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Track 2: Novel Strategies to Advance Biotherapeutic Development

Category: Poster Abstract

(T1130-02-09) Evaluation of Sample Preparation Techniques for Protein Quantification

Tuesday, May 14, 2024
11:30 AM – 12:30 PM PT


Purpose: In recent decades, the pharmaceutical industry has undergone a significant transformation, adopting a modality-agnostic approach to identify the most effective therapeutics for specific diseases.1 This evolution encompasses pivotal changes in traditional bioanalytical workflows crucial for generating data across various therapeutic areas. With the emergence of new analytical workflows that offer enhanced ease of use, efficiency, precision, and accuracy, it is imperative to ensure these methods are fit for purpose. For instance, when quantifying proteins (bottom-up), data quality depends on the selection of surrogate peptides which is essential to the accuracy of the analysis.2 Additionally, sample preparation methods need to be both efficient and practical.3 In this work, we have developed and evaluated four sample preparation methods utilized in the proteomics field to build quantitative LC-MS/MS workflows specifically designed for the bioanalysis of large therapeutic molecules, such as proteins and monoclonal antibodies (mAbs).

Methods: We extracted a proprietary protein drug from mouse plasma using four sample preparation methods: pellet digestion, filter-aided sample preparation (FASP), suspension traps (S-traps)4, and immunoprecipitation (IP). To increase throughput, the last three methods were evaluated using a 96-well plate format. For pellet digestion, the samples were reduced with tris (2-carboxyethyl) phosphine (TCEP) and alkylated with chloroacetamide (CAM). Then, protein pellets were formed via acetone precipitation. The pellets underwent overnight trypsin digestion, followed by solvent drying and reconstitution for LC-MS/MS analysis. In FASP, samples were reduced and alkylated as mentioned above. Proteins were further denatured with 8M urea and triethylammonium bicarbonate (TEAB) in 10% isopropanol (IPA) solution and transferred to a pre-washed 30 kDa molecular weight cut-off (MWCO) filter plate. After washing with 8M urea and TEAB in IPA solution samples were digested overnight with trypsin. Post-centrifugation, peptides were recovered for LC-MS/MS analysis. For S-trap, proteins, after reduction and alkylation, were denatured with phosphoric acid and suspended/precipitated in a trapping/binding buffer with TEAB and methanol. After trapping in suspension traps (S-traps), proteins were washed and digested with trypsin for 2 hours, and the peptides were eluted, dried, and reconstituted for LC-MS/MS analysis. In the IP method, antibody immobilized on magnetic beads were mixed with samples to form immunocomplexes. After washing, the captured proteins on magnetic beads were reduced with TCEP, alkylated with CAM, and digested with trypsin for 1 hour. After quenching the reaction and removing magnetic beads, the sample was subjected to LC-MS/MS analysis. LC-MS/MS separated peptides using a 2.1 mm x 150 mm (3µm) reversed-phase C18 column (Luna Omega Polar C18, Phenomenex) at 40˚C. The UHPLC (Shimadzu) was directly coupled to a qtrap mass spectrometer (Sciex 6500+). A 5 µL sample was injected at a flow rate of 450 µL/min with a total gradient time of 15 minutes.

Results: Key bioanalytical parameters guide the choice of assay for accurate protein analysis. Specificity ensures analytical precision, recovery reflects extraction efficiency, and matrix effects assess the impact of interferences. Linearity enables adaptability across concentration ranges, and sensitivity establishes the limit of quantification (LOQ). Precision (%CV) ensures consistent results, while accuracy (%bias) focuses on the closeness of measured values to the true concentrations. Also, high throughput is beneficial in scenarios where many samples must be analyzed efficiently. The chromatograms in Figure 1 show the specificity of our four extraction methods. Pellet Digestion: Ideal for scenarios where high throughput is not a priority and the sensitivity requirement is not critical. Despite its lower throughput and notable interferences at 100 ng/mL (137% matrix effects, Figure 2), this method is a practical option during the early stages of method development. FASP: Although prone to extraction variability that led to interferences at 100 ng/mL (156% recovery, Figure 2), FASP can be effectively utilized in cases where such interference is manageable, particularly with higher analyte concentrations. This method's adaptability makes it suitable for specific analytical conditions. IP: With its exceptional sensitivity and a lower LOQ of 7.21 ng/mL (Figure 2), IP is constrained by limited reagent availability and a narrower linearity range (7.21 – 9379 ng/mL). Nonetheless, it is the method of choice in scenarios demanding high sensitivity within its operational range. S-trap: Featuring a wide linearity range (26.2 – 19,992 ng/mL, Figure 2), it is a robust choice for scenarios requiring a broad dynamic range. Also, its ability to accurately quantify lower concentrations in the ng/mL range renders it a versatile tool for diverse analytical needs.

Conclusion: A suite of sample preparation methods, including pellet digestion, FASP, S-trap, and IP were developed for LC-MS/MS quantification of large molecules in complex biological matrices.  Each method can be strategically selected for bioanalysis of therapeutic or biomarker proteins based on unique analytical scenarios such as expected analyte concentrations, sample volume, availability of capture reagents, and throughput requirements. Notably, antibody-free approaches, such as FASP and S-trap, offer an unbiased way to quantify total protein analytes, particularly beneficial in scenarios where capture reagents are unavailable.

References: 1. Blanco, M. J.; Gardinier, K. M.; Namchuk, M. N., Advancing New Chemical Modalities into Clinical Studies. ACS medicinal chemistry letters 2022, 13 (11), 1691-1698.
2. Palshikar, M.; Jadav, T.; Rajput, N.; Sahu, A. K.; Sengupta, P., Surrogate peptide selection and internal standardization for accurate quantification of endogenous proteins. Bioanalysis 2022, 14 (13), 949-961.
3. Ludwig, K. R.; Schroll, M. M.; Hummon, A. B., Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies. Journal of Proteome Research 2018, 17 (7), 2480-2490.
4. Kuras, M.; Woldmar, N.; Kim, Y.; Hefner, M.; Malm, J.; Moldvay, J.; Döme, B.; Fillinger, J.; Pizzatti, L.; Gil, J.; Marko-Varga, G.; Rezeli, M., Proteomic Workflows for High-Quality Quantitative Proteome and Post-Translational Modification Analysis of Clinically Relevant Samples from Formalin-Fixed Paraffin-Embedded Archives. Journal of Proteome Research 2021, 20 (1), 1027-1039.

Figure 1. Chromatograms of surrogate peptides from the analyte protein for four different extraction methods from mouse plasma at 100 ng/mL analyzed by LC-MS/MS with a 15-minute liquid chromatography gradient (retention time = 7.4 minutes).

Figure 2. Key bioanalytical parameters of the assays (n=3): recovery, matrix effects, linearity, limit of quantification (LOQ), precision (%CV), and accuracy (%bias). The data were obtained using a 15-minute gradient.