Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Infrared-Tandem Mass Tags.

Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4-5-fold increases in reporter signal compared to conventional SPS-MS3 approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.

Infrared Photoactivation Boosts Reporter Ion Yield in Isobaric Tagging.

Isobaric tagging facilitates multiplexed experiments that can determine sequences and relative amounts of peptides in biological samples using tandem mass spectrometry (MSn). Limited reporter ion generation limits quantitative accuracy and precision. As reporter ions are susceptible to unintended fragmentation and scattering by high-energy collisions, we activated peptides with IR photons and prevented successive dissociation of generated reporter ions with ion parking, which altogether boosted reporter ion yield by up to 55%. Even so, unintended co-isolation of contaminating peaks in MS2 experiments distorts reporter ion intensities and can distort quantitative information. MS3 experiments address contamination by generating reporter ions via collisional activation (HCD) of one or more peptide product ions rather than the isolated peptide precursor ion. Because HCD performance is related to m/z, activation of multiple synchronously isolated product ions generates less than optimal reporter ion intensities. In this work, we show that using infrared multiphoton dissociation, which is not dependent on m/z, to generate reporter ions from 10 synchronously isolated peptide product ions results in a 2.4-fold increase in reporter ion intensities, significantly enhancing the sensitivity and dynamic range of quantitation via isobaric tagging.