Mass Spectrometry-Based Quantification of Proteins and Post-Translational Modifications in Dried Blood: Longitudinal...

The proteomic analysis of blood is routine for disease phenotyping and biomarker development. Blood is commonly separated into soluble and cellular fractions. However, this can introduce pre-analytical variability, and analysis of a single component (which is common) may ignore important pathophysiology. We have recently developed methods for the facile processing of dried blood for mass spectrometry-based quantification of the proteome, N-glycoproteome, and phosphoproteome. Here, we applied this approach to 38 patients in Tanzania who presented to the hospital with sepsis. Blood was collected on Mitra devices at presentation and 1, 3, and 28-42 days post-enrollment. Processing of 96 total samples was performed in plate-based formats and completed within 2 days. Approximately 2000 protein groups and 8000 post-translational modifications were quantified in 3 LC-MS/MS runs at ∼1.5 h per sample. Analysis of differential abundance revealed blood proteome signatures of acute phase response and neutrophilic inflammation that partially resolved at the 28-42 day timepoint. Numerous analytes correlated with clinical laboratory values for c-reactive protein and white blood cell counts, as well as the Universal Vital Assessment illness severity score. These datasets serve as proof-of-concept for large-scale MS-based (sub)phenotyping of disease using dried blood and are available via the ProteomeXchange consortium (PXD060377). SUMMARY: For the first time, we report the integrated quantitative analysis of proteins, N-glycopeptides, and phosphopeptides from dried blood specimens in a disease context. Sample collection on Mitra devices is easily incorporated into existing biobanking protocols and provides a convenient solution for sample storage and preparation for downstream mass spectrometry analysis. Signatures of sepsis are reflected in each of the analyzed proteomes and decline between presentation to the hospital and 1 month post. In addition to well-described markers, these analyses identify mediators of inflammation and innate immune signaling that would be missed in the more common analysis of cell-free plasma.