Glycan Analysis by MSⁿ Spectral Matching

 Place 0.5 μL of a sample solution* onto a target plate (specular surface stainless steel plate, 2 mm diameter) and allow it to dry.

* To obtain reliable data, the sample solution should contain analyte glycan at a concentration of 0.5–2.0 pmol/μL. In addition, the analyte needs to be desalted.

 Cover the dried analyte on the target plate with 0.5 μL of the matrix solution (10 mg/mL of 2,5-dihydroxybenzoicacid in 30% ethanol) and allow it to dry.

 Recrystallize the dried material by adding 0.15 μL of 99.5% ethanol to the matrix-analyte mixture on the target plate.

 Acquire a MS spectrum of the analyte in positive ion mode.

 Acquire MS² spectra of sodium adductions* derived from the glycan analytes. For the acquisition of CID spectra, adjust the CID energy so that the signal of the precursor ion almost disappears. If the intensity of the precursor ion in the spectrum is more than 15% of the base peak, the spectrum must be discarded and reacquired with a larger CID energy. Use an automatic acquisition function** with a regular raster that governs the laser shot patterns.

*Dope 0.5 μL of 10 mM NaCl solution onto the dried analyte when proton adduct ions are predominant.

**Automated acquisition is critical for obtaining reliable data.

 Search for candidate structures of the analyte glycan using GMDB.

 Compare the spectra of the glycan analyte and the various candidates.

 Sometimes multiple spectra display similarity to the analyte MS² spectra. In such cases, manually find signals that can be used to distinguish the spectra by comparing their MS³ spectra.

 Acquire MS³ spectra of the signals in the MS² spectra using the same procedure as described above for MS² acquisition (#5).

 Compare the MS³ spectra between analyte glycan and the candidates.

 Click a link for “m/z” on the left hand column in the top page.

 Input m/z value of a precursor ion of your MS² spectrum, and enter an appropriate value* in the box of “tolerance”.

*The appropriate value is generally from 0.5 to 1.0.

 Click a “Search” button. (Fig. 1)

 “Search Result” window will open. Relevant glycan structures are shown with a tree-structured list of m/z values of precursor ions, in which relevant precursor ions are highlighted in yellow.

 Click the m/z values of MS² precursor in the tree-structured list.

 Click the button “Show Spectra”. (Fig. 2)

 The spectral images appear with the corresponding glycan strucures in the “Spectrum” window. (Fig. 3)

 Where multiple spectra show similarity to the analyte MS² spectra, go back to the “Search Result” window and click the m/z values of MS³ precursor in the tree-structured list of candidates. Find the precursor ions that can be used to discriminate the spectra by comparing their MS³ spectra.

 Click the button “Show Spectra”.