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

Ariana E. Shannon, Rachael N. Teodorescu, No Joon Song, Lilian R. Heil, Cristina C. Jacob, Philip M. Remes, Zihai Li, Mark P. Rubinstein and Brian C. Searle ()
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Ariana E. Shannon: The Ohio State University Comprehensive Cancer Center
Rachael N. Teodorescu: The Ohio State University Comprehensive Cancer Center
No Joon Song: The Ohio State University Comprehensive Cancer Center
Lilian R. Heil: Thermo Fisher Scientific
Cristina C. Jacob: Thermo Fisher Scientific
Philip M. Remes: Thermo Fisher Scientific
Zihai Li: The Ohio State University Comprehensive Cancer Center
Mark P. Rubinstein: The Ohio State University Comprehensive Cancer Center
Brian C. Searle: The Ohio State University Comprehensive Cancer Center

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-58757-8

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