BioPharmaSoft

GlycoPro - for automatic glycan MS2 spectrum interpretation

Protein glycosylation is important, yet N-glycan structure characterization is extremely challenging due to glycan structure complexity, as agreed by experts attending the Frontiers in Glycomics: Bioinformatics and Biomarker in Desease (USA, September, 2006, or the publication: Proteomics, 2008, 8, 8-20). 

Currently, many researchers painstakingly interprete MS or MS2 spectra of N-glycans manually.  The world needs high throughput tools for glycoprotein characterization.

BioPharmaSoft took up the challenge and developed a software tool, GlycoPro, which helps users to find N-Glycan structures attached to peptides from thousands of tandem mass spectra of glycopeptides of human or mouse origin, automatically, in minutes or hours on a personal computer.  GlycoPro represents a significant step toward true high throughput glycomics.    

GlycoPro features

Currently, GlycoPro has a proprietary, built-in N-glycan structure library with over 140,000 entries.  Included in the database are glycans with acetylated, phosphorylated and sulfated monosaccharides.  The library will be expanded later to include glycans with other modified monosaccharides.

GlycoPro uses a special, proprietary process to access the large library and matches correct N-glycan structures to MS2 spectra.  It scores each hit and displays a graphical drawing of the N-glycan fragment for each and every of the matched MS/MS peaks.  Allowing quick, graphical inspection of the matched fragment peaks is crucial for establishing confidence in high throughput N-glycan structure determination by mass spectrometry.

GlycoPro takes an MS2 data folder name and the average MH+ of the peptide backbone (or the peptide sequence) as its input and finds N-glycans attached to the peptide.  The MS2 spectra (in DTA format) can be ALL those that are obtained from an LC-MS/MS run of a tryptic digest of one or a mixture of a couple of glycoproteins, under the same experimental conditions as in a typical proteomics experiment using mass spectrometry.

Figure 1 (below) shows a possible N-glycan structure found by GlycoPro, and automatic peak assignments of the glycan fragment ions to the peaks of an MS2 spectrum.   The peptide attached to the glycan has an average MW (MH+) of 1129.3 Da.  GlycoPro also identifies B and Y ion peaks due to re-arrangements. 

Note that for a Y ion, a black "+" is drawn below the fragment to show that the peptide is attached to the fragment. For a B ion, no black "+" is drawn.  Also on the plot, one red "+" means that it is an 1+ ion, and two red "+" means that it is a 2+ ion,  etc..  All the symbols on the plot are drawn  automatically by GlycoPro.  The MS2 spectrum was identified from thousands of MS2 spectra obtained from one LC-MS run, with the loading of less than 1 pmol of a tryptic digest of a glycoprotein.

Similarly, Figure 2 shows the N-glycan structure found on a human IgG peptide, EEQYNSTYR. 

Figure 3 shows the N-glycan structures found on a bovine fetuin peptide, LCPDCPLLAPLNDSR.

Figure 1. Glycan fragment ions assigned automatically to peaks of an MS2 spectrum of a glycopeptide.  The average MW of the peptide (MH+) is 1129.3 Da.  Note the B and Y ion peaks due to re-arrangement.

Figure 2. The N-Glycan structure found on the human IgG peptide "EEQYNSTYR" which has an average MW (MH+) of 1190.2 Da.

Figure 3.  The N-glycan structure found on the bovine fetuin peptide “LCPDCPLLAPLNDSR”that has an average MW (MH+) of 1742.0 Da.

NOTE: about the MS2 data used above

The MS2 data were obtained on a Thermo Finnigan's LTQ ion trap mass spectrometer with less than 1 pmol of tryptic digests of pure glycoproteins from commercial sources, using exactly the SAME conditions as in a typical  proteomics setup: the same sample preparation procedure, the same LC-MS setup, the same C18 column and the same elution gradient, and the same mass spectrometer parameters (e.g., collision energy).  If you have older MS2 data of pure glycoproteins (such as IgG), you can use them to test the performance of GlycoPro without generating more MS2 specifically.

Contact us today

BioPharmasoft is located in Boston, MA, USA.  We invite you to try GlycoPro for 30-days for free.  Please contact us.