Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos
Zhou Li, Rachel M. Adams, Karuna Chourey, Gregory Blake Hurst, Robert L. Hettich, and Chongle Pan
2012 March 01, Journal of Proteome Research 11 (3): 1582-1590
available online: 22 December 2011
A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification; isobaric chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; and (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. On the basis of the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.
To enable studies of differentially abundant proteins across various experimental conditions in the PMI, we are implementing methods for stable-isotope-based quantification. Using a model soil bacterial species, Pseudomonas putida F1, we have compared figures of merit such as depth of proteome coverage, quantification accuracy, precision, and reproducibility for several quantitative proteomics methods using a high-performance hybrid mass spectrometer, the LTQ Orbitrap Velos. Each approach has particular merits, and the final choice of approach depends on the requirements of the experiment at hand. Our results indicate that isobaric chemical labeling has the highest quantification precision, label-free quantification provides the largest number of protein identifications, and metabolic labeling is intermediate in both measures
Experiment design. Three P. putida cultures were grown in parallel, except that the culture 3 was metabolically labeled with 15N. Proteins were extracted from cell cultures and digested into peptides, which were measured using LCï¿½MS/MS. In the label-free method, the cultures 1 and 2 were prepared and measured separately. In metabolic labeling, the cultures 1 and 3 were mixed at the beginning. In isobaric chemical labeling, peptides from the cultures 1 and 2 were mixed after isobaric chemical labeling with TMT or iTRAQ.
Peptide quantification results at different reporter ion intensities of iTRAQ and TMT. Panels A (iTRAQ) and B (TMT) show 2-dimensional histograms of peptide log2 ratio versus the associated log2 intensity for reporter ions. The color encodes the frequency of peptides at a given log2 ratio and log2 intensity. Then, the entire intensity range was split into eight bins. Median and median absolute deviation were calculated and plotted for each bin (Panel C). As reporter ion intensity increased, quantification accuracy was improved. The value of MAD was independent of reporter ion intensity.
Distributions of quantified protein log2 ratios and peptide log2 ratios. Density plots were generated for log2 ratios quantified by each method at the protein level (A) and at the peptide level (B). iTRAQ and TMT produced narrower log2 ratio distributions than metabolic labeling and label-free at both the protein level and the peptide level, which indicates higher quantification precision.
Quantification reproducibility. Two-dimensional histograms were plotted to represent log2 ratios measured from the two technical replicates of each method (A: label-free (R2 = 0.2); B: metabolic labeling (R2 = 0.77); C: iTRAQ (R2 = 0.87); D: TMT (R2 = 0.87)). The color encodes the frequency of proteins quantified at log2 ratios in the two replicates. Quantification reproducibility was significantly improved in the labeling-based approaches.
Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos Zhou Li, Rachel M. Adams, Karuna Chourey, Gregory B. Hurst, Robert L. Hettich, and Chongle Pan Journal of Proteome Research 2012 11 (3), 1582-1590 PMID:22188275