High-throughput plasma proteomics with cap-flow LC separation and dia-PASEF-MS/MS detection

Plasma represents a major source for clinical diagnosis and research which is obtained minimally invasive. Proteomics analysis of plasma is challenged by the high dynamic range of protein abundance as well as the variability between individual donations and donors. Major issues that hinder widespread application of mass spectrometry-based plasma proteome analysis is the low sample throughput of nanoLC-MS applications and lower sensitivity of HPLC separation at high flow rates. Capillary flow rate chromatography (1-10ul/min) provides a good option to combine high sensitivity and high sample throughput. The protein depth of cap-LC is similar to that observed with nanoLC applications using similar column dimensions and gradient length, however durations for sample loading and column equilibration are minimized due to the increased flow rate.

Two sample preparations of plasma proteins were performed for 15 individual plasma donations, direct trypsination for a "neat" proteome composition and the ENRICH-iST kit for a deep plasma proteome. HPLC and MS settings were optimized at a flow rate of 2 µl/min to surpass 110 samples/day. MS acquisition parameters for dia-PASEF on the TimsTOF HT mass spectrometer were adapted to maintain reliable quantitation and to maximize the number of peptide and protein hits. For cap-LC separation, the separation gradient and the injection amount were programmed for highest sample throughput at highest level of data coverage as well as minimal sample carry-over and column loading and equilibration periods.

A pooled sample was generated for LC method optimization. First, dia-PASEF parameters were optimized with nanoLC separation by probing the number of IM ramps, the position and extraction width of the windows within a 600 Th range for precursor isolation and limiting the ion mobility range to allow for optimal positioning of the windows or the precursor ion cloud. Using the optimized dia-PASEF cycle, injection amount and gradient length were tested for nanoLC and cap-LC flow rates. For nanoLC, we aimed at highest proteome coverage achievable to generate a reliable precursor library for subsequent fast gradient analysis. For cap-LC measurements, gradients for 75 and 140 samples/day were programmed and the reproducibility and throughput were confirmed. Results were searched in Spectronaut 19 against the spectral library generated by nanoLC analysis as well as in the directDIA+ node. For neat plasma, more than 400 protein groups with 140 SPD were quantified reliably. Enriched plasma samples covered more than 2000 protein groups in 7 min active gradient time.