona fide secreted proteins covering a broad abundance range. In the analyzed primary cells, that is, mesenchymal stromal cells and normal human dermal fibroblasts, more than 70% of the identified proteins are linked to classical secretion pathways. Furthermore, 4–12% are predicted to be released by unconventional secretion pathways. Interestingly, evidence of release by ectodomain shedding in a large number of the remaining candidate proteins is found. In summary, it is convinced that comparative secretomics is currently the method of choice to obtain high‐confident secretome data and to identify novel candidates for unconventional protein secretion which have been neglected so far.

Secretomics—A Key to a Comprehensive Picture of Unconventional Protein Secretion

“…Quantitative protein analysis by mass spectrometry is the method of choice in proteomics to characterize cellular compartments ( Itzhak et al, 2017 ), interaction with proteins ( Bensimon et al, 2012 ), nucleotides ( Brillen et al, 2017 ), or drugs ( Savitski et al, 2014 ), as this, in combination with specific fractionation steps, allows the determination of a significantly enriched protein population. We and others have shown that bona fide secreted proteins can be identified regardless of the secretion pathway when secretome and proteome data are compared by so-called “comparative secretomics” approach ( Poschmann et al, 2021 ). Thus, we demonstrated that, depending on the cell line analyzed, comparative secretomics results in a high proportion of bona fide secreted proteins, with more than 30–70% being classically secreted proteins and 4–29% being candidate proteins released via unconventional secretion pathways ( Figure 1A ).…”

“… High-confident secretomes and the human secretome. (A) By means of comparative secretomics approach we were able to generate lists of high-confident secreted proteins of NHDF, MSC and A549 cells including 72–88% of proteins which were predicted to be secreted ( Poschmann et al, 2021 ). (B) Our prediction tool OutCyte was used to estimate the number of candidates LLSP in the human secretome to 3,475 ( Zhao et al, 2019 ).…”

Identification of secreted proteins by comparison of protein abundance in conditioned media and cell lysates

“…We independently confirm the utility of this approach by using TMT-based labeling of lysate and media proteins, followed by label-based quantification of TMT data. Previous analyses have classified proteins as secreted if their mean abundance in the conditioned media was at least 1.5-fold higher relative to the cell lysate [17]. To identify candidate UCPS cargoes, we applied more stringent criteria based on prioritizing signal-peptide lacking, BFA-resistant proteins with the highest M/L ratios.…”

The secrets of protein secretion: what are the key features of comparative secretomics?

“…Therefore, comparative secretomics was developed to allow the direct experimental identification of secreted proteins by omitting bioinformatic annotations as a decisive step. Comparative secretomics have shown to be robust and give access to high-confident data with a high proportion (>80%) of bona fide secreted proteins and a fivefold higher identification rate if consideration was not limited to proteins exclusively identified in the secretome [6]. Nevertheless, further research is necessary to better understand the composition of the secretome, because it depends on different criteria, such as cell type and culture condition.…”

“…Thus, alternative approaches have been developed and applied to unravel these mechanisms of protein secretion. In this context, comparative secretomics has proven to be a versatile tool to allow full characterization of the secretome [6].…”