Pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis

Schira‐Heinen, Jessica; Grube, Leonie; Waldera-Lupa, Daniel M.; Baberg, Falk; Langini, Maike; Etemad-Parishanzadeh, Omid; Poschmann, Gereon

doi:10.1016/j.bbapap.2019.06.004

Cited by 16 publications

(13 citation statements)

References 117 publications

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“…Systematic identification and quantification of secretome proteins are commonly done using conditioned medium of a (primary) cell type and its analysis by mass spectrometry-based proteomics. A major challenge is the low concentration of secreted proteins within the conditioned medium (Schira-Heinen et al, 2019). Therefore, many studies concentrate medium from tens of millions of cells (Kleifeld et al, 2010(Kleifeld et al, , 2011Kuhn et al, 2012;Wiita et al, 2014;Schlage et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

An optimized quantitative proteomics method establishes the cell type‐resolved mouse brain secretome

et al. 2020

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To understand how cells communicate in the nervous system, it is essential to define their secretome, which is challenging for primary cells because of large cell numbers being required. Here, we miniaturized secretome analysis by developing the "highperformance secretome protein enrichment with click sugars" (hiSPECS) method. To demonstrate its broad utility, hiSPECS was used to identify the secretory response of brain slices upon LPSinduced neuroinflammation and to establish the cell type-resolved mouse brain secretome resource using primary astrocytes, microglia, neurons, and oligodendrocytes. This resource allowed mapping the cellular origin of CSF proteins and revealed that an unexpectedly high number of secreted proteins in vitro and in vivo are proteolytically cleaved membrane protein ectodomains. Two examples are neuronally secreted ADAM22 and CD200, which we identified as substrates of the Alzheimer-linked protease BACE1. hiSPECS and the brain secretome resource can be widely exploited to systematically study protein secretion and brain function and to identify cell type-specific biomarkers for CNS diseases.

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Section: Introductionmentioning

confidence: 99%

An optimized quantitative proteomics method establishes the cell type‐resolved mouse brain secretome

et al. 2020

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“…Moreover the secretion rate varies among cell types and amounts of proteins in the CM additionally depend on incubation length, protein degradation, and factors like cytokines and nutrients in the medium. [ 20 ]…”

Section: Discussionmentioning

confidence: 99%

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

2020

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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.

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“…For mass spectrometry analysis, we used four independent biological replicates of MSCs. Confluent MSCs were washed three times with PBS in order to remove the FBS and were then incubated for another 48h in serum-free N2-medium [22,70,71] consisting of Dulbecco's modified Eagle's medium (DMEM, Gibco Cell Culture, Thermo Fisher Scientific, Darmstadt, Germany) containing 5 mg/mL insulin, 30 nM sodium selenite, 100 µM putrescine, 20 nM progesterone, and 5 µg/mL transferrin (all Sigma-Aldrich Chemie GmbH). Medium was changed again after 48 h to further exclude FBS effects, and was then incubated for another 48 h in N2 medium.…”

Section: Preparation Of Mesenchymal Stem Cell Secretome and Proteomementioning

confidence: 99%

“…The corresponding factors are likely secreted or shedded, and their identification might therefore provide important information on how existing limitations regarding successful myelin repair might be overcome. However, the nature of MSC-derived differentiation and maturation factors acting on the oligodendroglial lineage have not been disclosed thus far [21] and might therefore to be deciphered by sophisticated strategies such as mass spectrometry-based secretome analysis [22]. Although MSC secretome and proteome data already exist, most of them deal with their anti-inflammatory properties [23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Secretome Analysis of Mesenchymal Stem Cell Factors Fostering Oligodendroglial Differentiation of Neural Stem Cells In Vivo

Agrelo

Schira‐Heinen

Beyer

et al. 2020

IJMS

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Mesenchymal stem cell (MSC)-secreted factors have been shown to significantly promote oligodendrogenesis from cultured primary adult neural stem cells (aNSCs) and oligodendroglial precursor cells (OPCs). Revealing underlying mechanisms of how aNSCs can be fostered to differentiate into a specific cell lineage could provide important insights for the establishment of novel neuroregenerative treatment approaches aiming at myelin repair. However, the nature of MSC-derived differentiation and maturation factors acting on the oligodendroglial lineage has not been identified thus far. In addition to missing information on active ingredients, the degree to which MSC-dependent lineage instruction is functional in vivo also remains to be established. We here demonstrate that MSC-derived factors can indeed stimulate oligodendrogenesis and myelin sheath generation of aNSCs transplanted into different rodent central nervous system (CNS) regions, and furthermore, we provide insights into the underlying mechanism on the basis of a comparative mass spectrometry secretome analysis. We identified a number of secreted proteins known to act on oligodendroglia lineage differentiation. Among them, the tissue inhibitor of metalloproteinase type 1 (TIMP-1) was revealed to be an active component of the MSC-conditioned medium, thus validating our chosen secretome approach.

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Pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis

Cited by 16 publications

References 117 publications

An optimized quantitative proteomics method establishes the cell type‐resolved mouse brain secretome

An optimized quantitative proteomics method establishes the cell type‐resolved mouse brain secretome

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

Secretome Analysis of Mesenchymal Stem Cell Factors Fostering Oligodendroglial Differentiation of Neural Stem Cells In Vivo

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