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QUANTITY: An Isobaric Tag for Quantitative Glycomics.

Yang S, Wang M, Chen L, Yin B, Song G, Turko IV, Phinney KW, Betenbaugh MJ, Zhang H, Li S - Sci Rep (2015)

Bottom Line: Quantitative glycomics--analysis of glycans at global level--however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity.Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples.This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.

ABSTRACT
Glycan is an important class of macromolecules that play numerous biological functions. Quantitative glycomics--analysis of glycans at global level--however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity. As a result, technologies that can facilitate global glycan analysis are highly sought after. Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples. This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation. Our results demonstrated QUANTITY is an invaluable technique for glycan analysis and bioengineering.

No MeSH data available.


Related in: MedlinePlus

QUANTITY isobaric tandem mass tags for glycan labeling and quantitation.(a) Molecular structure and isotope positions of QUANTITY. (b) N-acetylglucosamine (GlcNAc, the first residue on the reducing end of N-glycans) labeled with QUANTITY and its fragmentation in MS2. A water molecule is lost during labeling, probably through a six-membered ring formation (neighboring group participation reaction). The arrow indicates fragmentation site in MS2. (c) Workflow of N-glycan extraction from solid-phase for labeling of N-glycans by isobaric tandem mass tags and analysis of peptide using LC-MS/MS. Proteins are immobilized on beads via reductive amination. Sialic acids are stabilized via carbodiimide coupling. The released N-glycans by PNGase F are labeled with QUANTITY. The global proteins are further analyzed by direct digestion from beads.
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f1: QUANTITY isobaric tandem mass tags for glycan labeling and quantitation.(a) Molecular structure and isotope positions of QUANTITY. (b) N-acetylglucosamine (GlcNAc, the first residue on the reducing end of N-glycans) labeled with QUANTITY and its fragmentation in MS2. A water molecule is lost during labeling, probably through a six-membered ring formation (neighboring group participation reaction). The arrow indicates fragmentation site in MS2. (c) Workflow of N-glycan extraction from solid-phase for labeling of N-glycans by isobaric tandem mass tags and analysis of peptide using LC-MS/MS. Proteins are immobilized on beads via reductive amination. Sialic acids are stabilized via carbodiimide coupling. The released N-glycans by PNGase F are labeled with QUANTITY. The global proteins are further analyzed by direct digestion from beads.

Mentions: As other isobaric tags for peptides and small molecules, the 4-plex QUANTITY reagents are a set of four molecules with identical chemical structures and molecule weight, yet they contain different stable isotope nuclei like 13C and 2H in various positions (Synthesis of QUANTITY is described in Supporting Materials Figures S1&2). Their structures consist of a reporter with molecular mass ranging from 176 to 179 Daltons in the series, a balancer that compensates the mass difference of the reporters, and a reactive primary amine to conjugate with glycans via reductive amination (Fig. 1). This labeling chemistry is the same as that used by 2-AA/2-AB16, so well-established protocols for 2-AA/2-AB labeling can be applied without much modification. A noticeable difference between our tags and 2-AA/2-AB, however, is that a water molecule is lost spontaneously and stoichiometrically from QUANTITY-labeled glycans, while 2-AA/2-AB labeled glycans only show the partial loss of a water molecule17. This phenomenon might be proceeded through an energetically favored six-membered ring formation in a neighboring group participation mechanism (a.k.a. neighboring participation reaction)18. Upon MS2 fragmentation, QUANTITY-labeled glycans yield strong reporter ions for accurate quantification without the need of extra positive ions such as Na+ or metal ions, thereby eliminating ion suppression effect and preventing the formation of multiple H+/Na+ adducts. Furthermore, outfitting glycans with a permanently positive-charged quaternary amine can enhance their ionization in MS. Therefore, the detection sensitivity of glycans is considerably enhanced, which is advantageous when analyzing low abundance glycans or limited samples.


QUANTITY: An Isobaric Tag for Quantitative Glycomics.

Yang S, Wang M, Chen L, Yin B, Song G, Turko IV, Phinney KW, Betenbaugh MJ, Zhang H, Li S - Sci Rep (2015)

QUANTITY isobaric tandem mass tags for glycan labeling and quantitation.(a) Molecular structure and isotope positions of QUANTITY. (b) N-acetylglucosamine (GlcNAc, the first residue on the reducing end of N-glycans) labeled with QUANTITY and its fragmentation in MS2. A water molecule is lost during labeling, probably through a six-membered ring formation (neighboring group participation reaction). The arrow indicates fragmentation site in MS2. (c) Workflow of N-glycan extraction from solid-phase for labeling of N-glycans by isobaric tandem mass tags and analysis of peptide using LC-MS/MS. Proteins are immobilized on beads via reductive amination. Sialic acids are stabilized via carbodiimide coupling. The released N-glycans by PNGase F are labeled with QUANTITY. The global proteins are further analyzed by direct digestion from beads.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663469&req=5

f1: QUANTITY isobaric tandem mass tags for glycan labeling and quantitation.(a) Molecular structure and isotope positions of QUANTITY. (b) N-acetylglucosamine (GlcNAc, the first residue on the reducing end of N-glycans) labeled with QUANTITY and its fragmentation in MS2. A water molecule is lost during labeling, probably through a six-membered ring formation (neighboring group participation reaction). The arrow indicates fragmentation site in MS2. (c) Workflow of N-glycan extraction from solid-phase for labeling of N-glycans by isobaric tandem mass tags and analysis of peptide using LC-MS/MS. Proteins are immobilized on beads via reductive amination. Sialic acids are stabilized via carbodiimide coupling. The released N-glycans by PNGase F are labeled with QUANTITY. The global proteins are further analyzed by direct digestion from beads.
Mentions: As other isobaric tags for peptides and small molecules, the 4-plex QUANTITY reagents are a set of four molecules with identical chemical structures and molecule weight, yet they contain different stable isotope nuclei like 13C and 2H in various positions (Synthesis of QUANTITY is described in Supporting Materials Figures S1&2). Their structures consist of a reporter with molecular mass ranging from 176 to 179 Daltons in the series, a balancer that compensates the mass difference of the reporters, and a reactive primary amine to conjugate with glycans via reductive amination (Fig. 1). This labeling chemistry is the same as that used by 2-AA/2-AB16, so well-established protocols for 2-AA/2-AB labeling can be applied without much modification. A noticeable difference between our tags and 2-AA/2-AB, however, is that a water molecule is lost spontaneously and stoichiometrically from QUANTITY-labeled glycans, while 2-AA/2-AB labeled glycans only show the partial loss of a water molecule17. This phenomenon might be proceeded through an energetically favored six-membered ring formation in a neighboring group participation mechanism (a.k.a. neighboring participation reaction)18. Upon MS2 fragmentation, QUANTITY-labeled glycans yield strong reporter ions for accurate quantification without the need of extra positive ions such as Na+ or metal ions, thereby eliminating ion suppression effect and preventing the formation of multiple H+/Na+ adducts. Furthermore, outfitting glycans with a permanently positive-charged quaternary amine can enhance their ionization in MS. Therefore, the detection sensitivity of glycans is considerably enhanced, which is advantageous when analyzing low abundance glycans or limited samples.

Bottom Line: Quantitative glycomics--analysis of glycans at global level--however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity.Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples.This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.

ABSTRACT
Glycan is an important class of macromolecules that play numerous biological functions. Quantitative glycomics--analysis of glycans at global level--however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity. As a result, technologies that can facilitate global glycan analysis are highly sought after. Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples. This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation. Our results demonstrated QUANTITY is an invaluable technique for glycan analysis and bioengineering.

No MeSH data available.


Related in: MedlinePlus