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Direct comparison of MS-based label-free and SILAC quantitative proteome profiling strategies in primary retinal
Juliane Merl1, Marius Ueffing, Stefanie M Hauck
1Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany.
Abstract:
To better understand the involvement of retinal Müller glial (RMG) cells in retinal diseases, we phenotyped primary porcine RMGs in dependence of cultivation time using different quantitative proteomic strategies. A well-established LC-MS/MS-based quantification method was employed: stable isotope labeling by amino acids in cell culture (SILAC) and directly compared to label-free (LF) quantifications, based on total peak intensities using two different programs (MaxQuant and Progenesis LC-MS). The overall numbers of detected proteins were largely similar (overlap of 1324 proteins), only a total of 173 proteins were significantly altered between the different culture conditions. However, among these, only 21 proteins were shared between the three analytical strategies. Hence, the majority of altered proteins only reached significance thresholds in one of the applied analyses with a larger overlap between the two LF approaches. Among the shared, differentially abundant proteins were known RMG markers as well as new proteins associated with glial cell transition. However, proteins correlated to cellular transitions and dedifferentiation were also found among the proteins only significant in one or two of the applied strategies. Consequently, the application of different quantification and analytical strategies could increase the analytical depths of proteomic phenotyping.
Insights
Comparing proteomic strategies for retinal Müller glial (RMG) cells revealed that different quantification methods identify unique proteins. Combining analyses enhances the depth of proteomic phenotyping for retinal disease research.
Area of Science:
- Ophthalmology
- Proteomics
- Cell Biology
Background:
- Retinal Müller glial (RMG) cells play a crucial role in retinal health and disease.
- Understanding RMG cell behavior requires accurate phenotyping.
- Proteomic strategies are essential for detailed cellular analysis.
Purpose of the Study:
- To compare different quantitative proteomic strategies for phenotyping primary porcine RMG cells.
- To assess the impact of cultivation time on RMG cell proteomes.
- To identify differentially abundant proteins and understand cellular transitions.
Main Methods:
- Utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS).
- Employed stable isotope labeling by amino acids in cell culture (SILAC) and label-free (LF) quantification.
- Compared MaxQuant and Progenesis LC-MS software for LF analysis.
Main Results:
- High overlap in detected proteins across strategies (1324 proteins).
- Only 173 proteins showed significant alterations between culture conditions.
- A small subset (21 proteins) was consistently identified across all three analytical strategies.
- Label-free approaches showed greater overlap than SILAC.
- Identified known RMG markers and novel proteins related to glial cell transition and dedifferentiation.
Conclusions:
- Different proteomic quantification and analysis strategies yield distinct results.
- Combining multiple strategies increases analytical depth for RMG cell phenotyping.
- This approach can reveal subtle changes related to cellular transitions relevant to retinal diseases.
