Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry

The human relaxins belong to the
Insulin/IGF/Relaxin
superfamily
of peptide hormones, and their physiological function is primarily
associated with reproduction. In this study, we focused on a prostate
tissue-specific relaxin RLN1 (REL1_HUMAN protein) and a broader tissue
specificity RLN2 (REL2_HUMAN protein). Due to their structural similarity,
REL1 and REL2 proteins were collectively named a ‘human relaxin
protein’ in previous studies and were exclusively measured
by immunoassays. We hypothesized that the highly selective and sensitive
immunoaffinity-selected reaction monitoring (IA-SRM) assays would
reveal the identity and abundance of the endogenous REL1 and REL2
in biological samples and facilitate the evaluation of these proteins
for diagnostic applications. High levels of RLN1 and RLN2 transcripts
were found in prostate and breast cancer cell lines by RT-PCR. However,
no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM
in cell lines, seminal plasma, or blood serum. The IA-SRM assay of
REL2 demonstrated its undetectable levels (<9.4 pg/mL) in healthy
control female and male sera and relatively high levels of REL2 in
maternal sera across different gestational weeks (median 331 pg/mL; N = 120). IA-SRM assays uncovered potential cross-reactivity
and nonspecific binding for relaxin immunoassays. The developed IA-SRM
assays will facilitate the investigation of the physiological and
pathological roles of REL1 and REL2 proteins.