Detection and Quantitation of Small Proteins Using Mass Spectrometry

Small proteins or microproteins, despite long being ignored, have important roles in the regulation of larger protein complexes, metabolic pathways, and gene expression. However, these proteins remain under-represented in proteomics studies because of low detection efficiency in traditional mass spectrometry (MS) workflows. Their inherent characteristics often lead to depletion during sample preparation and a low detection efficiency in LC-MS. To improve detection and quantitation, we took advantage of the large set of documented small proteins in Escherichia coli and systematically compared and optimized different sample preparation and LC-MS approaches. We evaluated different top-down and bottom-up approaches, including data-dependent acquisition, data-independent acquisition, and parallel reaction monitoring. Our data highlight the benefit of top-down proteomics for identifying new small proteins and bottom-up preparation coupled with parallel reaction monitoring acquisition for quantitation of protein levels in different growth conditions. Our systematic comparison can serve as a guideline for MS detection of small proteins in all organisms.