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Abstract INTRODUCTION:
Human relaxin peptides are a group of seven closely related peptide hormones that have been shown to be part of various physiological processes such as pregnancy, fertility and cardiovascular adaptations. Thus, peptides of this family are believed to be a promising diagnostic marker for several correlated clinical fields such as heart disease, fibrosis and infertility. Low expression levels in the pmol/ml range and the lack of robust proteotypic peptides make these hormones difficult peptides for identification and quantification using conventional proteomic approaches and may explain inconsistent information on their expression. To date there are only a few commercial immunoassays targeting human relaxin peptides for routine application, with varying quality. A routine LC-MS/MS method for the quantification of relaxin peptides from biological matrices does not yet exist. So far, only two approaches have been described to quantify relaxin from biological matrices, and in both cases the choice of internal standard was not ideal.1,2 Furthermore, there is only very limited mass spectrometric data that verifies immunogenic methods and results, as cross reactivity between the different members of the peptide family can occur. We addressed the aforementioned problems as well as the lack of a fast routine LC-MS/MS based method for human relaxin peptides, and decided to develop a precise method for quantification of relaxin peptides by LC-MS/MS.
METHODS:
A solid phase extraction method optimized for RLX-2 was compared to an immunoaffinity enrichment from serum or plasma by different anti-relaxin antibodies. As LC-MS/MS detection of the A-chain of RLX-2 was more sensitive than the B-chain or the intact RLX-2, the A-chain of RLX-2 was set as target and measured in positive ion mode by ESI-MS/MS or nanoLC-ESI-MS/MS. A 13C6-15N-leucine labeled endogenous RLX-2 peptide was used as an internal standard with both the A-chain and B-chains labeled, allowing the flexibility to quantify the individual RLX2 chains or fragments thereof separately. Aside from RLX-2, transitions for relaxin-1, relaxin-3 and insulin were investigated as well.
RESULTS:
The limit of quantification for RLX-2 was around 100 pg/ml (approx. 16 fM) using an ExionLC coupled with a QTRAP 5500 (AB Sciex) or 8 pg/ml (approx. 1.3 fM) using EASY-nLC 1000 coupled with an Orbitrap Velos Pro (Thermo Fisher). Thereby the sample preparation for the quantification of RLX-2 by LC-MS/MS was based on immunoaffinity enrichment. Efficient, non-denaturing dissociation of the A-chain and B-chain during the reduction step at a lower pH improved the further quantification of RLX-2. In addition, the detection of the A-chain could be enhanced by adding DMSO to the mobile phases.
CONCLUSION:
The UPLC-MS/MS and nanoLC-MS/MS allows the specific quantification of RLX-2 in the endogenous range. Proof-of-principle experiments with the first human samples from pregnant women showed a clear identification and quantification of relaxin-2. Furthermore, it was shown that CKD patients have higher RLX-2 values compared to healthy volunteers.3
REFERENCES:
1. Rais, Y. and Drabovich, A. P., Identification and quantification of human relaxin proteins by immunoaffinity-mass spectrometry. J. Proteom Res. 2024. 23(6): p. 2013-2027.
2. Fülöp, A., Armbruster, F.P., Grön H.J., Dschietzig T.B. and Diesner M., Development of a LC-MS/MS Quantification Assay for Human Relaxin H2 and Extraction of Human Relaxin H1 and Relaxin H3. Clin Lab. 2024. 1;70(2).
3. Dahlke, M., Ng, D., Yamaguchi, M., Machineni, S., Berger, S., Canadi, J., Rajman, I., Lloyd, P. and Pang, Y. Safety and tolerability of serelaxin, a recombinant human relaxin-2 in development for the treatment of acute heart failure, in healthy Japanese volunteers and a comparison of pharmacokinetics and pharmacodynamics in healthy Japanese and Caucasian populations. J. Clin. Pharmacol. 2015. 55(4): p. 415-422. |
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
