|
Abstract This presentation gives an overview of our efforts made to develop the concept of “smart samplers” for affinity capture or tryptic digestion of proteins. Several examples of “smart sampling” will be highlighted and its potential in advanced protein analysis will be discussed through demonstration of the sampler’s performance.
INTRODUCTION:
The use of microsampling is evolving from newborn screening protocols to a growing applicability across various fields. Traditionally, dried matrix spots (DMS), especially dried blood spots (DBS), have served as an efficient method for collecting and transporting biological samples—due to their minimal volume requirements, ease of at-home collection, and robustness in ambient conditions. Within this context, the presentation focusses on the emerging strategy of "smart sampling” within the field of LC-MS based protein determination. When determining proteins from conventional DMS with LC-MS, often tedious and time-consuming sample preparation steps need to be carried out. Examples of these steps are immunocapture and tryptic digestion. “Smart sampling” can be defined as the integration of tedious sample preparation steps in the initial stage of the sampling workflow on filter paper. It saves both time and simplifies the work to be carried out in the laboratory. Our work aims to revolutionize the pretreatment process in protein analysis by embedding sample preparation steps directly into the microsampler.
METHODS:
By immobilizing either mAbs or trypsin to the filter-paper, in this way essential steps like immunocapture or enzymatic digestion (bottom-up) are embedded in the microsampler. In this way these samplers have the ability to start the sampling at the moment the sample is applied. The chemical strategy to do this is first to functionalize the filter paper, making it reactive. Sodium periodate oxidation and divinyl sulfone modification are examples of these functionalization strategies. In the second step the mAbs or trypsin is immobilized through covalent binding.
RESULTS:
We have successfully immobilized both mAbs and trypsin on filter paper, with the purpose to start sample preparation for LC-MS based protein determination. The “smart affinity samplers” show the ability to capture biomarkers from serum and whole blood allowing sub ng/mL quantification while “smart proteolytic samplers” are able to digest complex biological samples, for qualitative and quantitative determination. In both ”smart samplers”, the majority of sample preparation can take place outside the laboratory setting.
CONCLUSION:
”Smart sampling” allows to perform tedious sample preparation steps in the initial stage of the sampling workflow on filter paper. This strategy is a new direction within bioanalysis of proteins with LC-MS.
References:
1. L. Reubsaet and T.G. Halvorsen, Advancements in clinical approaches, analytical methods, and smart sampling for LC–MS-based protein determination from dried matrix spots. J. Sep. Sci., 47(9-10)(2024) p. 2400061.
2. A. Mrsa, et al., One-step functionalization of paper and simplified antibody immobilization for on-the-spot immunocapture from dried serum in LC-MS/MS based targeted protein determination. J. Mass Spec., 59(1)(2024) p. e4989.
3. C. Johannsen, et al., Smart sampling as the "Spot-on" Method for LC-MS protein analysis from DBS. J. Sep. Sci. (2023) 46:2300394.
4. T.G. Halvorsen and L. Reubsaet, Is this the end of dried blood spots as we know it? Anal. Sci. Adv., (2023), 4 (7-8) p. 267-274.
5. M.T. Nguyen, et al., Smart proteolysis samplers for pre-lab bottom-up protein analysis - Performance of on-paper digestion compared to conventional digestion. Sep. Sci. Plus, (2022) p. 1-13.
|
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
