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.
|Chemical structures of the Thermo Scientific aminoxyTMTsixplex Label Reagents.
A. Functional regions of the reagent structure including MS/MS fragmentation sites by higher energy collision dissociation (HCD) and electron transfer dissociation (ETD).
B. aminoxyTMTsixplex Reagent structures and isotope positions (red asterisks).
|Reaction scheme for labeling of reducing-end sugars with aminoxyTMT Reagent. For more information about alkoxyamine and hydrazide chemistries, see our review of Carbonyl Reaction Chemistry.
The Tandem Mass Tag (TMT) Reagent family consists of TMTzero, TMTduplex, TMTsixplex and TMT10plex sets which are specially designed to enable a rapid and cost-effective transition from method development to high-throughput protein quantitation. The TMTzero tag allows testing and optimization of sample preparation, labeling, fractionation and MS fragmentation for peptide identification and reporter detection without using the more costly isotope-labeled compounds. The TMTsixplex reagent set allows sixplex protein profiling for multiple conditions, including time courses, dose responses, replicates or multiple sample comparisons. Each TMT tag is based on the same chemical structure, eliminating the need to modify labeling conditions or HPLC separation conditions between experiments.
|Workflow schematic for sixplex reagent experiment. Intact proteins or proteolytic digests of proteins extracted from cells, tissue, or fluids are treated with PNGase F/A glycosidases to release N-linked glycans from the peptides. Glycans are separated from the protein or peptide material using a hydrophobic clean-up column (C18 or polymer-based resin), and purified free glycans are labeled at the reducing end with the aminoxyTMT Reagents. After a quenching step with acetone, individually labeled samples are combined and quenched reagent is removed using a HILIC column. Labeled samples are analyzed using a mass spectrometer to identify glycoforms in the sample and quantify reporter ion relative abundance at MS2-level.
|Relative quantitation of glycans from monoclonal antibodies. N-glycans were released from three different mouse monoclonal antibodies (approx. 100μg each) using PNGase F glycosidase. Each of the three glycan samples was split into two equal parts and labeled with a different mass tag,indicated in the figure. After labeling, quenching and clean-up, the samples were mixed and analyzed by direct infusion ESI-MS in the positive ion mode on a Thermo Scientific Velos Pro mass spectrometer. Glycoforms of interest were identified in the MS spectra, and were subjected to HCD MS/MS. Reporter ion relative peak intensities provide information on relative glycoform abundance in the three samples (insets).
- Atwood III, J.A., et al. (2008). Quantitation by Isobaric Labeling: Applications to Glycomics. J Proteome Res 7(1), 367–374.
- Prien JM, et al. (2010). Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics. Anal Chem. 82(4) 1498-508.
- Snovida et al., Applications of Aldehyde-Reactive Thermo Scientific Tandem Mass Tag (TMT) Reagents for Mass Spectrometry-based Quantitative Glycomics. ASMS 2013 conference proceedings.
Mass Spec Sample Prep Workflow (selection guide)
Mass spectrometry technical guide
Quantitative proteomics technical guide
Service providers that use our mass tagging products
TMT Technology - opens Proteome Sciences website
TMT Amine-reactive Tandem Mass Tag Reagents
SILAC Isotope Labeling Reagents and Kits