Testing Smart Proteolytic Samplers for Protein Quantification
University of Oslo researchers recently tested new types of smart proteolytic samplers for liquid chromatography-mass spectrometry (LC–MS)-based protein quantification. Their approaches and findings were published in the Journal of Chromatography A (1).
For over 100 years, scientists have been practicing sampling biological matrices on filter paper. Since then, various sampling applications have been developed, but the first report on liquid chromatography–mass spectrometry (LC–MS)-based determination of a protein from a dried biological matrix was not published until the mid-1980s. A critical step in bottom-up sample preparation is the enzymatic digestion of proteins (mainly trypsin), as this treatment can enable good mass spectrometric sensitivity. However, this approach can typically be time-consuming when analyzing dried blood spots, making sample preparation potentially rate-limiting when pursuing fast diagnoses.
Filter paper in laboratory. Scientists are chemical filtration by filtering through filter paper in a glass funnel, Close up. Pharmacist filtering substance in the lab. | Image Credit: © mehmet - stock.adobe.com
One effort to make total workflows more efficient is the introduction of smart sampling, where tedious aspects of sample preparation are integrated into the filter paper used to collect samples. Enzymatic digestion and affinity-based capture/clean-up are examples of sample preparation steps that have successfully been integrated on filter paper. This approach is fundamentally different from conventional digestions of protein from dried samples; in smart proteolytic sampling, proteins are dried onto filter paper while conventional workflow proteins need to be redissolved before digestion.
Producing smart samples is a two-stage process: cellulose is functionalized, and afterward, the reagent protein is covalently bound in a process known as immobilization. Several functionalization chemistries have been tested for this purpose, including 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (HEMA-VDM) chemistry, tosylation, divinyl sulfone (DVS), and periodate reduction (IO4- reduction). The latter two functionalization chemistries typically yield filter paper quality similar to those of untreated filter paper samples. For the covalent binding of trypsin, only HEMA-VDM chemistry and IO4- reduction have been described. Shotgun proteomics, which is a sensitive bottom-up approach for studying complex protein mixtures through combined high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS), can be carried out using the smart sampling strategy (2). When combined with post-proteolytic immunoextractions, there is potential for LC–MS-based target protein determination. However, thorough assessments of the use of smart proteolytic samplers in quantitative determination had not been carried out before this study.
In this report, the scientists attempted to determine if smart proteolytic samplers can be used for LC–MS-based quantitative protein determination from paper. The samplers were tested on a range consisting of simple solutions and complex biological samples, and then quantitatively assessed. Smart proteolytic samplers with varying properties are often tested for this purpose, including different combinations of paper quality and functionalization chemistry. As such, this paper also included a premiere description of using divinyl sulfone (DVA) to produce smart proteolytic samplers.
The divinyl sulfone functionalization in combination with trypsin immobilization was shown to be as successful as periodate functionalization. With Whatman Grade CF12 paper being used, smart proteolytic samplers with high tryptic activity could be produced. These proved capable of producing tryptic peptides with good linearity in buffered solutions: up to 0.998 for BSA and 0.990 for Cytochrome C. The repeatability of peptides produced from complex samples is around 45–50% on average, and when internal correction is commenced, relative standard deviation (RSD) values drop considerably. In the best case (DVS functionalized paper, immobilized with trypsin), the average RSD improved from 40% to below 20%. Overall, the scientists concluded that reliable quantification can be feasible when either internal correction or internal standards are used.
Future research will focus on describing the quantitative performance of smart proteolytic samplers, keeping in mind quantification limits and linearity ranges. Additionally, the scientists want to put effort towards using protein internal standards in robust and reliable quantification.
References
(1) Al-Rubaye, N.; Mrsa, A. Garrastacho, M. R.; et al. Smart Proteolytic Samplers for Liquid Chromatography-Mass Spectrometry Protein Quantification: Assessing Trypsin Immobilization via Divinyl Sulfone or Periodate Functionalization. J. Chromatogr. A 2025, 1747, 465825. DOI: 10.1016/j.chroma.2025.465825
(2) Shotgun Proteomics. Science Direct 2020. Available at: https://www.sciencedirect.com/topics/medicine-and-dentistry/shotgun-proteomics (accessed 2025-3-19)
