Rapid assay development for low input targeted proteomics using a versatile linear ion trap

Abstract Advances in proteomics and mass spectrometry enable the study of limited cell populations, where high-mass accuracy instruments are typically required. While triple quadrupoles offer fast and sensitive low-mass specificity measurements, these instruments are effectively restricted to targeted proteomics. Linear ion traps (LITs) offer a versatile, cost-effective alternative capable of both targeted and global proteomics. Here, we describe a workflow using a hybrid quadrupole-LIT instrument that rapidly develops targeted proteomics assays from global data-independent acquisition (DIA) measurements without high-mass accuracy. Using an automated software approach for scheduling parallel reaction monitoring assays (PRM), we show consistent quantification across three orders of magnitude in a matched-matrix background. We demonstrate measuring low-level proteins such as transcription factors and cytokines with quantitative linearity below two orders of magnitude in a 1 ng background proteome without requiring stable isotope-labeled standards. From a 1 ng sample, we found clear consistency between proteins in subsets of CD4+ and CD8+ T cells measured using high dimensional flow cytometry and LIT-based proteomics. Based on these results, we believe hybrid quadrupole-LIT instruments represent a valuable solution to expanding mass spectrometry in a wide variety of laboratory settings.