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One strategy for improving the throughput of human plasma proteomic discovery analysis while maintaining good depth of analysis is to multiplex using isobaric tags. At present, the greatest multiplexing that is commercially available uses the TMT10plex kit. As an example of this approach, we describe efficient shotgun discovery proteomics of large numbers of human plasma to identify potential biomarkers. In the analysis strategy, a common pooled reference was used to enable comparisons across multiple experiments. Duplicate samples showed excellent overall reproducibility across different TMT experiments. Data filters that improved the quality of individual peptide and protein quantitation included using a filter for purity of the targeted precursor ion in the isolation window and using only unique peptides.

Quantitative Comparisons of Large Numbers of Human Plasma Samples Using TMT10plex Labeling