Ultra-deep MS based plasma proteomics in a hand foot and mouth disease cohort

Hand foot and mouth disease (HFMD) is usually a mild illness affecting children ≤5 years old, commonly caused by coxsackieviruses A16, A10 and A6, and enterovirus A71 (EV-A71). However, since 1997, large outbreaks of severe EV-A71 associated HFMD, involving millions of infections and substantial numbers of deaths, have been reported in the Asia Pacific region. Increased detections of EV-A71 and associated neurological disease have also been documented outside of Asia. Currently, there are no clinically proven effective antivirals to treat severe HFMD. Inactivated EV-A71 vaccines have been successfully developed in China and most recently in Taiwan, but have only been implemented in China. The identification of biomarkers that could differentiate between clinical severity, predict disease progression and help in prognosis and patient stratification has become one of the main goals for this project.

Recent developments in mass spectrometry-based plasma proteomics which combines new protein enrichment strategies with more sensitive and fast instruments, address the major analytical challenge in mass spectrometry-based analysis of the deep plasma proteome by decreasing the dynamic range of protein abundance in the analysed sample. This enables the detection of proteins beyond the functional plasma proteome and get into the realm of tissue leakage and cell signalling, now addressing biological pathways relevant to disease.

We have profiled the proteomes of 20 HFMD samples from two different severity groups (n of 10 per group) in a direct comparison of neat plasma to bead-based enrich plasma using the PreOmics ENRICH plasma and the new ENRICHplus. Samples have been analysed by LC-MS/MS using the EvosepOne Tims-Tof-Pro platform using the 60 samples per day (spd) and DIA-PASEF acquisition. DIA-NN library free has been used for data analysis. We show that we can detect around 3200 and 4200 protein groups when using the ENRICH plasma and ENRICHplus, respectively. This means we can increase the plasma proteome depth by a 4.5 and 6-fold change compared to neat plasma when using the PreOmics ENRICH plasma and ENRICHplus, respectively.