Human Insulin Like Growth Factor 1 (IGF1)

Insulin-like growth factor 1 (IGF1) is an important biomarker for the treatment of growth hormone disorders. Recently, there has been increased interest in using mass spectrometric (MS) detection methods to measure IGF1. However, widespread clinical deployment of an MS-based IGF1 assay requires increased throughput and speed to justify analysis costs, as well as robust industrial platforms that are reproducible across laboratories.

Presented here is an MS-based quantitative IGF1 assay with a performance evaluation of >1,000 samples/day and an ability to quantify IGF1 point mutations and post-translational modifications. The throughput of the IGF1Mass spectrometry immunoassays (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a cutting-edge format, and high-throughput MALDI-TOF-MS analysis. The resulting assay had a limit of detection and limit of quantification of 1.5 μg/L and 5 μg/L, respectively, with intra- and inter-assay precision CVs of less than 10% and good linearity and recovery. The IGF1-MSIA was compared to the commercially available IGF1-ELISA via the Bland-Altman method comparison test, resulting in a slight positive bias of 16%. 

The IGF1 MSIA was deployed in an optimized parallel workflow using two pipetting robots and MALDI-TOF MS instruments synchronized into 1-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data acquisition and data processing. With this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This testing rate is a significant improvement over existing MS-based IGF1 assays. and is on par with enzyme-based immunoassays. In addition, a mutation was detected in ~1% of the samples (SNP: rs17884626,creating an A→T substitution at position 67 of IGF1), demonstrating the ability of IGF1-MSIA to detect point mutations and post-translational modifications .

introduction

Insulin-like growth factor 1 (IGF1) is an important biomarker for the treatment of growth hormone disorders. IGF1 is produced by the IGF1 gene, located on chromosome 12 in humans, and is a critical mediator involved in cell growth, differentiation and transformation. Human IGF1 is a 70 amino acid protein containing three intra-disulfide bonds with a mass of 7648.7 Da. 

• Serum IGF1 reference values ​​in healthy people are 20–600 μg/L. Most of the IGF1 produced acts as an endocrine hormone via secretion from the liver, but the molecule can also serve as a paracrine hormone in certain tissues, including cartilage cells, and even as an oncogene in autocrine mode. 
• IGF1 exerts its effects by binding to the IGF1 receptor on target tissues. In the blood, 99% of IGF1 is bound to IGFBPs (Insulin-like Growth Factor Binding Proteins), with 80% of IGF1 circulating in a ternary complex consisting of one molecule of IGF1, one molecule of IGFBP3, and one molecule of an acid labile subunit .  Therefore, the presentation of circulating IGF1 has created the need for methods to disrupt these complexes for accurate IGF1 quantification. 
• In the last thirty years, IGF1 has been generally quantified using radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme-linked immunosorbent assay (ELISA) and chemiluminescence. These methods use various sample preparation steps to break down and remove IGFBPs (thus providing free IGF1 for testing), including low pH, size exclusion chromatography, and acid-ethanol extraction. Some methods also involve the addition of an IGFBP blocker, typically IGF2 . 
• Because of these variability, commercial assays have not provided comparable measurements of serum IGF1.
• Since 2001 there has been increasing interest in the use of mass spectrometric detection methods to measure IGF1. Such methods can be categorized as either bottom-up (enzymatic digestion followed by analysis of surrogate peptides representative of IGF1) or top-down (no digestion – the intact IGF1 is analyzed directly) and in some cases use immunoprecipitation as a separation step before detection.
• In 2004, our group used a top-down mass spectrometry immunoassay (MSIA) to quantify IGF1 in human samples. The approach used a novel sodium dodecyl sulfate (SDS) treatment to disrupt IGFBPs prior to immunoaffinity capture of IGF1, followed by direct detection of intact IGF1 using MALDI-TOF mass spectrometry.

Endogenous IGF1 was quantitated by introducing a mass-shifted IGF1 analog (internal standard) into the samples prior to any processing.

The internal standard was present throughout the workflow/analysis and was finally registered as a mass-resolved signal in the mass spectra used for normalization and quantification of the IGF1 signal (via a working curve method). In 2008, Thevis  et. Al. used immunoprecipitation coupled to liquid chromatography/electrospray ionization tandem mass spectrometry (LC/MS) to quantify IGF1 and related analogues. This approach monitored productions arising from dissociations of terminal IGF1 peptide fragments (the molecule was fragmented from its intact form, limiting sequence coverage due to the three remaining intact disulfide bonds). Recently, MS-based non-immunoprecipitation methods for IGF1 quantification have gained traction. In 2009, Kay  et. al  implemented an acetonitrile precipitation extraction using a bottom-up LC/MS /MS-SRM approach . This approach monitored MS  ³ ions of the N-terminal IGF1 fragment (residues 1-21), provided a dynamic range of 16-2000 μg/L and had CVs <13%. Solid phase extraction methods have also been found to work for IGF1 quantification. Bystrom  et. Al. demonstrated top-down quantification of IGF1 via solid-phase extraction coupled with narrow mass extraction (via Q-TOF) of a single IGF1 isotope in the 7  ⁺  charge state (m/z 1093.5209) yielded excellent analytical metrics (CVs <5.2%, LOD 3.7). μg/l) . Also Kay  et. al . further refined their approach by now introducing solid phase extraction (SPE) at the front end of the workflow and extended the method to monitor two IGF1 tryptic fragments (1-21 and 38-50). Each of these approaches has its own merits and taken together they clearly demonstrate the value of IGF1 quantification with MS detection.

However, apart from providing an unequivocal detection of IGF1 with high analytical performance, the widespread clinical adoption of an MS-based IGF1 assay requires increased throughput and speed to justify the cost of the analyzes (mainly due to time unit and consumables) , robust industrial platforms that are reproducible across laboratories (turnkey systems), and the long-term sustainability of the supply chains associated with the IGF1 assay (as well as any additional assays). In addition, and considering the ability of mass spectrometry to easily detect molecular variants of a target protein, the current status of IGF1 mass spectrometry assays couldalso benefit from improved configurations that account for protein microheterogeneity across populations. Such heterogeneity could potentially cause currently used top-down and bottom-up MS approaches to fail when a patient is phenotypically heterozygous via a single nucleotide polymorphism or when N- or C-terminal truncations affect the monitored m/z signals change in LC/MS. In addition, such protein heterogeneity may have undiscovered pathophysiological implications and potential clinical utility.

In this work, we present an MS-based quantitative IGF1 assay that meets all of the above requirements by achieving these two goals: 1) To rigorously quantify IGF1 in human plasma samples at a rate of >1,000 samples/day over time and to objectively assess economic considerations associated with the transfer of such targeted mass spectrometric assays from research laboratories to clinical use, and 2) to consider the IGF1 heterogeneity discovered during the analysis of large populations in order to intelligently design IGF1 mass spectrometric assays that avoid errors avoid (or allow for the study of variants in alternative disease situations) due to structural variants.To the best of our knowledge, none of these aspects of assay/biomarker translation have previously been tested and reported to any significant extent.