Comparison of protein expression ratios observed by sixplex and duplex TMT labeling method

Abstract

Stable isotope labeling via isobaric derivatization of peptides is a universally applicable approach that enables concurrent identification and quantification of proteins in different samples using tandem mass spectrometry. In this study, we evaluated the performance of amine-reactive isobaric tandem mass tag (TMT), available as duplex and sixplex sets, with regard to their ability to elucidate protein expression changes. Using rat brain tissue from two different developmental time points, postnatal day 1 (p1) and 45 (p45), as a model system, we compared the protein expression ratios (p45/p1) observed using duplex TMT tags in triplicate measurements versus sixplex tag in a single LC-MS/MS analysis. A correlation of 0.79 in relative protein abundance was observed in the proteins quantified by these two sets of reagents. However, more proteins passed the criteria for significant fold change (-1.0 ≤ log(2) ratio (p45/p1) ≥ +1.0 and p < 0.05) in the sixplex analysis. Nevertheless, in both methods most proteins showing significant fold change were identified by multiple spectra, increasing their quantification precision. Additionally, the fold change in p45 rats against p1, observed in TMT experiments, was corroborated by a metabolic labeling strategy where relative quantification of differentially expressed proteins was obtained using (15)N-labeled p45 rats as an internal standard.

Figure 1

Schematic diagram showing the design workflow used for the TMT experiments. The protein extracts from rat brain at two different postnatal developmental time points, p1 and p45, were digested using trypsin, and peptides were labeled with sixplex or duplex TMT reagents. Labeled peptides were combined and subjected to MudPIT analysis in LTQ Orbitrap Velos.

Figure 2

Venn diagram depicting the overlapped and unique proteins quantified by the sixplex and duplex TMT labeling experiments.

Figure 3

Frequency histogram of the p45/p1 ratios depicting distribution of log₂ protein expression ratios observed in the sixplex and duplex TMT experiments. The data were normalized as described under Experimental Procedures to compensate for differences in the sample preparation, handling and labeling. Positive values for the log₂ ratios correspond to up-regulated proteins (proteins “enriched”) in p45 samples whereas negative values are for downregulated proteins in p45 (proteins “enriched” in p1 samples). * 6 proteins less in the histogram for scaling of y-axis

Figure 4

Scatterplot of correlation of postnatal day 45 versus postnatal day 1 (p45/p1) protein expression ratios determined by sixplex and duplex. The log₂-transformed ratios of p45/p1 from the sixplex experiment were plotted against those from the duplex experiment.

Figure 5

Scatter plots examining the comparison of fold change observed by the TMT-based quantitation and ¹⁵N metabolic labeling approach (SILAM) approach. The modest correlation of the log₂-transformed p45/p1 protein ratios observed by the two varied quantification approach illustrate the reliability of detected changes in the overlapping proteins in sixplex vs SILAM and duplex vs SILAM, respectively.

Figure 6

A base2 logarithmic representation of spectral counts observed for 357 proteins commonly detected in duplex and sixplex TMT experiments and demonstrate statistically significant regulation difference (-1.0 ≤ log₂ (p45/p1 ratio) ≥ +1.0 and p value of < 0.05).