Title : Global and site-specific detection of human
integrin alpha 5 beta
1 glycosylation using tandem mass spectrometry and the StrOligo algorithm
Abstract :
- Glycans are oligosaccharides associated with proteins , and are known to confer specific functions and conformations on glycoproteins
- As protein tridimensional structures are related to function, the study of glycans and their impact on protein folding can provide important information to the field of proteomics
- The subdiscipline of glycomics (or glycoproteomics) is rapidly growing in importance as glycans in proteins have shown to be involved in protein-protein or protein-(drug, virus, antibody) interactions
- Glycomics studies most often aim at identifying glycosylation sites , and thus are performed on deglycosylated proteins resulting in loss of site-specific details concerning the glycosylation
- In order to obtain such details by mass spectrometry (MS), either whole glycoproteins must be digested and analyzed as mixtures of peptides and glycopeptides , or glycans must be isolated from glycopeptide fractions and analyzed as pools
- This article describes parallel experiments involving both approaches, designed to take advantage of the StrOligo algorithm functionalities with the aim of characterizing glycosylation microheterogeneity on a specific site
- A hybrid quadrupole-quadrupole-time-of-flight (QqTOF) instrument equipped with a matrix-assisted laser desorption/ionization (MALDI) source was used
- Glycosylation of alpha 5 beta 1 subunits of human integrin was studied to test the methodology
- The sample was divided in two aliquots, and glycans from the first aliquot were released enzymatically, labelled with 2-aminobenzamide, and identified using tandem mass spectrometry (MS/MS) and the StrOligo program
- The other aliquot was digested with trypsin and the resulting peptides separated by reversed-phase high-performance liquid chromatography (HPLC)
- A specific collected fraction was then analyzed by MS before and after glycan release
- These spectra allowed, by comparison, detection of a glycopeptide (several glycoforms) and elucidation of peptide sequence
- Com positions of glycans present were proposed, and identification of possible glycan structures was conducted using MS/MS and StrOligo