Title : Identification of N-glycosylation
sites of the murine neural cell adhesion molecule
NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry
Abstract :
- Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation
- For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans , because functions of proteins may be modulated or depend on the presence of glycans at specific sites
- Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not
- Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain
- Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans
- The resulting peptides were analyzed by MALDI-TOF-MS
- In addition, high-resolution Fourier transform-ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion
- By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site