Abstract
High resolution proteomics approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics an open question still remains, which quantification strategy is best suited for identification of biologically relevant changes, especially in clinical specimens. In this study, a thorough comparison of a label-free approach (intensity-based) and 8-plex iTRAQ was conducted as applied to the analysis of tumor tissue samples from non-muscle invasive and muscle-invasive bladder cancer. For the latter, two acquisition strategies were tested including analysis of unfractionated and fractioned iTRAQ-labeled peptides. To reduce variability, aliquots of the same protein extract were used as starting material, whereas to obtain representative results per method further sample processing and MS analysis were conducted according to routinely applied protocols. Considering only multiple-peptide identifications, LC-MS/MS analysis resulted in the identification of 910, 1092 and 332 proteins by label-free, fractionated and unfractionated iTRAQ, respectively. The label-free strategy provided higher protein sequence coverage compared to both iTRAQ experiments. Even though pre-fraction of the iTRAQ labeled peptides allowed for a higher number of identifications, this was not accompanied by a respective increase in the number of differentially expressed changes detected. Validity of the proteomics output related to protein identification and differential expression was determined by comparison to existing data in the field (Protein Atlas and published data on the disease). All methods predicted changes which to a large extent agreed with published data, with label-free providing a higher number of significant changes than iTRAQ. Conclusively, both label-free and iTRAQ (when combined to peptide fractionation) provide high proteome coverage and apparently valid predictions in terms of differential expression, nevertheless label-free provides higher sequence coverage and ultimately detects a higher number of differentially expressed proteins. The risk for receiving false associations still exists, particularly when analyzing highly heterogeneous biological samples, raising the need for the analysis of higher sample numbers and/or application of adjustment for multiple testing.
Highlights
Application of mass spectrometry-based quantitative approaches has largely contributed to the emerging role of proteomics [1]
Three experimental approaches were evaluated aiming to select the optimal strategy for determination of protein differential expression in highly complex samples employed in clinical proteomics (i.e. non-muscle invasive in comparison to muscle-invasive bladder cancer)
In the case of iTRAQ some minor modifications of the classical FASP protocol were necessary to ensure compatibility with the subsequent labeling, as suggested by the manufacturer and described earlier [39,40,41]. These include a reduction of the initial amount of protein processed by FASP, and substitution of the ammonium bicarbonate buffer with triethylammonium bicarbonate to avoid interference of the former with labeling via interactions with the iTRAQ reagents
Summary
Application of mass spectrometry-based quantitative approaches has largely contributed to the emerging role of proteomics [1]. Label-based methods allow for the simultaneous analysis of multiple samples in a single MS run (multiplexing), resulting in reduced analytical variability. This is relevant for the application of TMT and iTRAQ labeling, since up to eight (for iTRAQ) [10] or ten (for TMT) [11] samples can be analyzed simultaneously during a single experiment. In these cases, due to the isobaric nature of labels, labeled peptides appear as a single peak in the full MS scan. Upon peptide fragmentation at the MS/MS level, the isotope-containing reporter ions are released and distinguished according to their masses based on the label composition
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