Sample Preparation Strategies for Antibody-Free Quantitative Analysis of High Mobility Group Box 1 Protein

Abstract

Sickness behavior and fatigue are induced by cerebral mechanisms involving inflammatory cytokines. High mobility group box 1 (HMGB1) is an alarmin, and a potential key player in this process. Reliable quantification methods for total HMGB1 and its redox variants must be established in order to clearly understand how it functions. Current methods pose significant challenges due to interference from other plasma proteins and autoantibodies. We aimed to develop an antibody-free sample preparation method followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to measure HMGB1 in human plasma. Different methods were applied for the removal of interfering proteins and the enrichment of HMGB1 from spiked human plasma samples. A comparison of methods showed an overall low extraction recovery (<40%), probably due to the stickiness of HMGB1. Reversed-phase liquid chromatography separation of intact proteins in diluted plasma yielded the most promising results. The method produced an even higher degree of HMGB1 purification than that observed with immunoaffinity extraction. Detection sensitivity needs to be further improved for the measurement of HMGB1 in patient samples. Nevertheless, it has been demonstrated that a versatile and fully antibody-free sample preparation method is possible, which could be of great use in further investigations.

Cytokines in the Brain and Neuroinflammation: We Didn’t Starve the Fire!

“…They, therefore, set out to develop an antibody-free liquid chromatography coupled with a tandem mass spectrometry-based detection method for HMGB-1. In particular, Kvivik et al showed that, notwithstanding suboptimal recovery, the method developed enabled "the identification of several unique HMGB-1 peptides" [140] (p. 12). This accomplishment, in turn, has the potential to allow for "the measurement of different redox variants" in the future [140] (p. 12).…”

High Mobility Group Box 1: Biological Functions and Relevance in Oxidative Stress Related Chronic Diseases

“…Recently, an antibody-free sample preparation method followed by liquid chromatography joined with tandem mass spectrometry (LC-MS/MS) to measure HMGB1 in human plasma has been developed. The reversed-phase liquid chromatography separation of intact HMGB1 in diluted plasma produced a higher degree of HMGB1 purification than immunoaffinity extraction, which is useful for overcoming its low recovery in body fluids [ 32 ]. Currently, the analytical methods represent a compromise between isoform resolution (LC/MS) and ease of use with high throughput measurement of total HMGB1 (ELISA) with the possibility of reducing interference (PCA-ELISA).…”