Thank you for your response!

Thank you for your question! Unfortunately, I do not have much experience with plant cells, but my understanding is that the field is much less developed for this type of samples, while most focus has been put on the characterization of eukaryotic cells. However, I believe it is possible to apply this protocol - with due modifications and optimizations - to plant cells. And more in general lines, any sample you can extract proteins from could be used as an input for glycoproteomics workflows.

Thank you for your question! In theory, any sample you can extract proteins from can be used as an input for glycoproteomics workflows. Typically these could be cell pellets, tissues, organs or even entire organisms (flies, zebrafish, etc.). For more complex samples you might need to homogenize the input sample for a optimal solubilization with detergents. I sadly do not have experience with plant cells, but my guess is that it is possible to adapt published protocols (mainly developed for eukaryotic cells) to plant cells. Regarding the equipment/supplies, we will go through this part during the webinar, but please feel free to reach out after the presentation should anything remain unclear.

Thanks for the question. I sadly do not have experience with GAGs or pentosan. Nevertheless, the genetic engineering approach introduced in this webinar allowed also the study of GAGs from a biosynthesis perspective. If you are interested, I would recommend the work from my colleagues at the Copenhagen Center for Glycomics (https://glycomics.ku.dk), eg. PMID: 30104636 or PMID: 34936441.

Thank you for your question! Unfortunately, I do not have direct experience with plant cells, but I believe it is possible to apply this protocol - with due modifications and optimizations - to plant cells. In general lines, any sample you can extract proteins from can be used as an input for glycoproteomics workflows.

Thank you for your question. During the webinar, I will provide a practical example of a glycoproteomics workflow used for the characterization of O-mannosylation in humans, where I will refer to the instruments and major reagents used to this aim. However, if you have any further specific questions about this after the talk, please do not hesitate to intervene during the Q&A session.

Thank you for your question. Glycans can be used to purify the protein they are conjugated to, and there are several methods that could be explored for this purpose. During the webinar I will discuss about strategies for enrichment of glycosylated peptides (and proteins) - for example using chemical tools, lectins and antibodies - and I think that part of the talk might give you an idea of what are the tools currently available to this aim.

Thank you for your question. During the webinar, I will aim to put these techniques in context, both in terms of the limitations they attempt to address and the impact they have on characterizing this fascinating and complex modification. However, if you have any further questions about this after the talk, please do not hesitate to intervene during the Q&A session.

Thanks for the question. We will see in the webinar that glycoproteomics methods can shed light on the glycosylation status not only of "healthy" cells, but also in presence of disease. For example, glycoproteomics has helped understanding if alterations to the glycosylation landscape are at the basis of an observed human disease, guiding the selection of possible candidate proteins to further investigate, while proposing a molecular mechanism for pathogenesis. These topics will be covered during the webinar, where I will bring the example of how protein O-mannosylation is a crucial modification in human health and disease.

Thank you for the question! Main workflows for proteome-wide glycoproteomics are rather versatile and could indeed be used to elucidate if glycosylation changes are associated with cell dynamics. For this aim, you could consider using quantitative MS/MS approaches based on protein- (eg. SILAC) or peptide-level labelling (eg. dimethyl, diethyl, TMT,...) to compare the glycosylation status between two (or more) conditions, treatments and so on. The attached reference could give you an example of how glycoproteomics (combined with glycomics in this case) can be applied in a similar setup. I hope this can be of help, please do not hesitate to raise the point during the Q&A session!

Thank you for your question. In summary, glycoproteomics is arguably the only technique that allows for the study of protein glycosylation, the most complex and possibly most abundant modification found in a cell, also on a proteome-wide scale. This provides information on a cell/tissue's glycosylation status both in its native state and during disease progression, allowing researchers to determine whether changes in glycosylation patterns underpin disease mechanisms. During the webinar, I will provide a broader perspective on the need for this tool and its potential applications in scientific research, which may shed more light on this.

Thank you for your question! I am not an expert on this topic, but I am aware there are increasingly more tools to study glycosylation in microbial organisms. For example, directly related to microbial glycoproteomics, I would refer you to this review: PMID: 28365498; while for a more general overview I would recommend you check out the chapter of the Essentials of Glycobiology (PMID: 35536956), which also contains several references that might be of help.

Thank you for reaching out and for your interest! Please do not hesitate to read more about our research and contact me or any of my colleagues at the Copenhagen Center for Glycomics using the contacts at https://glycomics.ku.dk/.

Thank you for your question. Analyzing a cell's glycosylation status may help determine whether a specific type of protein glycosylation (even at a specific site) is required for further "regulation" of a protein, such as turnover, folding, or modification. The crosstalk between (O-GlcNAc type) glycosylation and protein phosphorylation, as well as the role of glycosylation in protein folding, have all been extensively studied, and might represent examples of this.

Thank you for your question. In summary, glycoproteomics is arguably the only technique that allows for the study of protein glycosylation, the most complex and possibly most abundant modification found in a cell, also on a proteome-wide scale. This provides information on a cell/tissue's glycosylation status both in its native state and during disease progression, allowing researchers to determine whether changes in glycosylation patterns underpin disease mechanisms. During the webinar, I will provide a broader perspective on the need for this tool and its potential applications in scientific research, which may shed more light on this.

Thanks for the question! The best way to get hands-on practice is to join someone with experience during the preparation method and data analysis process. This webinar might not be the best format to see the process "in the making", however I will be very happy to provide details, eg. on the instruments and reagents used, should you be interested to introduce a similar workflow in your research.