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Techniques & Tools Sample Preparation

Wanted: (Much) Better Bioanalytical Sample Prep

In recent years, protein analysis has – rightly – caught the attention of scientists all over the world. Medical and pharmaceutical scientists alike have recognized that various proteins present in biological samples can serve as potential biomarkers and drug targets for numerous diseases, including cancer, neurodegenerative diseases, and immune-mediated disorders. More recently, targeted proteomics of intact molecules has emerged as a promising and rapidly growing facet of this field.

Based on LC and MS, these advanced workflows host many benefits for proteomic applications versus traditional immune-based methods (1, 2). The major benefits include high accuracy, precision, and selectivity, as well as the ability to analyze several intact proteins in one run. But these approaches are not without their challenges. Low sensitivity due to poor ionization and fragmentation efficiencies, alongside non-specific binding and adsorption of proteins to surfaces and other molecules, are the primary struggles faced by the field. An efficient sample preparation protocol is therefore vital to the development of a successful workflow for intact protein analysis.

During my recent research stay at the laboratory of Kevin Schug (University of Texas at Arlington, USA), I worked to develop an analytical approach based on LC-triple quadrupole MS for the direct quantitation of multiple intact proteins (growth factors and cytokines) in complex biological matrices. The fundamental principles of single and multidimensional setup for such an approach have been published previously (3, 4, 5). During our work, however, we hit a stumbling block: the extremely limited commercial availability of sample preparation options for intact proteins – especially for cases with limited amounts of samples.

Sample preparation workflows for intact proteins generally involve many complex, laborious, and costly steps, namely immuno-affinity purification, 2D-gel electrophoresis, use of magnetic beads and nano-particles, and size-exclusion chromatography, or any combination of these (2). Therefore, we really need a strong focus on the development of new prospective sample preparation methods with possible use for isolation and enrichment of multiple intact proteins before instrumental analysis. The need for such sample pretreatment techniques is undeniable, especially when you consider that potential proteomic biomarkers – which can have variable physicochemical properties – are present in the complex biological matrices at trace concentrations.

Even with such super-sensitive instrumentation, we are not currently able to reliably target multiple intact proteins in complex biological samples due to the lack of appropriate sample preparation approaches.

In my view, solid-phase extraction (SPE) and monolithic SPE spin columns are just some of the options worth testing, and may even provide a simpler and cheaper alternative for sample preparation of intact proteins. Nowadays, SPE is commonly used for sample preparation of small peptides after protein digest, but it has also been applied for the analysis of a smaller intact proteins (up to 7.5 kDa) (6). The use of organic polymer monoliths with different chromatographic mechanisms have been reported for the separation of intact proteins in traditional LC column formats, thanks to their high permeability, macropore structure and better resistance to extreme pH conditions – which are desirable characteristics when dealing with proteins that have a very high or very low isoelectric point (7).

Another interesting area in the sample preparation field is coacervation – the aggregation of amphiphilic molecules to form a separate liquid phase in aqueous media. The application of coacervates to the preparation of biological samples offers the potential to increase the concentration of low abundance analytes and the selective extraction of hydrophobic analytes from a complex matrix. Looking ahead, we plan to study the detailed application potential and conduct a systematic evaluation of these non-immunobased sample preparation methods for a larger set of variable intact proteins (different pI, GRAVY, molecular weight, abundance, and tendency to aggregate) in biological samples.

Every year, we see improvements across various performance parameters in analytical instrumentation, particularly in sensitivity. However, even with such super-sensitive instrumentation, we are not currently able to reliably target multiple intact proteins in complex biological samples due to the lack of appropriate sample preparation approaches. My hope is that, in the future, we will see more development and focus on this integral and crucial part of every bioanalytical approach.

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  1. A Arora, K Somasundaram, “Targeted Proteomics Comes to the Benchside and the Bedside: Is it Ready for Us?”, BioEssays. 41 (2019) 1800042. DOI: 10.1002/bies.201800042.
  2. D Donnelly, et al., “Best practices and benchmarks for intact protein analysis for top-down mass spectrometry”, Nat. Methods. 16 (2019) 587–594. DOI: 10.1038/s41592-019-0457-0.
  3. E.H. Wang, P.C. Combe, K.A. Schug, Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer, J. Am. Soc. Mass Spectrom. 27 (2016) 886–896. DOI: 10.1007/s13361-016-1368-2.
  4. D.D. Khanal, Y.Z. Baghdady, B.J. Figard, K.A. Schug, Supercharging and multiple reaction monitoring of high‐molecular‐weight intact proteins using triple quadrupole mass spectrometry, Rapid Commun. Mass Spectrom. 33 (2019) 821–830. DOI: 10.1002/rcm.8418.
  5. Y.Z. Baghdady, K.A. Schug, Online Comprehensive High pH Reversed Phase × Low pH Reversed Phase Approach for Two- Dimensional Separations of Intact Proteins in Top-Down Proteomics, Anal. Chem. 91 (2019) 11085–11091. DOI: 10.1021/acs.analchem.9b01665.
  6. K Bronsema et al., “A quantitative LC-MS/MS method for insulin-like growth factor 1 in human plasma”, Clin. Chem. Lab. Med. 56 (2018) 1905–1912. DOI::10.1515/cclm-2017-1042.
  7. J Masini, F Svec, “Porous monoliths for on-line sample preparation: A review”, Anal. Chim. Acta. 964 (2017) 24–44. DOI: 10.1016/j.
About the Author
Katarina Maráková

Assistant Professor, Faculty of Pharmacy, Comenius University, Bratislava, Slovakia

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