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Analytical Chemistry

Simple Tag Could Help Decode Glycoproteome

Proteomics: Technique makes it easier to identify and characterize cellular glycoproteins

by Stu Borman
April 22, 2015

TRAIL MARKERS
A reaction scheme showing the IsoTaG method.
Credit: Adapted from Nat. Chem. Biol.
IsoTaG identifies cellular glycoproteins by marking them with dibromide tags. The tagged glycopeptides are then analyzed by tandem mass spectrometry.

Glycosylation, the addition of sugar groups to proteins, is the most varied form of modification that cells make to proteins. And largely because of this diversity, glycosylation remains the least well understood protein modification.

A new technique called isotope-targeted glycoproteomics (IsoTaG) takes a major step toward helping researchers understand the glycoproteome, the diversity of glycoproteins found in cells. IsoTaG provides a systematic method for determining sequences of chains of sugars and the sites on proteins to which those sugars attach—information that has been difficult or impossible to obtain until now.

Glycosylation regulates a wide spectrum of cellular processes, such as cell signaling and the delivery of new proteins to correct sites in cells. The modification also plays a role in disease states, including Alzheimer’s and cancer progression. For these reasons, scientists want to learn much more about it.

For N-linked glycoproteins, one of the two glycoprotein types, existing glycoproteomics methods could map sequences of sugar chains, called glycans, or protein-attachment locations but not both at the same time. And attachment sites and sequences have been nearly impossible to analyze for O-linked glycoproteins, the other type of modifcation, except with specialized mass spectrometry (MS) techniques accessible in only a handful of laboratories.

IsoTaG, developed by Carolyn R. Bertozzi and coworkers at the University of California, Berkeley, is the first glycoproteomics technique that identifies locations in proteins at which glycans attach and determines the sequence of those glycans at the same time (Nat. Meth. 2015, DOI: 10.1038/nmeth.3366).

IsoTaG uses bioorthogonal chemistry, metabolic reactions that don’t interfere with cellular chemistry, to mark each N- and O-linked glycan in cellular glycoproteins with a tag complex containing both biotin and a dibromide group. The biotin allows the researchers to grab the glycoproteins from a cellular mixture, immobilize them on a surface, and then chop the protein down to just the sequence containing the attached glycan. After freeing these glycopeptides from the surface, the scientists analyze them using tandem MS.

Normally researchers get the identity of a protein by matching its MS data to that of known proteins in a database. But most glycoproteins have not been characterized by MS and are not in these databases. And trying to identify these glycoproteins requires methods that destroy important information about the glycan sequence. The dibromide tag allowed Bertozzi’s team to get around this issue because they could easily spot the glycopeptides via the unique MS signal created by the dibromide group.

With the dibromide tags, the researchers could characterize the locations to which N-linked glycans attach and the corresponding glycan sequences. IsoTaG still can’t fully characterize O-linked glycoproteins but narrows down O-glycan attachment sites to small peptide regions, which wasn’t possible before.

IsoTaG’s ability to identify O-linked attachment locations for the first time “is a major technological step forward” for glycoproteomics, says Matthew R. Pratt of the University of Southern California, who specializes in glycosylation and other protein modifications.

IsoTaG “is a novel way to capture information on both peptide sequence and glycostructures” at the same time, comment Sergey Vakhrushev and Henrik Clausen of the University of Copenhagen’s Center for Glycomics in an e-mail message. The two point out some limitations of the method, such as the complexity of carrying out metabolic labeling and unresolved questions about how quantitative and sensitive the method will eventually turn out to be. “But it is a big first step toward solving a major problem in the field,” they write.

Bertozzi says her group has filed a patent application on the method, and that the protein-tagging reagents and data analysis software used in the technique eventually may be commercially available.

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