The early detection of disease poses a substantial challenge in the field of human health, necessitating the development of more accurate biomarkers, enhanced patient stratification techniques, and predictive methods for treatment response. Proteins present in the circulatory system reflect an individual’s physiological state, and protein levels are typically measured using singleprotein immunoassays. However, recent breakthroughs in plasma proteomics show great potential in improving the comprehension of disease in clinical practice. The use of high-throughput, quantitative analysis through mass spectrometry (MS)-based proteomics of blood, plasma, and serum has been shown to be highly effective in improving the detection and quantification of low-abundance proteins and posttranslational modifications (PTMs).¹

Comprehensive analysis of the plasma proteome provides valuable insights into an individual’s health status. Research has demonstrated the efficacy of a rapid and highly reproducible proteomic workflow that allows for the quantitative analysis of hundreds of plasma proteomes obtained from just one µL of blood from a single finger prick. This innovative three hour workflow utilises a single-run shotgun proteomic method that eliminates the need for protein depletion and produces protein data that reflects allele differences, metabolic risk, and inflammatory status.²

Nevertheless, this approach presents analytical challenges due to the wide range of protein abundances found in these samples. Protein expression is influenced not only by an individual’s genetic background but also by various factors such as time, location, and physiological responses to external stimuli like stress, disease, aging, and physical activity. Furthermore, a single protein or gene expression product can manifest in multiple proteoforms due to alternative splicing, point mutations, PTMs, and endogenous proteolysis — each with distinct biological activities.^3-4

To address this complexity, the implementation of orthogonal approaches in plasma proteomics has demonstrated the ability to facilitate more accurate and comprehensive data analysis. Three recent technological advancements open new possibilities for enhancing early disease detection and personalised treatment strategies with plasma proteomics.

Advanced Proteomics Analysis with prm-PASEF

Parallel accumulation serial fragmentation (PASEF) is an MS-based proteomics method employed for peptide sequencing and identification. A novel advancement called parallel reaction monitoring-PASEF (prmPASEF) integrates ion mobility as an additional dimension to liquid chromatography-MS (LCMS) proteomics. This innovative approach enhances proteome coverage while reducing analysis time. By combining trapped ion mobility spectrometry (TIMS) with a highspeed time-of-flight (TOF) mass spectrometer, the sensitivity for proteomics is significantly improved, enabling the detection of minute protein quantities present in single cells.

Using a dual ion mobility trap, prm-PASEF facilitates highly multiplexed targeted acquisition. This technology enables a shorter chromatographic separation while maintaining the number of targeted peptides.⁵ Furthermore, the design of TIMS allows for the consistent measurement of collisional cross section (CCS) values for all detected ions. The enhanced selectivity of the system enables researchers to obtain more reliable relative quantitation information from complex samples and short gradient analyses (Figure 1).⁶