Developing a targeted quantifying method for immune cell markers with the Stellar mass spectrometer

Advances in proteomics and mass spectrometry enable the study of limited cell populations such as performing mouse models. Low-protein load studies often require two steps to perform differential expression analysis between biological conditions. Initial sample characterization experiments to determine proteins and corresponding peptides have been almost exclusively dependent on high-mass accuracy instruments. The discovery results are then used to develop targeted quantitation experiments to better define biological changes based on biology. While triple quadrupoles offer fast and sensitive low-mass accuracy measurements, these instruments are effectively restricted to targeted proteomics and require tremendous effort to create effective methods. Linear ion traps (LITs) offer a versatile, cost-effective alternative capable of both targeted and global proteomics.

 

Here, we describe a workflow using the newly released Thermo Scientific^TM Stellar^TM mass spectrometer (MS) that rapidly develops targeted proteomics assays from global data-independent acquisition (DIA) measurements without needing high mass accuracy. 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 an economical solution to democratizing mass spectrometry in a wide variety of laboratory settings.

 

Learning Objectives