Optimal analytical strategies for sensitive and quantitative phosphoproteomics using TMT‐based multiplexing

In large‐scale quantitative mass spectrometry (MS)‐based phosphoproteomics, isobaric labeling with tandem mass tags (TMTs) coupled with offline high‐pH reversed‐phase peptide chromatographic fractionation maximizes depth of coverage. To investigate to what extent limited sample amounts affect sensitivity and dynamic range of the analysis due to sample losses, we benchmarked TMT‐based fractionation strategies against single‐shot label‐free quantification with spectral library‐free data independent acquisition (LFQ‐DIA), for different peptide input per sample. To systematically examine how peptide input amounts influence TMT‐fractionation approaches in a phosphoproteomics workflow, we compared two different high‐pH reversed‐phase fractionation strategies, microflow (MF) and stage‐tip fractionation (STF), while scaling the peptide input amount down from 12.5 to 1 μg per sample. Our results indicate that, for input amounts higher than 5 μg per sample, TMT labeling, followed by microflow fractionation (MF) and phospho‐enrichment, achieves the deepest phosphoproteome coverage, even compared to single shot direct‐DIA analysis. Conversely, STF of enriched phosphopeptides (STF) is optimal for lower amounts, below 5 μg/peptide per sample. As a result, we provide a decision tree to help phosphoproteomics users to choose the best workflow as a function of sample amount.