Glycosylation is an important post-translational modification associated with many proteins that have a regulatory function. Several LC-MS/MS approaches have been used for analysis and structural elucidation of glycoproteins. Gargi Choudhary, Jae Schwartz, Diane Cho report.
Most commonly, a glycoprotein is enzymatically digested, and the resulting fragments are fractionated by reversed phase LC. The peptide fractions may be either analysed by on-line MS/MS, or collected and analysed off-line by MALDI.
Peptides that do not correspond to predicted masses may represent glycosylated forms. These peptides are subsequently treated with a glycosidase to cleave off the oligosaccharide.
The difference in the mass following a cleavage is used to infer the carbohydrate constituents.
Generally, this method does not allow determination of the oligosaccharide structure or of the exact site of its attachment to the peptide. Glycoproteins can be challenging to analyse because they are generally present in low concentration in cells.
In addition, glycopeptides are often hydrophilic and do not bind well to the reversed phase column used in analysis, making determination of the position of elution difficult. This report describes a method for using a Data Dependent Neutral Loss experiment and the high sensitivity MSn capabilities of the Finnigan LTQ linear ion trap mass spectrometer to characterise glycoproteins.
The goal is to develop a selective and sensitive LC-MS method for unambiguous identification and characterisation of glycoproteins.
The experimental conditions are:
Sample Prep. Ribonuclease B was reduced with dithiothrietol (DTT), alkylated with iodoacetic acid, and then enzymatically modified.
HPLC. The digested protein was separated on a
100 x 0.15mm column packed with 5µM Vydac C-18 stationary phase. The Finnigan Surveyor MS Pump was used with the following gradient conditions:
Solvent A: water/0.1percent formic acid
Solvent B: acetonitrile/0.1percent formic acid
Gradient: 5percent to 60percent B in 20 min.
60percent to 80percent B in 2 min.
80percent B for 5 min.
Eluted peptides were analysed on a Finnigan LTQ linear ion trap mass spectrometer, equipped with a Nanospray ion source operated at 1.7kV spray voltage and 150oC heated capillary temperature. A Data Dependent Neutral Loss experiment was performed with a collision energy of 25percent and a neutral loss mass width of 0.2µ. The Data Dependent acquisition parameters are shown in Fig.1.
Results and discussion
When glycopeptides are fragmented in tandem mass spectrometry, they typically exhibit neutral losses corresponding to the mass of a monosaccharide moiety.
Table 1 shows common monosaccharide components of glycoproteins and their corresponding neutral loss masses. High mannose glycoproteins such as ribonucleaseB show losses of 162.1µ for a singly-charged ion, or 81.05µ for a doubly-charged ion. Therefore, Data Dependent settings were chosen to trigger an MS3 scan when a peptide showing a neutral loss of 81.05µ is detected.
Fig.2 shows a flow chart of scan events in the Data Dependent Neutral Loss experiment. First, an MS survey scan is taken. The five most intense peaks from the survey scan are chosen for MS/MS scans. If an MS/MS scan detects a neutral loss of 81.05µ, and the parent ion is among the three most intense peaks, an MS3 scan is triggered. When the MS3 scans are complete, or if no neutral loss is detected, the process begins again with another MS survey scan.
A total of 19 MS3 scans were acquired, of which 13 corresponded to the observed glycoforms of ribonucleaseB. This illustrates the specificity of the method. All five known glycoforms of ribonucleaseB were successfully identified.
Conclusion
These results demonstrate that the ultra-high sensitivity and high spectral quality offered by the Finnigan LTQ mass spectrometer make it ideal for the analysis of enzymatically modified glycoproteins. The Data Dependent Neutral Loss experiment presented here enables determination of the position of elution, whereas the high sensitivity MS/MS and MS3 spectra generate information-rich data for structural elucidation of the various glycoforms of ribonucleaseB.
Gargi Choudhary, Jae Schwartz and Diane Cho are with Thermo Electron Corporation, Boston, Massachusetts, USA. www.thermo.com
