Detection of IGF-1 protein variants by use of LC-MS with high-resolution accurate mass in routine clinical analysis.

Low or increased serum insulin-like growth factor 1 (IGF-1) concentrations might indicate growth hormone (GH) deficiency or overproduction, respectively. Currently, IGF-1 is mostly measured by automated immunometric assays. However, reagent availability issues prompted us to evaluate LC-MS with high-resolution accurate mass measurement (HRAM) as an alternative (1 ). We developed and validated an IGF-1 LC-MS HRAM method on a Q Exactive mass spectrometer (Thermo Scientific) using 1:70000 resolution (at m/z 200) with a mass accuracy of 5 ppm. A similar method has been published for a qTOF instrument (Agilent) (2 ). We compared our LC-MS-HRAM method initially in 459 patient samples with the iSYS IGF-1 automated immunoassay (IDS). This yielded a least square linear fit of LC-MS HRAM 0.84 iSYS 3.5, with an r of 0.966. During further studies involving 1720 samples to generate reference ranges, the 2 methods continued to be highly correlated, with a reproducible systematic bias. However, we also identified 16 outliers for which the LC-MS HRAM method gave dramatically lower results than the iSYS immunoassay (Fig. 1). Of the 16 samples examined, 15 had LC-MS HRAM IGF-1 concentrations of approximately 50% of those obtained by the iSYS assay, whereas 1 sample had a MS result of 5% of the immunoassay measurement. Reexamination of the HRAM data of these 16 samples led to the discovery of a protein variant with a mass m/z 1098 that showed a spectrum with similar isotopic ratio as the monitored m/z 1093 charge envelope for IGF-1 (most-abundant observed m/z 1093.5214, [M 7H] ). When the protein variant peak was quantitated and summed with the IGF-1 results, the LC-MS HRAM IGF-1 (method I) values closely matched those of the iSYS immunoassay, leading us to hypothesize that the protein variant was related to IGF-1. Additional studies included a comparison with a reference LC-MS HRAM method (method II) (2 ) (Quest Diagnostics), which showed good correlation with our m/z 1093 data in the normal population and in the variant samples (both approximately 50% of iSYS in those with 1098 present) (Fig. 1). Ongoing LC-MS HRAM IGF-1 testing revealed that this phenomenon occurred in approximately 0.6% of our patient population. We also confirmed that the mass spectrum for the IGF-1 variant was identical between various patients with as little as 2 ppm error. Reduction studies with dithiothreitol revealed no difference of mass between reduced and nonreduced protein. Fragmentation of wild-type IGF-1 (wtIGF1) predominantly produced b65 and b68 ions. The fragments from the wtIGF1 and IGF-1 variant were identical, implying that the difference in IGF-1 species mass most likely came from a C-terminal variant with possible changes in amino acids at positions 69 or 70. The difference between the observed monoisotopic mass of 1093[M 7H] 7 (1093.0917) and 1098[M 7H] 7 (1097.3802) was 4.2885. We performed in silico analysis of all variant possibilities for IGF-1. Only 1 of these variants, A70T, was likely among the possible © 2015 American Association for Clinical Chemistry 1 Nonstandard abbreviations: IGF-1, insulin-like growth factor 1; GH, growth hormone;HRAM, high-resolution accuratemassmeasurement; wtIGF1, wild-type IGF-1; SNP, single nucleotide polymorphism. Fig. 1. Test results for the 16 disparate samples and 1 wild-type patient, tabulated along with representative spectra for wtIGF-1 (1093 [M+7H]) and A70T-IGF1 (1098 [M+7H]). Top, patient without A70T-IGF1 present (wild-type); middle, patient with both wtIGF1 and A70T-IGF1 present; bottom, patient with A70T-IGF1 exclusively, no wtIGF1 present. *Confirmed A70T with DNA sequencing. NP, not performed. Clinical Chemistry 61:7 990–996 (2015) Letters to the Editor