In the clinical laboratory, identification and quantification of intact proteins by high-resolution, accurate-mass (HRAM) mass spectrometry is a highly specific and robust technique. However, protein variants with mass-to-charge ratios (m/z) differing from that of the wild type (WT) are not reported.
In the case of insulin-like growth factor-1 (IGF-1), all intact variants produce wide isotopic envelopes of up to 14 detectable peaks, most of which are not unique for any variants at the level of the instrument’s resolution. Knowing the width of isotopic envelopes and their overlap can help identify different variants; failure to account for width can results in false positives.
In this study, we further develop a system to differentiate IGF-1 variants by mass spectrometry (MS), using Isotopic Peak Index (IPi), relative retention time (rRT) and tandem MS.
Previously, we developed a novel naming convention for isotopic peaks, called the IPi, wherein peaks within any isotopic envelope are designated by a subscript. The monoisotopic peak is designated as IP0, and heavier isotopic peaks are identified as IP1 through IP13. To make the detection more efficient, we selected as few m/z as possible to detect all the variants. This resulted in 4 variant groups (VGs), each monitored at a single m/z. The presence of a peak at a VG m/z indicates the presence of a variant from that group.
For variants within VGs, we have 3 ways to distinguish between them. The first is using IPi concept, which can distinguish variants within each VG, as long as they have different IPis. The second way is using the relative retention time (rRT), which is defined as the difference between the RT of a variant and that of the WT. This value can help distinguish variants from the same VG that have the same IPi, such as A38V and A67V; R55K and R36Q. The third way is tandem MS, which can distinguish between the most abundant pair of variants, A67T and A70T, owing to specific y ions generated during their fragmentation. We recently developed such a method.
Of the 307,269 samples we analyzed, 1,266 (0.4%) variants were identified. The following variants (and their count) were identified: R50W/T4M/A67T/A70T (1,210), A67V (23), A38V (9), P66A (6), R36Q/R50Q (5), V17M/V44M (5), A67S (4), R55K/R56K (3), T29I/T41I (1), S34N (1), S33P (1), Y31H (1).
For 2 samples, IPi and rRT did not match WT or known variants; follow-up by DNA sequencing identified new variants: S33P and Y31H. In addition, IPi and rRT results matched known variants, but follow-up DNA sequencing identified 3 new point mutations: R50Q, R56K, and T41I. We tested 15 samples in the R50W/T4M/A67T/A70T group using tandem MS, and were able to distinguish 9 A67T variants and 6 A70T variants. The MS/MS technique will be applied to this patient set.
HRAM, IPi, rRT, and tandem MS can be used together to identify and differentiate IGF-1 variants in routine clinical analysis.