Glyquest is a software tool for mass spectrometry (MS) analysis of N-glycan structures (see References at the end).

Glyquest has a large, built-in N-glycan structure database that can be searched by a built-in database search engine. The database can be customersized to include user specified or specially modified N-glycan structures. Glyquest quickly searches thousands of tandem (MS/MS) mass spectra of glycopeptides against the database, finds glycopeptide spectra, assigns N-glycan structures to the spectra and annotates fragment peaks graphically.

Glyquest finds the N-glycan structures attached to glycopeptides based on CID MS/MS spectra (collision induced dissociation tandem mass spectrometry spectra). Glyquest's built-in mammalian N-glycan structure database is compresive and can include ALL known N-glycan structures.

As inputs, Glyquest accepts 1) a folder containing many MS/MS spectra from one LC-MS run, and 2) the amino acid sequence of the peptide part of a glycopeptide (or the molecular weight of the chemical label used for derivatization). Then Glyquest

- searches through all the experimental MS/MS spectra
- finds all potential glycopeptide spectra
- for each of the potential spectra, finds a list of N-glycan structure candidates from the built-in database
- for each of the candiates, finds a similarity score between the theoretical fragment spectrum of the candidate and the experimental glycopeptide spectrum
- reports the top three N-glycan structures with the highest scores (for each of the experimental glycopeptide spectrum)
- finally, draws a fragment N-glycan structure graph for EACH of the matched peaks on each of the experimental glycopeptide spectum that a user chooses to view

All the N-glycan structures on one glycosylation site can be found in one search.

Glyquest does all the above automatically. Processing 5000 spectra takes about 30 minutes on a typical personal computer. Glyquest saves hours, days, or even weeks of manual searching, matching and annotating by highly skilled researchers.

Glyquest works with folders containing thousands of MS/MS spectra. You do not have to find the glycopeptide spectra first. You do not have to input the spectra one by one.

The success of proteomics was largely due to the availability of protein databases. Protein N-glycosylation has been a daunting and challenging task because the complexity of N-glycan structures and the very limited availability of N-glycan structure databases. The few available databases (such as CarbBank) cannot be accessed easily for mass spectrometric data analysis. Glyquest greatly simplifies this challenging task, making high throughput glycomics and protein glycosylation research accessible to most proteomics labs.

Glyquest finds or predicts N-glycan structures attached to glycopeptide based on LC-MS/MS data (liquid chromatography tandem mass spectrometry). Glyquest can also find N-glycan structures released from glycoproteins, non-derived or derived by chemical modifications or chemical labeling, such as using 2-aminobenzamide (2-AB). Current version does not work with permethylated glycans.

If desired, the peptide sequence can be further confirmed by using ETD together with CID in the same LC-MS analysis (ETD: electron transfer dissociation). On an ETD equipped LTQ, the instrument method could be setup as the following:

Event 1: full MS scan;
Event 2: CID on the most intense peak;
Event 3: ETD on the most intense peak;
Event 4: CID on the second most intense peak;
Event 5: ETD on the second most intense peak;
......

Glyquest accepts data in Thermo's (or ThermoFisher Scientific's) DTA format, in the raw file format, mzData and mzXML format.

The figure below shows an N-glycan structure found on the peptide NEEYNK of human alpha-1-acid glycoprotein. The MS/MS scan number is 1543 with a precursor charge of 3+ in the raw file of a six protein mixture acquired on a Thermo's LCQ mass spectrometer. The raw file can be found at http://bioinformatics.icmb.utexas.edu/OPD (The Acc# is opd00099_PROTS. The raw file name is 6prot_75pmol_each).

The screen shot below shows the user interface of Glyquest.

Glyquest is commercially available now. For more information, please contact: info@biopharmasoft.com or h.gao@biopharmasoft.com For information on the published research, please contact the author at h.gao@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 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.

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 labs are 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).

References:

Glyquest is commercially available now. For more information, please contact: info@biopharmasoft.com or h.gao@biopharmasoft.com For information on the published research, please contact the author at h.gao@biopharmasoft.com