To compare two samples quantitatively, you need to use isobaric tagging.

https://en.wikipedia.org/wiki/Isobaric_tag_for_relative_and_absolute_quantitation

you can do it using label free quantitative methods. And also perform without enrichment as the conditions will be same for both glycated and non-glycated peptides it would be most reliable way to determine their levels. 

Hi Gnanesh,Thanks for your suggestion. If I perform both glycated and non-glycated peptides, how should I identify them in the same run? In addition, the glycated peptides may be covered by the non-glycated peptides due to the high abundant of non-glycated pepetides.

Hi Adam, Thanks for your answer. Do you know how much the collision energy is needed for isobaric tagging labeling? The glycated peptides fragment at relatively low collision energy. If the isobaric tagging labeling need high collision energy, I am afraid that I cannot get a good fragmentation.

Hi Lina,

Sorry to say, I don't know the answer to that question. I suggest you consult with your local MS/proteomics experts.

Hi,

You can check with the following link and references therein

https://en.wikipedia.org/wiki/Label-free_quantification.

As you are doing LC my opinion is that both of them separates well. 

Hi Lina,

firstly, I think you may be referring to 'glycosylation' [post-translational addition of glycans] rather than 'glycation' [formulation/ chemical modification of the protein not in the cell]? The levels of oligosaccharide addition are very different and their behaviour is very different, so for the rest of my answer i will be assuming that you are referring to 'glycosylation' - apologies if I made the wrong assumption.

We specialise in software that can do this task at Protein Metrics, and one of our founders is a world expert in glycosylation analysis. At the PEPTIDE level we can compare all peptides that are modified versus peptides that are not modified, and produce a table or other graphics for the entire protein. 

However that certainly is not the only answer:

- the peptides being compared will have different ionisation efficiencies, and therefore comparing the mass spectrometry signal - whether with isobaric tags or not - needs to be carefully managed if you want to get extremely accurate

- all glycosylated peptides are labile - fragment easily - so you will need orthogonal techniques to make sure that you capture all of the structures. This is a factor for any electrospray technique, and for most MALDI techniques as well. Note that even if you apply low collision energy you are likely to get some fragmentation

- orthogonal techniques for profiling all of the glycosylation structures include a released glycan analysis - e.g. with PNGaseF - which will allow you to analyse all of the glycans on their own, but you will lose positional information. In the biopharma industry this is a standard way of doing a profile though and can dig very deeply with labelled glycans and Fluorescent tags. Quantification can be by area under the curve at the frequency of the tag.

- if you are looking at proteins that have both N-linked and O-linked glycosylations, then you will need to cope with a number of different structures, and a wide variety of possible sites - and that will add to the complexity and perhaps make it difficult to know where you have placed an isobaric tag

- isobaric tags in our software are treated like a modification though, and so you could compare tagged and non-tagged species that way.

Lastly, the type, purity, and size of the protein will make a huge difference to the study, and how you decide to enzymatically digest it will be a factor. So for example if you use multiple enzymes, you can make sure that you localize the sites properly which should help you to then make sure you are comparing the right species.

We have a number of videos and studies available for you to read, and we absolutely love doing this stuff because it is such fun! I have included a link to a publication we did recently with the Heck lab on previously unattested glycosylations in the HIV pathway

http://www.ncbi.nlm.nih.gov/pubmed/26280087

http://www.proteinmetrics.com/solutions/

Regards

St John