Differentiation of Common IGF-1 Variants Using HRMS COM Determination with Follow-Up MS/MS Verification.

Insulin-like growth factor 1 (IGF-1), a peptide hormone regulator of growth hormone (GH), has common variants with differing functionality. These variants are a result of single amino acid changes in the peptide that can lead to significant changes in the resulting protein. The standard method of evaluating any of these variants is by using tandem mass spectrometry (MS/MS) methods. A novel method has been developed to evaluate some variants solely by high-resolution mass spectrometry (HRMS) of the intact peptide by calculating the center of mass (COM) of the [M + 7H] isotopic distribution. This has allowed differentiation between the nonfunctional V44M variant and the A67T/A70T functional variants without the need for MS/MS. However, MS/MS is still needed to differentiate between the A67T and A70T variants. In this chapter we outline the LC-HRMS method for IGF-1 analysis with the inclusion of COM calculations and subsequent MS/MS differentiation.