The carbonyl-reactive Thermo Scientific™ aminoxyTMT™ (Tandem Mass Tag™) Label Reagents enable multiplexed characterization and quantitation of carbonyl-containing biomolecules (carbohydrates, steroids, oxidized proteins) by mass spectrometry (MS).
The six compounds of the aminoxyTMTsixplex™ Reagent Set have the same mass (i.e., isobaric) and chemical structure (carbonyl-reactive aminoxy group, spacer arm, and mass reporter). However, the specific distribution of 13C and 15N isotopes on either side of the HCD or ETD MS/MS fragmentation site in each reagent results in a unique reporter mass (126-131Da) in the low mass region. This set of reporter ions is used to measure the relative abundance of labeled molecules in a combined (multiplexed) MS sample representing six different treatment conditions. For glycobiology MS applications, the reagents enable quantitative profiling of glycan isoforms and discovery of glycan biomarkers; they provide improved ionization of labeled glycans for increased sensitivity and better retention of labeled glycans by reversed-phase liquid chromatography (RPLC).The aminoxyTMT reagents may be used to quantify a broad range of biologically important molecules including carbohydrates, steroids, or oxidized proteins.
Quantitative – enables relative quantitation of glycans or other carbonyl-containing biomolecules from multiple samples derived from cells, tissues or biological fluids
Stable – oxime bond and products formed by labeling reaction are stable and don't require an additional reduction step
Efficient – achieve labeling efficiency greater than 90% in one hour
Sensitive – 20-fold increase in signal-to-noise compared to unlabeled native glycan MS analysis
Multiplex – able to identify and characterize up to six samples concurrently
Optimized – procedure and reagents optimized for excellent labeling efficiency and recovery of glycans
Label reagent sets contain sufficient aminoxyTMT tags for the labeling of 6 control samples (Part No. 90400), one sixplex experiment (Part No. 90401), or five sixplex experiments (Part No. 90402)
PNGase F (or PNGase A) glycosidase and Waters Oasis™ HLB columns
Multiplex up to 6 different samples concurrently in a single experiment
Relative quantitation of glycans
Study of structural diversity of protein glycosylation
Study of glycosylation in cell signaling and regulation
Study of cancer progression, biomarker discovery and analysis of biotherapeutics
Native glycans are difficult to study by mass spectrometry because of their poor ionization efficiency. Quantitation of glycans is particularly challenging due to the lack of standards for all naturally-occurring glycans and difficulties reproducibly quantifying multiple samples. The aminoxy group has better reactivity with carbonyls and better stability of the labeled product compared to hydrazides. Labeling with the aminoxyTMT reagents improves ionization of glycans, thus improving sensitivity, and enables relative quantitation of glycans for up to six samples concurrently.
The aminoxyTMTzero™ Label Reagent contains no heavy isotopes and can be used to evaluate the reagent in different workflows. The aminoxyTMTsixplex Label Reagents share an identical structure with aminoxyTMTzero Reagent but contain different numbers of 13C and 15N isotopes in the mass reporter. The optimized labeling procedure can be completed in one hour. After tag labeling, samples are quenched and cleaned-up using HILIC solid-phase extraction before mass spectrometry analysis.