Lysosomes are composed of soluble and transmembrane proteins that target lysosomes in a signal-dependent manner. Our engineers use a wide range of methods including subcellular isolation, liquid chromatography tandem mass spectrometry (LC-MS/MS) for lysosomal protein identification. Here, CD BioSciences provides specialized lysosomal luminal proteins identification services to support lysosomal proteomic studies.
Background of Lysosomal Luminal Proteins Identification
Lysosomes are acidic organelles capable of degrading most biological macromolecules. They rely on the synergistic action of about 50 soluble acid hydrolases such as glycosidases, proteases, lipases, nucleases, phosphatases and sulfatase enzymes at low pH. Most soluble acid hydrolases are modified with mannose-6-phosphate (Man6-P) residues on their N-linked oligosaccharides, allowing them to be recognized by Man6-P receptors in the Golgi complex, mediating their interaction with the secretory pathway Isolated and delivered to the endosomal/lysosomal system. Man6-P modification is removed differently depending on tissue and cell type. Other soluble enzymes and non-enzyme proteins are transported to the lysosome in an M6P-independent manner. Studies have shown that any abnormality in the system results in the secretion of lysosomal enzymes, and the accumulation of macromolecules within the lysosomes leads to severe lysosomal storage disorders (LSDs). Therefore, many studies have focused on understanding the structural and functional properties of lysosomal enzymes.
Fig. 1. Receptors involved in targeting of soluble lysosomal proteins. (Braulke T, et al., 2019)
Our Lysosomal Luminal Proteins Identification Services
We have successfully obtained high yields of Man6-P lysosomal lumenal protein by an affinity chromatography purification strategy. Currently, CD BioSciences focuses on the identification of lysosomal proteins by mass spectrometry (MS). Here, our engineers take different strategies to provide customers with satisfactory lysosomal luminal protein identification services. In addition, we can also identify the binding site of Man6-P receptors to Man6-P.
- Mass spectrometry-based method
We combined two-dimensional gel electrophoresis (2D-PAGE) with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or static nanoelectrospray ionization tandem mass spectrometry (nano-ESIMS/MS) to analyze lysosomal luminal proteins. This method is commonly used to identify known lysosomal luminal proteins. - Semi-quantitative method
We differentiated specific Man6-P affinity matrix binders from non-specific binders by manual selection, and used specific Man6-P affinity matrix binders for analysis in a variety of different tissues. - Man6-P receptor genes knockout
We induced defective sorting or dephosphorylation of lysosomal hydrolases by knocking out the Man6-P receptor genes in cells or mice, LC-MS/MS was further used to identify lysosomal matrix proteins and potential novel lysosomal proteins.
Our Advantages
- Combining multiple advanced techniques, such as 2D-PAGE, MALDI-MS, nano-ESIMS/MS, gene knockout, etc.
- Experienced team.
- Successful identification of multiple Man6-P glycoproteins and novel lysosomal intraluminal proteins.
- Complete detailed experimental evidences.
- Definition of Man6-P-containing lysosomal proteins based only on their enrichment behavior and/or after deglycosylation allowing analysis of intact Man6-P glycopeptides.
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.
Reference
- Braulke T, Bonifacino J S. (2009) Sorting of lysosomal proteins[J]. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1793(4): 605-614.