Protein glycosylation is important (Cell, 126, 855-867, 2006; Nat. Rev. Cancer., 5, 526-542, 2005), yet N-glycan structure characterization at high throughput is extremely challenging due to their structure complexity and lack of databases (Proteomics, 8, 8-20, 2008).

Glyquest is a software tool for mass spectrometry (MS) analysis of N-glycan structures. It has a large N-glycan structure database that can be searched by a built-in database search engine. The database can be expanded or customersized to include user specified or modified N-glycan structures. Glyquest can quickly search thousands of tandem (MS/MS) mass spectra of glycopeptides against the database, find glycopeptide spectra, assign N-glycan structures to the spectra and annotate fragment peaks graphically.

The work has been published on line by JASMS, http://dx.doi.org/10.1016/j.jasms.2009.05.012 and will be published in print in the July 2009 issue of the journal (Journal of the American Society for Mass Spectrometry).

(Glyquest is commercially available now. For more information, please contact: info@biopharmasoft.com )

We are eager to serve the glycomics community and we are looking for collaborators.

Probably one way to start our cooperation is:

1: you send us one DTA (MS/MS spectrum) which you've annotated manually (i.e., you know the N-glycan structure and the attached peptide (or other moiety)).
2: we analyze the spectrum using Glyquest and send back the annoted spectrum.
3: if you like the result, we could send you an evaluation version of Glyquest (valid for 30-60 days).
4: if you like the evaluation version, we could discuss further.

Though Glyquest is easy to install and easy to use, the ideal scientist to evaluation Glyquest would be someone who has experience manually annotating tandem mass spectra of N-glycan structures and (preferably) has some computer skills.

Use of ETD/ECD for peptide sequence confirmation:

Glyquest searches for various N-glycan structure attached to a given peptide based on the molecular weight of the peptide (or other moiety). The spectra are collision induced dissociation (CID) tandem mass spectra (MS/MS) of glycopeptides. To verify or determine the peptide sequence, electron transfer dissociation (ETD) or electron capture dissociation (ECD) can be used. For example, with an LTQ mass spectrometer (Thermo Scientific) with ETD capability, a typical top 10 data dependent MS method can be modified as below:

Myths about N-glycan structure analysis by mass spectrometry:

- Myth 1: N-glycans do not ionize well, so chemical derivatization is necessary for ESI (electrospray ionization) analysis. This may be true for bare N-glycans, but tryptic glycopeptideds have basic residues which enable proper ionization by ESI.

- Myth 2: One has to find new chromatographic separation methods (such as HILIC) to separate N-glycans. Again, it may be true for bare N-glycans, but glycopeptides can be separated into groups based on their peptide part by regular C18 columns. Modern mass spectrometers may be fast enough to obtain tandem mass spectra of different N-glycan forms attached to the same peptide.

- Myth 3: Sialic acid residues fall off the N-glycan structures easily, therefore, information on the intact N-glycan structure cannot be obtained. This may be true for MALDI, but may not be true for ESI.

In short, we think that many proteomics laboratories may be suited for high throughput N-glycan structure analysis using existing ion trap mass spectrometers and existing experimental setup. One could also determine/confirm the peptide sequence if ETD or CID is available.

To test what we said above, one can digest a pure glycoprotein (for example, IgG, immunoglobulin G, in the picomole range) using trypsin and run it just like a regular protein digest (using a C18 column, the regular reverse phase LC gradient, positive ESI, an ion trap mass spectrometer, and the same collision energy).

(This site is under construction. Your suggestions are greatly appreciated.)