Background
Blood analysis is one of the most commonly performed procedures in medicine, where clinical parameters are used for diagnosis and for decision on treatment options. If LC-MS/MS based proteomics has long been a powerful research tool but has not provided the robustness and throughput to decipher new biomarkers in large cohort studies of blood plasma. Here, we have combined the robustness and speed of a tims-Q-TOF instrument operated in PASEF mode with PASEF together with the a robust Hight-Trouhghput EvoSep One LC system and a 4D feature alignment-based processing
Methods
192 blood plasma samples from patients with a severe infection were digested with trypsin after depletion of the 12 most abundant proteins. 100ng of each digest were separated on an EvosepOne system at a sampling rate of 100 runs per day (11.5 min gradient). After every 10th we include QC sample. LC-MS/MS measurements were performed on a timsTOF Pro system using a reduced cycle time (0,5 seconds instead of 1.1) PASEF method adapted to short gradients. Post processing was performed with PEAKS studio (version X, Bioinformatics Solutions Inc.). Search results were corrected to 1% PSM FDR and quantification was performed based on MS1 feature intensities. A match of feature vectors in retention time, m/z, 1/K0 was applied to reduce missing values and to transfer protein identifications between runs.
Findings
We found high quantitative reproducibility across the study (R2 > 0.97, median CV 9.3%). Using the 4D feature alignment-based processing allowed to improve the number of quantified proteins from 188 to 500 protein groups on average per run. In the sample set we found several proteins of intermediate or lower abundance (CRP, PSA, IFN-γ)
Conclusion
The combination of these latests technologies offers a robust and high throughput LC-MS/MS solution for plasma screening.