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Pharmaceutical Discovery, May 1, 2005 
Identification of Glycosylated Peptides Using a Linear Ion Trap Mass Spectrometer
By Gargi Choudhary , Jae Schwartz , Diane Cho
Identification of Glycosylated Peptides Using a Linear Ion Trap Mass Spectrometer
In proteins that have regulatory functions, glycosylation is an important yet challenging post-translational modification. This report describes a novel glycoprotein characterization method, designed to determine structure and position of elution.
May 1, 2005
By: Gargi Choudhary, Jae Schwartz, Diane Cho
Pharmaceutical Discovery

Glycosylation is an important post-translational modification associated with many proteins that have a regulatory function. Several liquid chromatography and tandem mass spectrometry (LC–MS-MS) approaches have been used for analysis and structural elucidation of glycoproteins. Most commonly, a glycoprotein is enzymatically digested, and the resulting fragments are fractionated by reversed-phase LC. The peptide fractions can be analyzed either by on-line MS-MS or collected and analyzed off-line by matrix-assisted laser desorption ionization (MALDI) MS. Peptides that do not correspond to predicted masses might represent glycosylated forms. These peptides subsequently are 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 analyze because they generally are present in low concentration in cells. In addition, glycopeptides often are 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 (Thermo Electron Corp., Waltham, Massachusetts, USA) to characterize glycoproteins. The goal is to develop a selective and sensitive LC–MS method for unambiguous identification and characterization of glycoproteins.

Experimental Conditions Sample preparation. Ribonuclease B was reduced with dithiothrietol (DTT), alkylated with iodoacetic acid and then enzymatically modified.

HPLC. The digested protein was separated on a 100 × 0.15 mm column packed with 5 µM Vydac® C-18 stationary phase (MicroTech Scientific, Vista, California, USA). The Finnigan Surveyor™ MS Pump was used with the following gradient conditions:

Solvent A: water/0.1% formic acid; Solvent B: acetonitrile/0.1% formic acid; Gradient: 5% to 60% B in 20 min.; 60% to 80% B in 2 min. and 80% B for 5 min.

 

Figure 1. Data Dependent parameters for analysis of enzymatically modified ribonuclease B.
Mass spectrometry. Eluted peptides were analyzed on a Finnigan LTQ linear ion trap mass spectrometer, equipped with a nanospray ion source operated at 1.7 kV spray voltage and 150 °C. heated capillary temperature. A Data Dependent Neutral Loss experiment was performed with a collision energy of 25% and a neutral loss mass width of 0.2 u. The Data Dependent acquisition parameters are shown in Figure 1.

 

Table I. Neutral loss markers for glycopeptides
Results and Discussion When glycopeptides are fragmented in tandem MS, they typically exhibit neutral losses corresponding to the mass of a monosaccharide moiety. Table I shows common monosaccharide components of glycoproteins and their corresponding neutral loss masses. High mannose glycoproteins, such as ribonuclease B, show losses of 162.1 u for a singly-charged ion or 81.05 u for a doubly-charged ion. Therefore, Data Dependent settings were chosen to trigger an MS3 scan when a peptide showing a neutral loss of 81.05 u is detected.

 

Figure 2. Flowchart of scan events in the Data Dependent Neutral Loss experiment.
Figure 2 shows a flowchart 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 81.05 u 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.

 

Figure 3. Base peak chromatogram generated by LC–MS-MS analysis of enzymatically modified ribonuclease B.
Figure 3 shows the base peak chromatogram generated by LC–MS-MS analysis of the ribonuclease B digest using the Finnigan LTQ mass spectrometer. The position of elution of the glycopeptides was determined from the MS3 scans. A total of 19 MS3 scans were acquired, of which 13 corresponded to the observed glycoforms of ribonuclease B. This illustrates the specificity of the method. All five known glycoforms of ribonuclease B were identified successfully.

 

Figure 4. Single MS-MS and MS3 spectra of the 5 mannose glycoform of ribonuclease B.
Figure 4 shows representative MS-MS and MS3 spectra for one of the five glycoforms of ribonuclease B. All spectra shown are single spectra form, illustrating the high sensitivity of the Finnigan LTQ mass spectrometer.

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 ribonuclease B.

Gargi Choudhary is a product marketing specialist, Jae Schwartz is principal scientist and Diane Cho is ion trap product marketing manager at Thermo Electron Corporation. Gargi Choudhary can be reached at Thermo Electron Corporation, 355 River Oaks Parkway, San Jose, California 95134 USA.