Neutron-encoded mass signatures for multiplexed proteome quantification

Abstract

We describe a protein quantification method called neutron encoding that exploits the subtle mass differences caused by nuclear binding energy variation in stable isotopes. These mass differences are synthetically encoded into amino acids and incorporated into yeast and mouse proteins via metabolic labeling. Mass spectrometry analysis with high mass resolution (>200,000) reveals the isotopologue-embedded peptide signals, permitting quantification. Neutron encoding will enable highly multiplexed proteome analysis with excellent dynamic range and accuracy.

Figure 1. NeuCode feasibility and scan sequence

(a) Theoretical mass calculations of the 39 isotopologues for a +8 Da lysine amino acid. Shown in solid black are the isotopologues used for the experiments presented here. (b) Theoretical calculation depicting the percentage of peptides that are resolved (FWOM) when spaced 12, 18, or 36 mDa for resolving powers 15 thousand to 1 million. (c) MS1 scan collected with 30,000 resolving power from an nLC-MS/MS analysis of yeast LysC peptides and inset of a selected precursor having m/z at 827 (black trace). The signal recorded in a subsequent high resolution MS1 scan (480,000 resolving power) is shown in red – only at this high resolution is the quantitative data revealed. Presented below the MS1 scan is an MS/MS spectrum following CAD and ion trap m/z analysis of the neutron encoded SILAC pair. The inset displays that the SILAC pair is concealed at typical resolution.

Figure 2. NeuCode SILAC quantitative results

(a) Boxplots showing the measured (box and whiskers) and true (dashed lines) values for both methods at mixing ratios of 1:1 and 1:5. Boxplots demarcate the median (stripe), the 25^th to 75^th percentile (interquartile range, box), 1.5 times the interquartile range (whiskers), and outliers (open circles) for both SILAC (black) and NeuCode SILAC (red). (b) NeuCode SILAC and SILAC demonstrate a strong correlation for quantifying protein changes during the myogenic differentiation of mouse-derived C2C12 myoblasts (R² = 0.78). (c) The combination of NeuCode SILAC with mTRAQ labeling affords six channels of MS1-centric quantification.