A sweet revolution in glycopeptide analysis
Posttranslational modification (PTM) of proteins with glycan groups to form glycoproteins is a common biological motif. Glycans are involved in a wide variety of biological and physiological processes, including recognition and regulatory functions, cellular communication, immunological defense, gene expression, and cell growth and development. Aberrant glycosylation of proteins is connected with cell immortalization, transformation, and metastasis. Due to its many biological functions, glycosylation is one of the most-studied PTMs of eukaryotic cell proteins.
To elucidate glycopeptide localization and composition, a combination of high-resolution accurate-mass (HRAM) mass spectrometry and multiple dissociation techniques is required. Such versatility is needed for thorough glycopeptide characterization. HCD or CID fragmentation provides glycan composition information, while ETD and EThcD is used to sequence the peptide and determine glycosylation sites.
Thermo Scientific Pierce Glycoprotein Isolation Kits, Concanavilin A (ConA) and Wheat Germ Agglutinin (WGA), enable the isolation of glycoproteins at the protein level and from complex mixtures such as serum, tissue, and cultured cell lysates. These complete kits contain the immobilized lectins, binding and wash buffers, as well as the columns required to process up to 10 mg of total protein.
Reversed-phase separation on Thermo Scientific low flow systems is an integral component of glycoproteomics workflows and combines seamlessly with Thermo Scientific Orbitrap mass spectrometers. The Thermo Scientific EASY-nLC 1200 System and UltiMate 3000 RSLCnano System are the LC systems of choice for glycoproteomics, with the EASY-nLC 1200 ensuring operational simplicity and high performance, and the UltiMate 3000 RSLCnano offering versatility and precision.
The Thermo Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer is HRAM-capable and offers multiple dissociation techniques (CID, HCD, ETD, and EThcD) for thorough glycopeptide characterization. The system's intelligent data acquisition strategies simplify workflows while improving duty cycle and dynamic range. The Orbitrap Fusion Lumos MS is also capable of synchronous precursor selection (SPS) MS3 acquisition, supporting multiplexed quantification of glycopeptides from various biological samples. This workflow is only available on tribrid mass spectrometers.
Glycopeptide data interpretation is challenging because it requires both peptide sequencing and characterization of the glycosylation site(s) and overall glycan composition. Thermo Scientific Proteome Discoverer Software (2.0 and higher) offers Byonic™ Software from Protein Metrics as a node within its architecture. Byonic Software uses HCD data to determine peptide sugar composition, while corresponding ETD/EThcD data elucidate the peptide backbone. This results in an accurate portrayal of the peptide sequence, its site(s) of glycosylation, and its glycan composition.