Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification

• Objective comparison between iTRAQ, DML and LF for relative quantitative proteomics.
• Experimental design of (i) proteins in different ratios, (ii) a two proteome model.
• The latter mimic the biomarker hypothesis with a majority of unregulated proteins.
• Evaluated by parameters important for expression proteomics using two MS setups.
• The overall performance of the 3 best methods were; DML FTICR > iTRAQ QTOF > LF FTICR.

The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55–1.16, r2: 0.61–0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR > iTRAQ QStar > LF LTQ-FTICR > DML QStar > LF QStar.