Advances in proteomics and mass spectrometry have enabled the study of limited cell populations, such as single-cell proteomics, where high-mass accuracy instruments are typically required. While triple quadrupoles offer fast and sensitive nominal resolution measurements, these instruments are effectively limited to targeted proteomics. Linear ion traps (LITs) offer a versatile, cost-effective alternative capable of both targeted and global proteomics. We demonstrate a workflow using a newly released, hybrid quadrupole-LIT instrument for developing targeted proteomics assays from global data-independent acquisition (DIA) measurements without needing high-mass accuracy. Gas-phase fraction-based DIA enables rapid target library generation in the same background chemical matrix as each quantitative injection. Using a new software tool embedded within EncyclopDIA for scheduling parallel reaction monitoring assays, we show consistent quantification across three orders of magnitude of input material. Using this approach, we demonstrate measuring peptide quantitative linearity down to 25x dilution in a background of only a 1 ng proteome without requiring stable isotope labeled standards. At 1 ng total protein on column, we found clear consistency between immune cell populations measured using flow cytometry and immune markers measured using LIT-based proteomics. We believe hybrid quadrupole-LIT instruments represent an economic solution to democratizing mass spectrometry in a wide variety of laboratory settings.

With the Q-LIT, we demonstrate how to generate nominal-mass targeted transition libraries using both DDA, composed of offline fractionated samples, as well as gas-phase fractionated (GPF) DIA libraries. We then show the quantitative accuracy of targeted PRMs utilizing a Q-LIT using matched-matrix calibration curves at 1, 10, and 100 ng of low abundant immune cell populations. To facilitate this, we developed an open-source software tool that directly schedule-optimized PRM assays from DIA libraries. Finally, we show the quantitative consistency measuring “real-world” biological targets in cytokine-stimulated CD8+ T cells with as little as 1 ng on column. These results suggest that a Q-LIT can perform as an inexpensive stand-alone instrument for quantitative proteomics, capable of a wide range of proteomics measurements without the need for high-resolution mass spectrometry.

Splenocytes were isolated from mouse spleens, then stimulated with IL-2 or IL-15. At the peptide level, the samples were combined to make a "pooled" sample.