Large-scale multiplexed quantitative discovery proteomics enabled by the use of an M ¹⁸O-labeled universal reference sample

The quantitative comparison of protein abundances across a large number of biological or patient samples represents an important proteomics challenge that needs to be addressed for proteomics discovery applications. Herein, we describe a strategy that incorporates a stable isotope ¹⁸O-labeled "universal" reference sample as a comprehensive set of internal standards for analyzing large sample sets quantitatively. As a pooled sample, the ¹⁸O-labeled "universal" reference sample is spiked into each individually processed unlabeled biological sample and the peptide/protein abundances are quantified based on ¹⁶O/ ¹⁸O isotopic peptide pair abundance ratios that compare each unlabeled sample to the identical reference sample. This approach also allows for the direct application of label-free quantitation across the sample set simultaneously along with the labeling-approach (i.e., dual-quantitation) since each biological sample is unlabeled except for the labeled reference sample that is used as internal standards. The effectiveness of this approach for large-scale quantitative proteomics is demonstrated by its application to a set of 18 plasma samples from severe burn patients. When immunoaffinrty depletion and cysteinyl-peptide enrichment-based fractionation with high resolution LC-MS measurements were combined, a total of 312 plasma proteins were confidently identified and quantified with a minimum of two unique peptides per protein. The isotope labeling data was directly compared with the label-free ¹⁶O-MS intensity data extracted from the same data sets. The results showed that the ¹⁸O reference-based labeling approach had significantly better quantitative precision compared to the label-free approach. The relative abundance differences determined by the two approaches also displayed strong correlation, illustrating the complementary nature of the two quantitative methods. The simplicity of including the ¹⁸O-reference for accurate quantitation makes this strategy especially attractive when a large number of biological samples are involved in a study where label-free quantitation may be problematic, for example, due to issues associated with instrument platform robustness. The approach will also be useful for more effectively discovering subtle abundance changes in broad systems biology studies.