Cell biology provides the methods needed to analyze the composition and properties of purified cellular components. Combining subcellular isolation and proteomics facilitates the enrichment and analysis of lysosomes and low-abundance multiprotein complexes. Here, CD BioSciences provides an efficient service for the analysis of lysosomal proteins in whole-cell subcellular separations to support lysosomal proteome research.
Background of Lysosomal Proteins in Whole Cell Subcellular Fractionation Analysis
Two general protein identification methods have been used to study the lysosomal proteome. One approach focuses on soluble proteins, using affinity purification to isolate proteins targeted to lysosomes via the mannose 6-phosphate (Man6-P) pathway. Another approach uses subcellular separation to isolate membranes rich in lysosomal proteins. Among them, subcellular separation is a flexible and tunable method that reduces sample complexity and can be effectively combined with high-resolution 2D gel, mass spectrometry techniques. Combined with MS-based protein identification methods, subcellular isolation has been very useful in identifying many candidate lysosomal proteins, and the distribution of subcellularly localized proteins has been studied at the whole proteome level.
Fig. 1. Subcellular fractionation and purification of lysosomal fractions for proteomic analysis. (Markmann S, et al., 2017)
Our Lysosomal Proteins in Whole Cell Subcellular Fractionation Analysis Services
One approach to lysosomal functional characterization is to use proteomic approaches to identify proteins in subcellular fractions rich in this organelle. Our engineers develop subcellular isolation methods to improve the accuracy of lysosomal proteomics. CD BioSciences is dedicated to combining subcellular isolation with mass spectrometry and protein sequencing for proteomic analysis of lysosomes and related organelles. Here, we provide the following strategies to analyze lysosomal proteins in whole-cell subcellular isolates.
- We use quantitative mass spectrometry methods to measure protein distribution over a gradient and label protein distribution by reference to the distribution location. We use quantitative isotope labeling or label-free methods to measure protein distribution, which can predict the localization of potentially new lysosomal proteins.
- Density variation is a specific marker of lysosomal proteins. We use methods that specifically alter lysosomal buoyant density changes, such as treating animals with Triton WR-1339 (Telosapol) or treating cultured cells with progesterone prior to tissue harvest and subcellular isolation. Our goal is to measure characteristic changes in their density by gradient centrifugation for use in identifying lysosomal candidates for proteomics experiments.
In the characterization of the lysosomal proteome by subcellular isolation, we can also distinguish novel lysosomal protein candidates from contaminants and other types of proteins by LC-MS/MS techniques.
Our Advantages
- Subcellular isolation provides a powerful tool to determine the subcellular localization of proteins of interest.
- Combining selectivity of changes in lysosome density with tandem mass spectrometry.
- Enables protein identification and quantification of isobarically labeled samples.
- Contributes to understanding lysosome complexity and function in biology and medicine.
- Enables tracking of the total distribution of candidate proteins in all subcellular and extracellular fractions.
We look forward to collaborating with you. You are always welcome to engage in discussions with us at any point of the project. If you are interested in our services, please feel free to contact us for more information.
References
- Markmann S, Krambeck S, Hughes C J, et al. (2017) Quantitative proteome analysis of mouse liver lysosomes provides evidence for mannose 6-phosphate-independent targeting mechanisms of acid hydrolases in mucolipidosis II[J]. Molecular & Cellular Proteomics. 16(3): 438-450.
- Della Valle MC, Sleat DE, et al. (2011) Classification of subcellular location by comparative proteomic analysis of native and density-shifted lysosomes. Mol Cell Proteomics.10(4): M110.006403.