Global and site-specific detection of human integrin α5β1 glycosylation using tandem mass spectrometry and the StrOligo algorithm

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 α5β1 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. Compositions of glycans present were proposed, and identification of possible glycan structures was conducted using MS/MS and StrOligo. Copyright © 2005 John Wiley & Sons, Ltd.

[1]  S. Hakomori,et al.  Functional role of N-glycosylation in alpha 5 beta 1 integrin receptor. De-N-glycosylation induces dissociation or altered association of alpha 5 and beta 1 subunits and concomitant loss of fibronectin binding activity. , 1994, The Journal of biological chemistry.

[2]  H. Perreault,et al.  Application of the StrOligo algorithm for the automated structure assignment of complex N-linked glycans from glycoproteins using tandem mass spectrometry. , 2003, Rapid communications in mass spectrometry : RCM.

[3]  Standing,et al.  A tandem quadrupole/time-of-flight mass spectrometer with a matrix-assisted laser desorption/ionization source: design and performance , 2000, Rapid communications in mass spectrometry : RCM.

[4]  R. Parekh,et al.  Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. , 1995, Analytical biochemistry.

[5]  F. Giancotti,et al.  A structural view of integrin activation and signaling. , 2003, Developmental cell.

[6]  H. Perreault,et al.  Site‐specific N‐glycosylation analysis: matrix‐assisted laser desorption/ionization quadrupole‐quadrupole time‐of‐flight tandem mass spectral signatures for recognition and identification of glycopeptides , 2004, Rapid communications in mass spectrometry : RCM.

[7]  R. Dwek,et al.  Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography. , 1997, Analytical biochemistry.

[8]  Richard O Hynes,et al.  Integrins Bidirectional, Allosteric Signaling Machines , 2002, Cell.

[9]  S. Hakomori,et al.  Detailed Oligosaccharide Structures of Human Integrin α5β1 Analyzed by a Three‐Dimensional Mapping Technique , 1996 .

[10]  D. Stupack,et al.  Control of Integrin Function , 1996, The Journal of Biological Chemistry.

[11]  Hélène Perreault,et al.  Automated structural assignment of derivatized complex N-linked oligosaccharides from tandem mass spectra. , 2002, Rapid communications in mass spectrometry : RCM.