MON-487 Simultaneous Monitoring of 13 IGF-1 Variants in a Clinical Mass Spectrometry Assay

In the clinical laboratory, identification and quantification of intact proteins by high resolution accurate mass (HRAM) mass spectrometry (MS) is a highly specific and robust technique. However, protein variants with mass-to-charge ratios (m/z) differing from the wild type are not measured. In addition, variants may not be resolved from the wild type or from each other. HRAM can enable resolution of IGF-1 and its variants at the level of isotopic masses. However, the choice of which isotope to monitor is critically important as intact IGF-I produces a wide isotopic envelope of up to 13 detectable peaks. For this purpose, we employ the concept of Peak Isotopic Index (PII) wherein peaks in the isotopic clusters are designated by a subscript. We designate IGF-1(0) for the monoisotopic peak, and IGF-1(1), IGF-1(2), etc., for heavier isotopic peaks. The same applies to IGF-1 variants. Unfortunately, most of the variant isotopic peaks are not resolved. Consequently, simultaneous monitoring of multiple variants is challenging. In the current study, our objective was to simultaneous monitor 13 IGF-1 variants in a routine clinical analysis employing HRAM MS. For the routine IGF-1 assay we used a Thermo Q Exactive™ Focus Hybrid Quadrupole-Orbitrap™ MS coupled to a multiplex high performance liquid chromatography (HPLC) system. To efficiently monitor all the variants, they were split into 4 groups, each corresponding to a single m/z for that group of variants: Group 1 (P66A(4), R36Q(6)) monitored at m/z 1089.8050; Group 2 (A67S(4), T29I(8)) monitored at m/z 1095.8065; Group 3 (S34N(2), A70P(3), A38V(1), M59R(4)) monitored at m/z 1097.0960; and Group 4 (V17M(4), V44M(4), T4M(6), A67T(6), A70T(6)) monitored at m/z 1098.0969. All specimens that were suspected to contain IGF-1 variants were submitted for DNA sequencing for identification and/or confirmation. We analyzed 77,231 samples and identified 302 patients (0.4%) with IGF-1 variants. Most (290, 96.0%) were Group 4 variants, compared to 8 (2.7%) from Group 3, 3 (1.0%) from Group 2, and 1 (0.3%) from Group 1. Of the samples analyzed, 299 corresponded to patients heterozygous for a particular IGF-1 variant; the amount of wild type IGF-1 reported for those patients was a half of the total IGF-1 present in the sample. We also identified 3 homozygous patients (all variants belonging to Group 4), in which no wild type IGF-1 was detected and all of the IGF-1 was expressed in a form of a variant. In this study we demonstrated that 13 IGF-1 variants can be routinely screened by HRAM MS assay using only 4 m/z ratios. To our knowledge, this is the largest number of IGF-1 variants monitored in a clinical setting. While the clinical significance of these variants may be presently unknown, knowledge that the variants are present may help avoid misdiagnosis on the basis of low wild type IGF-1 levels