The proteomic analysis of blood is routine for disease phenotyping and biomarker development. Whole 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 proteins, N-glycopeptides and phosphopeptides. 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 two days. Approximately 2,000 protein groups and 8,000 post-translational modifications were quantified in 3 LC-MS/MS runs at ~1.5 hours 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).

Samples on Evotips were analyzed on an Evosep One LC using a 60 sample-per-day method with a Pepsep 8 cm x 150 μm column (1.5 μm particle size) with a PepSep Sprayer and stainless steel (30 μm) emitter. The LC was interfaced to an Exploris 480 and Orbitrap Astral MS using a Nanospray Flex Source. Orbitrap DDA-MS/MS used a 60,000 resolution precursor mass range from 650-2000 m/z, 300% AGC and 25 ms IT, and Orbitrap MS/MS used 30,000 resolution, 200% AGC and auto IT, an isolation window of 1.2 m/z and stepped NCE of 20, 30 and 40%. Cycle time was 1 s. Astral MS/MS used an Orbitrap precursor scan at 240K resolution from 850-1550 m/z with 300% AGC and 50 ms IT and a 0.6 s cycle time. Astral DDA-MS/MS used an intensity threshold of 1E4, 300% AGC and 10 ms IT, isolation window of 1.2 m/z and NCE of 35%. Astral DIA-MS/MS used 139 x 5 m/z windows from 850-1550 m/z with a 500% AGC, 8 ms IT and 35% NCE. The Glyco-N-DIA workflow used a development build of FragPipe 23 was used for analysis of Astral DIA data, using Orbitrap or Astral DDA data for library generation.

HILIC enrichment was performed as previously described. Briefly, 250 mg of Polyhydroxyethyl A (12 µm, 300 Å; PolyLC cat# BMHY1203) was equilibrated with 25 mL of 0.1% TFA for 30 min, and 200 µL of slurry was added to P200 pipette tips plugged with 3 discs of MK360 (Ahlstrom) and arrayed in a 96-well format using a multichannel pipette. Tips were centrifuged at 200 xg for 3 min per step followed by washing with 2 x 200 µL of 80% (v/v) MeCN:H2O containing 0.1% TFA (wash buffer). Next, 80 µL of the digest was diluted with 320 µL of MeCN and 4 µL TFA. Samples were passed through tips in two rounds of 200 µL, and this was repeated 3 times and the flow-through reserved for phosphopeptides enrichment. Tips were washed with 3 x 200 µL wash buffer followed by elution with 2 x 200 µL of 0.1% TFA. An SPQC sample was prepared from equal volumes of all eluents, and 40 µL of each sample was diluted with 160 µL 0.1% FA on Evotips followed by loading and washing with 2 x 50 µL of 0.1% FA.