Posttranslational modifications of proteins, such as glycosylation, are widely studied as a possible way to detect human diseases and understand the complex biological processes of eukaryotic cells. N-linked glycosylation, attachment of glycans to asparagine residues in a protein, have been shown to be involved in various cellular processes. Although known to be important, studying N-linked glycopeptides has continued to be difficult by traditional proteomics methods due to the complex means of purification necessary to isolate N-linked glycoproteins from biological samples.
Asymmetrical flow field-flow fractionation (AF4) is a method used to separate complex biological macromolecules from diverse samples. AF4 differs from gel-based methods of protein separation in that AF4 does not require protein denaturation, allowing for the isolation of intact protein complexes. Chip-based AF4 has successfully been coupled with mass spectrometry analysis for intact protein characterization.1
Lectins are able to bind diverse glycans. The addition of lectins to N-linked glycoproteins allows for selective isolation of the lectin/glycopeptide complex from a complex biological sample, the inference of the makeup of the particular glycan based on its corresponding lectin binding partner type, and the use of compatible size sorting methods.
Kim et al.2 have developed a technique using AF4 for sorting lectin-glycopeptide complexes from biological samples. First, the N-linked glycoproteins are complexed with specific lectins, to create lectin-glycoprotein complexes. These complexes are digested with PNGase F and subjected to liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS) using the LTQ Orbitrap Velos ion trap mass spectrometer (Thermo Scientific) to identify the N-linked glycopeptides and their respective glycosylation sites. Kim et al. then used this technique to analyze, using Proteome Discoverer software (Thermo Scientific), and create a profile of N-linked glycopeptides found in lung cancer patients compared to those found in healthy individuals.
These results suggest that asymmetrical flow field-flow fractionation, combined with multiple lectin affinity and nLC-EDI-MS-MS, may be a viable alternative method for isolation and characterization of N-linked glycopeptides and provide means for quantitative analysis of biomarkers in cases of clinical disease.
1. Kim, K.H. and Moon, M.H. (2011) ‘Chip-type asymmetrical flow field-flow fractionation channel coupled with mass spectrometry for top-down protein identification‘, Analytical Chemistry, 83 (22), (pp. 8652-8658)
2. Kim, J.Y., et al., (2012) ‘Dual lectin-based size sorting strategy to enrich targeted N-glycopeptides by asymmetrical flow field-flow fractionation: Profiling lung cancer biomarkers‘, Analytical Chemistry, 84 (12), (pp. 5343-5350)
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