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Automated Identification of IGF‑1 Variants in a High-Throughput Clinical Laboratory


Author: Zengru Wu, Ph.D, DABCC Sr. Scientific Director, Mass Spectrometry and Clinical Chemistry, Quest Diagnostics




Circulating insulin-like growth factor-1 (IGF-1) level has been used for assessing growth-related disorders, such as acromegaly and growth hormone deficiency. Liquid chromatography−high resolution mass spectrometry (LC-HRMS) is the reference method for measuring IGF-1 due to its molecular specificity, quantitative performance, and detailed age/sex-specific reference intervals [1]. The approach eliminates the reagent lot-to-lot variability that impacts the performance of immunoassay platforms [2].


Dr. Wu’s group at Quest Diagnostics quantified IGF-1 as an intact protein in serum using a “top-down” approach. IGF-1 is separated from IGF binding protein (IGFBP) by acidified ethanol-induced protein precipitation; unbound IGF-1 remains in the supernatant while most proteins are precipitated. The supernatant is injected to LC-HRMS system directly. Online extraction and chromatographic separation further remove interfering substances. Unlike small molecules, intact protein and peptide biomarkers exhibit mass spectra with multiple charge states and complex isotopic envelopes. High-resolution mass spectrometers, such as Q-TOF and Orbitrap, provide the mass accuracy and resolution required to monitor the intensity of selected peaks within isotopic envelopes. For IGF-1, M4 peak (mass-to-charge ratio, m/z, 1093.5209) in the isotopic envelope of the [M+7H]7+ ion is used for quantification; and the peaks of M3 (m/z 1093.3778) and M5 (m/z 1093.6641) are used as qualifier ions to support correct peak assignment [3].


LC-HRMS based approach is effective for quantitation of wild-type IGF-1 protein. However, amino acid substitutions due to polymorphisms can lead to changes in m/z, essentially “hiding” the polymorphic variants. The possibility of having polymorphisms increases when larger proteins with more amino acid residues are monitored. Polymorphisms in the IGF-1 gene may cause mass shifts in the polypeptide, which can affect quantification and cause errors in clinical interpretation. Dr. Wu’s team built a library of 15 IGF-1 variants based on DNA database, literature case reports, and their previous discoveries [4]. Using this library, they (a) proposed a concept of “isotopic peak index (IPi)”, which allows simultaneous quantification of fifteen IGF-1 variants by monitoring only four ions (variant groups); (b) developed a “relative retention time” parameter that allows identification of new variants; and (c) utilized tandem MS (MS/MS) to distinguish the most common variants: A67T and A70T. All variants were confirmed by DNA sequencing [5]. Figure 1 illustrates how to differentiate a variant from the wild-type IGF-1 by monitoring differences in the IPi and relative retention time.


This approach allowed the identification of six variants from the ExAC database, namely P66A, A67S, S34N, A38 V, A67T, and A70T, two previously-reported V44M and A67V variants, and discovery of six new variants, Y31H, S33P, R50Q, R56K, T41I, and A62T. Major improvements in the workflow include enhanced automation, avoiding manual calculations that are prone to human error, and the ability to monitor more, and discover new IGF-1 variants. The workflow provides a profile of a patient’s IGF-1 status and can be used to investigate genotype−phenotype relationships in IGF-1 variants.


Figure 1. LC-HRMS data for specimens from two different patients. IPi can be used to distinguish between variants belonging to the same variant group (variants with isotopic peaks very close in m/z). Evidence that patient 1 contains an IGF-1 variant comes from the monitored peak being the sixth in the isotopic envelope after the monoisotopic peak (IP0) vs the fourth in the wild type. (A) EIC of WT4 for patient 1; (B) EIC of VG4 for patient 1; (C) [M+7H]7+ isotopic envelope for patient 1, where the rectangle indicates that IP6 was detected at VG4; (D) EIC of WT4 for patient 2; (E) EIC of VG4 for patient 2; and (F) [M + 7H]7+ isotopic envelope for patient 2, where the rectangle indicates that IP4 was detected at VG4. Abbreviations: IPi, isotope peak index; EIC, extracted ion chromatogram; IP6, isotope peak 6; IP4, isotope peak 4; LC-HRMS, liquid chromatography – high resolution mass spectrometry; VG4, variant group 4; WT4, wild type isotope peak 4.




高分辨率质谱自动检测和定量 IGF-1及其变体

胰岛素样生长因子1 (IGF-1)水平可用于评估和诊断与生长相关的疾病,例如肢端肥大症和生长激素缺乏症。液相色谱-高分辨率质谱测量 IGF-1 的优点在于分子特异性、定量准确性、和详细的年龄/性别特定正常值范围[1]。该方法不受试剂批次间差异的影响[2]。

吴博士在Quest检验的团队使用质谱全蛋白质分析方法测量血清中IGF-1及其变体的水平。与肽段分析方法相比,全蛋白质分析方法的样品处理更加简捷。通过加入酸化乙醇,胰岛素样生长因子1和结合蛋白分离,离心去除其它蛋白质沉淀。处理后的样品可直接进样,柱前抽提和高效液相色谱分离可进一步除去可能存在的干扰物质。与小分子不同,多肽和蛋白质标志物的ESI质谱图包含多个电荷,同位素峰质量差异很小,图谱复杂。QTOF 和 Orbitrap 等高分辨率质谱仪器提供了监测同位素峰所需的高精确性和高分辨率。对于 IGF-1,[M + 7H]7+离子的M4同位素峰 (m/z 1093.5209) 用于定量,M3 (m/z 1093.3778)和M5 (m/z 1093.6641) 峰辅助定性[3]。

高分辨率 LC-HRMS 的方法可准确定量样本中的野生型IGF-1。然而,蛋白质较大时,氨基酸突变和蛋白变体存在的可能性增加,蛋白质变体通常导致分子量和m/z的改变,从而导致相对应的IGF-1变体在定量中被“隐藏”起来,定量报告中浓度只代表野生型的浓度。基因多样性导致IGF-1变体的存在会引起定量偏差甚至造成检测错误。使用 DNA 数据库搜索结果、文献报告和既往发现,吴博士团队构建了一个包含 15 个 IGF-1 变体的数据库 [4]。同时,她们 (a) 提出了“同位素峰指数 (IPi)”的概念,仅监测 4个离子峰就可以检测出 15个IGF-1 变体; (b) 使用“相对保留时间”参数检测新的蛋白质变体; (c) 利用串联质谱区分最常见的变体, A67T 和 A70T。所有IGF-1变体均经过 DNA 测序验证[5]。图 1 说明了如何通过IPi和相对保留时间的差异将两种不同的IGF-1变体区分开来。

该方法可以从ExAC数据库中识别 6 个变体, 包括P66A、A67S、S34N、A38 V、A67T 和 A70T,以及 2 个先前报道的 V44M 和 A67V 变体,并发现了 6 个新的变体:Y31H、S33P、R50Q、R56K、T41I 和 A62T。优化工作流程提高了自动化水平,避免了易出错的手动计算,从而可以监控多种变体,并提高了发现新变体的能力。检验报告提供了患者 IGF-1及其变体的定性和定量检测结果。该方法可用于探索 IGF-1 及变体的基因型和表型关系。




References

1. Bystrom C, Sheng S, Zhang K, Caulfield MP, Clarke NJ, Reitz R. Clinical utility of insulin-like growth factor 1 and 2; determination by high resolution mass spectrometry. PLoS One 2012, 7(9):e43457.

2. Bonert V, Carmichael J, Wu Z, Mirocha J, Perez DA, Clarke NJ, Reitz RE, McPhaul MJ, Mamelak A. Discordance between mass spectrometry and immunometric IGF-1 assay in pituitary disease: a prospective study. Pituitary 2018,21(1):65−75.

3. Bystrom CE, Sheng S, Clarke NJ. Narrow mass extraction of time-of-flight data for quantitative analysis of proteins: determination of insulin-like growth factor-1. Anal Chem. 2011, 83(23):9005−9010.

4. Wu Z, Sanders H, Motorykin I, Caulfield MP, McPhaul MJ. Detection of insulin-like growth factor 1 variants by mass spectrometry: results from a clinical reference laboratory. Clin Chem. 2019, 65(8):1060-1061.

5. Motorykin I, Li H, Clarke NJ, McPhaul MJ, Wu Z. 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(34):11836-11842.


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