1 Mass Spectrometry of Glycosylation Sites and Structures of the Sugars
The attachment points of glycosylation sites N-linked (through asparagine) and O-linked (through serine), and the structures of the complex carbohydrates, can be determined by mass spectrometry.
1) Adjust the concentration of the sample to the optimum. This is probably between 20 and 100 pmol/μL for carbohydrates and glycosylated polypeptides (this will give a final concentration of 10–50 pmol/μL after mixing 1:1 with the matrix).
2) If the concentration is unknown, make a series of dilutions in water.
3) Prepare the stock DHB matrix solution (10 μg/μL) in 30% aq ACN containing 0.1% TFA.
4) Apply equal volumes of the sample (0.5 μL) and matrix solution (0.5 μL) to the MALDI plate and analyze by MALDI-TOF.
5) To verify the identity of the glycosylated peptides, compare the mass spectra of the digest mixture before and after deglycosylation of the peptides. Use (typically) 100 U PNGase F in 25 mM phosphate buffer, pH 7.4. Incubate at 37°C for 18 h.
6) Post source-decay (PSD)-MS can produce sequence information, and the loss of each monosaccharide unit of distinct mass can be interpreted from the spectra using the "glycomod" Website (see list at end of chapter) to reconstruct the glycosylation pattern.
2 Identification of Disulphide Linkages by Mass Spectrometry
1) Fragment the protein into peptides under low pH conditions to minimize disulphide exchange using pepsin, Glu-C, or cyanogen bromide.
2) Separate one-half of the digest and identify the disulphide-linked peptide fragments under mild oxidizing conditions (i.e., no reducing agents) by HPLC mass spectrometry (LC-MS).
3) Reduce an identical aliquot of the digested peptides with dithiothreitol (DTT) (100 mM) for 2 h at 37°C to cleave –S–S– bonds
4) Re-analyze the digest mixture by on-line LC-MS as before.
5) Identify the peptide(s) that disappear from the spectrum. These were disulphide linked.
6) Examine the peptides that appear at the appropriate positions for the individual components. The sum of their masses should correspond to the disulphide linked-peptides.
3 Selected Ion Monitoring (SIM)
Selected ion monitoring (SIM) is used to look for ions that are characteristic of a particular posttranslational modification in a peptide. This technique has particular application for on-line LC-MS, where the instruments can be set up to monitor selected ion masses as the components elute successively from the capillary LC or reversephase HPLC column. The precise parameters depend on the type of instrument and the software program available.
1) Use the instrument software to set up detection algorithms to carry out tandem MS on each component as it elutes from the chromatography column.
2) Adapt this to enable selective detection of the low-mass fragment ion(s) that are characteristic markers of the presence of particular posttranslational modifications.
3) Print out the chromatogram of the total ion current and, if measured, the ultraviolet (UV) trace (normally at 215 nm).
4) Overlay or superimpose the fragment ion chromatogram to identify the modified peptides.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.
