A missing link: How a protein lost a peptide bond

IGF-1 measurements are useful in the diagnosis of growth disorders. However, if a protein variant is present, its level is not included in the total concentration, thus providing falsely low results. Knowing that an insulin-like growth factor-1 (IGF-1) variant is present in a patient specimen is clinically relevant for diagnosis and can provide valuable information to physicians. Previously, we described a method for monitoring IGF-1 variants using liquid chromatography coupled with high-resolution, accurate-mass, mass spectrometry (LC-HRAM-MS)1. The variants are classified based on variant groups (VG), isotopic peak index (IPi), and relative retention time (rRT). Uncommon variants are analyzed by tandem mass-spectrometry (MS/MS) and DNA sequencing.

A potentially novel species was detected that did not match characteristics of any previously identified IGF-1 variant. The unusual feature of this variant was that its area under curve was 36% of that of the WT protein, which contrasts with the usual 100% found in a typical heterozygous patient.

MS/MS of the suspected variant showed two sets of b- and y-ions, which had never been see during our analysis. Sequencing the fragments revealed that one set of C- and N-terminal amino acids was from the Wild Type (WT) protein. The other set of b- and y-ions sequenced were manually partially identified as GL(or I)VD and PTGYG, respectively. A search of the human proteome did not identify proteins with these N- and C-termini.

Closer inspection revealed that identified sequences belong to the internal WT protein sequence, which is extremely unusual. Knowing this, both N-terminal positions 37-45, and C-terminal positions 27-36 were identified. This evidence suggested that this species is the WT protein but with loss of a peptide bond between 2 arginines at positions 36-37 and a gain of a water molecule to “cap” the newly formed termini, which was confirmed by its observed intact m/z. DNA sequencing showed only the WT sequence present, which indicates that the observed modified species is probably a posttranslational cleavage product.

To our knowledge, this post-translational modification of IGF-1 has not been previously reported.

The processes involved in generating this species (e.g. hydrolysis or enzymatic cleavage) and potential consequences for the patient are unknown. Because the broken bond is in the C-domain, activities such as IGF-1 binding to receptors and binding proteins are potentially affected. In addition, the total level of active WT IGF-1 is reduced. Consequently, the presence of this modified IGF-1 may contribute to the etiology of a patient’s condition, as in reported pathogenic variants V44M, R36Q, R50W, Y60H, and S35C. 

Reference

1. Motorykin I, et al. Isotopic peak index, relative retention time, and tandem MS for automated high throughput IGF-1 variants identification in a clinical laboratory. Anal Chem. 2021:93:11836-11842.