Exploring the Proteome II-Posters Presentations

Poster Number:

Presenter:

Rob Christian

Institute:	Lab or Branch
Waters Corporation, Life Sciences R&D (CRD), Milford MA

Title:

Glycosylation Detection and Characterisation Using an LC-MS/MS Precursor Ion Discovery Experiment on a Q-Tof Mass Spectrometer

Authors:

M. A. Ritchie, R. Christian, N. Johnson, J. B. Hoyes, A. Millar, R. Carruthers, C. Jones and J. Langridge

Abstract:

Asparagine (N-) linked glycosylation is one of the most common, complex, and highly variable post-translational modification. As glycoforms are the product of a series of biochemical modifications, perturbations within a cell can have profound effects on their structure. As glycosylation also plays an important role in cell signaling and recognition it's detection and characterisation are of great importance.

Here we present a method for the detection and characterisation of glycopeptides. A proteolytic digest is analysed by reverse phase HPLC-ESI using a Q-Tof mass spectrometer. The instrument is switched at one-second intervals between low and high collision energy on the collision cell. The quadrupole operates in the non-selective rf only mode. The first data set at low energy (7eV) shows only the normal pseudo molecular ions. The second at higher energy shows their fragments. Wherever a specified product occurs in the high-energy data all its possible precursors are revealed by the corresponding 7eV data. Upon detection of the carbohydrate oxonium ions at m/z 204 (HexNAc), 366 (HexHexNAc) and 274/292 (NeuAc) the instrument is switched into MS/MS mode and ions from the low energy spectra are selected by the quadrupole for fragmentation. The high mass of glycopeptides gives rise glycopeptide ions typically of 4 or more positive charges, a feature that is exploited for their preferential selection for MS/MS. The glycosidic bonds tend to be more labile than the peptide bonds hence MS/MS spectra produce predominately glycopeptide Y-type fragment ions. Interpretation of MS/MS data is facilitated through the use of software tools.

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