The accurate quantification of proteins, either absolute or relative, is a significant challenge in mass spectrometry.1 Several strategies that utilize differential labeling of samples with stable isotopes are currently in use. Isotopes can either be incorporated during in vivo protein synthesis, as is the case with stable-isotope labeling by amino acids in cell culture (SILAC), or in vitro by isobaric tagging with sulfhydryl or amine reactive reagents. Both of these common strategies have strengths and weaknesses. In the case of SILAC, the incorporation of the isotopes is relatively uniform in the sample, but the spectral complexity of the output increases with every isotope added, and the chemical nature of the individual amino acids limits the type of isotope that can be utilized. In vitro isobaric labeling can use up to eight different mass fingerprints for multiplex analysis, but this quantification is limited to peptides that undergo a tandem mass spectrometry scan. This makes replication a challenge, especially for low-abundance peptides such as protein post-translational modifications. In addition, isobaric labeling suffers from precursor interference, which can reduce quantitative accuracy. Both are significant disadvantages for this type of quantification.
Neutron encoding (NeuCode) is a technique that exploits subtle differences (in millidaltons) of stable isotopes. The differences in mass are related to the different nuclear binding energies of the extra neutrons.2,3 For example, two peptides with eight extra neutrons incorporated into various atoms have a slightly different mass. A peptide with six 13C atoms and two 15N atoms would be 8.0142 Da “heavy,” while a peptide with eight 2H atoms would be 8.0502 Da “heavy,” a difference of 36 mDa. This difference can be measured using high-resolution mass spectrometry such as Orbitrap Elite mass spectrometer (Thermo Scientific) or Fourier Transform Ion Cyclotron Resonance systems. By exploiting these subtle differences in mass, more than 30 different resolvable differences can be detected in a high-resolution quantification scan. In Hebert et al. (2013), this technique is compared to traditional SILAC in myoblasts. The NeuCode generated 5,747 quantifiable peptides, as compared to 3,400 from the traditional SILAC experiment; this is a 41% increase in the amount of quantifiable data.3 By varying the different isotopes used in the amino acids in combination with mass analysis using a high-resolution scanning mass spectrometer, dozens of samples can be quantified simultaneously using this technique.
NeuCode is an exciting new technique that utilizes the subtle differences in mass caused by the binding of energies of neutrons to create uniquely labeled peptides compressed into a narrow mass range that can be resolved using high-resolution mass spectrometry. Because the incorporation of these isotopes is similar to traditional SILAC, this technique does not suffer from precursor interference that hinders accuracy in isobaric tagging strategies. NeuCode is also more sensitive and accurate than traditional SILAC.3 Potential applications of this technology include increasing the multiplex analysis of SILAC-like experiments and increasing the resolution and coverage of quantitative proteomics.
1. Domon, B., and Aebersold, R. (2010) “Options and considerations when selecting a quantitative proteomics strategy,” Nature Biotechnology, 28(7) (pp. 710–21).
2. McAlister, G.C., et al. (2012) “Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses,” Analytical Chemistry, 84(17) (pp. 7469–78).
3. Hebert, A.S., et al. (2013) “Neutron-encoded mass signatures for multiplexed proteome quantification,” Nature Methods, 10(4) (pp. 332–4).
Post Author: Adam Humbard.