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

Abstract

In large-scale quantitative mass spectrometry (MS)-based phosphoproteomics, isobaric labeling with tandem mass tags (TMTs) coupled with offline high-pH reversedphase 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.

Hybrid-DIA: Intelligent Data Acquisition for Simultaneous Targeted and Discovery Phosphoproteomics in Single Spheroids

“…5A). Such a phosphoproteome coverage is on par with data obtained in other large-scale phospho-proteomics screenings that use significantly higher peptide input material 5,25,30 . This reflects that the improvements in our sample preparation processing and MS analysis pipeline makes it realistic to scale down input amounts for highly sensitive phosphoproteomics experiments, such as analyzing single spheroid, while preserving a considerable coverage of the quantifiable phosphoproteome.…”

“…3B). Additionally, we previously described how a second phosphopeptide-enrichment step in the Kingfisher platform is easily implemented by looping through the protocol, without the need to change buffers, but also reusing the beads and the elution buffer 25 . The implementation of the improved experimental protocol in combination with hybrid-DIA MS analysis, maximized the phospho-signaling information retrievable from single spheroids.…”

“…Transition Settings used in Skyline when importing hybrid-DIA or SureQuant data. .50 6.75 7.00 7.256.25 6.50 6.75 7.00 7.256.25 6.50 6.75 7.00 7.256.25 6.50 6.75 7.00 7.256.25 6.50 6.75 7.00 7 25. …”

Isobaric labeling: Expanding the breadth, accuracy, depth, and diversity of sample multiplexing

“…The hardware and software innovations mentioned above are complimented by vigorous and reproducible sample preparation strategies that aim to improve proteome depth and overall throughput. Numerous sample preparation strategies for isobaric labeling experiments are available, with some geared toward specific applications [37][38][39][40][41][42][43][44][45]. The streamlined TMT (SL-TMT) protocol [46], for example, was designed with the intent to establish a highly standardized workflow that minimizes the steps needed for efficient and reproducible sample preparation for TMT-based experiments.…”

“…In this issue, we highlight articles that focus on a diverse array of research areas including higher order multiplexing [57], optimal strategies for phosphorylation [39], predictions of TMTpro retention times [58], and plasma proteomics [59], among other topics. Please note, however, that we only scratch the surface of potential applications for isobaric tagging.…”