Sample Preparation: Are We Ready for a Booming Field of Intact Protein Analysis?

Proteins are biomolecules with many essential functions in a human body. Their varied expression in complex disorders, including cancer, predicts their potential use as biomarkers. Therefore, there is a need for reliable analytical workflows for their analysis in complex biological matrices. Despite the highly sensitive and advanced instrumentation available to us today for protein analysis, sample preparation remains one of the greatest challenges.

Targeted top-down proteomics based on mass spectrometry (MS) platforms connected to liquid chromatography (LC) or capillary electrophoresis (CE) as separation techniques is a growing field of research (1–3 ). Whether it is the pharmaceutical industry moving towards biopharmaceuticals or clinical researchers looking for new protein biomarkers, there is an immense demand for reliable bioanalytical workflows to target these biomolecules in biological matrices. complexes (4.5). Over 15,000 different proteins have been identified in major human body fluids. These circulating proteins may play an important role as biomarkers and potential drug targets in various disorders, including cancer, neurodegenerative and inflammatory diseases (1).

Biological fluids also contain large amounts of other chemical species, including salts, metabolites and lipids. Therefore, reliable quantitative analysis of intact proteins in complex biological samples using MS still presents significant challenges. Disadvantages of existing techniques include low overall sensitivity due to low ionization and fragmentation efficiency of intact proteins, non-specific binding of proteins to various analytical instrument surfaces, and sample preparation steps often time-consuming, expensive and labor-intensive steps required prior to analysis. . All of these concerns make it even more difficult to develop a successful analytical workflow for intact proteins, as an efficient sample preparation protocol is essential before using a powerful separation technique with sensitive and selective detection. On the other hand, today’s advanced analytical workflows bring several advantages to the field of proteomics, including high accuracy, precision, selectivity, and multiplexing capability that could be beneficial for determining a whole panel of multiple biomarkers in one. single analysis (2.6–8).

Sample preparation for intact proteins in biological matrices

The go-to sample preparation methods for proteomic analysis are highly selective and quite expensive immunoaffinity-based methods. Other frequently used sample pretreatment techniques for intact protein analysis primarily involve complex and laborious size exclusion chromatography (SEC), two-dimensional (2D) gel electrophoresis, or combinations thereof (1,2,9 ). In our recent work (6) investigating the development of an LC–triple quadrupole mass spectrometry (LC–MS/MS) method for the direct quantification of multiple intact growth factors and cytokines in human body fluids, we realized that the commercial availability of simpler, immunoaffinity-free sample preparation options for intact proteins. These could be different extraction techniques or coacervation systems (10-12), but few options could be found. Therefore, a strong focus on the development of these potential new sample preparation methods for the isolation and enrichment of multiple intact proteins present in complex samples should be of great interest.

Solid phase extraction by micro-elution (μSPE)

A common and relatively simple solution to eliminate interferences caused by the presence of cellular and extracellular matrix components in biological samples is protein precipitation (13,14). However, for proteomic analysis, precipitation can often result in deleterious protein loss. Another potential simple immunoaffinity-free sample preparation technique, already frequently used for the analysis of small peptides after protein digestion, is solid phase extraction (SPE) (15,16). SPE generally shows limited selectivity, which can be advantageous if one wishes to purify a wider range of proteins from the sample. Developments in the area of ​​new SPE sorbents have begun to focus on new materials and formats to target intact proteins as analytes, although commercially available SPE products of this type are still lacking on the market. SPE in microplate format (μSPE) is designed for efficient isolation and preconcentration of various substances. The SPE microplate has multiple advantages, including the use of small sample volumes, elution in volumes as low as 25 μL, and the absence of drying or reconstitution steps. The latter is particularly important for intact proteins, which can suffer severe recovery losses during such processes.

Although the success of adopting commercially available small molecule products for the preparation of intact proteins may be limited, and these methods still suffer from low recoveries and adverse matrix effects, in our latest work, we explored the practical opportunities and challenges of applying μSPE to the preparation of lower molecular weight intact proteins ( 65% in urine for all targeted proteins and >50% in serum and plasma for most proteins were obtained, as shown in Figure 1. Limits of quantification (LOQ) were at the concentration in ng /mL levels (corresponding to a range between approximately 0.35 and 97.6 nM). Preliminary results of this work were presented at the 69th ASMS Conference on Mass Spectrometry and Related Topics (17) and are currently being prepared for publication.


Each year, we witness the development of state-of-the-art analytical instrumentation and the improvement of the performance parameters, in particular of sensitivity, of these instruments. However, even with the most sensitive instrumentation, we are unable to reliably analyze multiple intact proteins in complex biological samples without improving the interface to create a highly efficient sample purification and fractionation strategy. , robust and reproducible. There are no simple universal strategies for sample preparation workflows for intact proteins and there is plenty of room to invent. We must continue to develop workflows capable of reaching proteins at very low concentration levels (pg/mL to ng/mL) in complex matrices, providing the appropriate selectivity for the proteins of interest and removing interferences potential. The systematic investigation of the potential application of non-immunoaffinity based sample preparation methods for the analysis of intact proteins in biological fluids will continue to be the focus of our future work. We hope others will also see this need and opportunity to contribute to an important area of ​​research.


I would like to warmly thank Professor Kevin A. Schug for his support throughout my research stays in his laboratory, his valuable discussions, comments and suggestions during the experimental work and the writing of the manuscript. Two research stays in Professor Schug’s laboratory at the University of Texas at Arlington were supported by the Fulbright Scholarship Program and the National Scholarship Program of the Slovak Republic. This work was also partially supported by the Science Grants Agency of the Ministry of Education, Science, Research and Sports of the Slovak Republic under the VEGA project 1/0483/20, and the article is based on the work of the Study Group on Sample Preparation and Network supported by the Analytical Chemistry Division of the European Chemical Society.


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Katarina Marakova is assistant professor at the Department of Pharmaceutical Analysis and Nuclear Pharmacy of the Faculty of Pharmacy of the Comenius University in Bratislava. His research focuses on the development of high performance separation methods (capillary electrophoresis, liquid chromatography and mass spectrometry), including multi-dimensional approaches for advanced pharmaceutical and biomedical applications. She completed two post-doctoral research stays at the University of Texas at Arlington (Professor KA Schug), supported by the National Scholarship Program of the Slovak Republic and the Fulbright Commission.

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