However, this equipment may not be readily available in clinical laboratories. Alternatively, quantitative IGF-1 assays based on high-resolution mass spectrometry have been described. Furthermore, despite the use of stable-isotope-labelled internal standards, small variations in enzymatic digestion conditions may cause significant differences in analytical results. A disadvantage of this workflow, however, is that these techniques are generally relatively time-consuming, making it difficult to implement these methods into clinical laboratories.
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This workflow, where, following enzymatic digestion, a unique peptide serves as surrogate for the protein is aimed at combating sensitivity-related issues that arise in protein mass spectrometry, such as poor transmission and fragmentation of intact protein ions.
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In order to reach sufficient analytical sensitivity for the quantitative analysis of proteins, the majority of LC-MS/MS methods for IGF-1 rely on immunoaffinity techniques as a mode of sample clean-up and are performed at the peptide level. Given the unique ability of mass spectrometry to use stable-isotope-labelled internal standards combined with highly specific detection based on mass-to-charge ratios, this technique has the possibility to separate and detect different forms of the same protein, is less prone to interferences and is characterised by an increased (interlaboratory) reproducibility compared to LBAs. In recent years, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has emerged as an alternative platform for the quantification of proteins in complex biological matrices.
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However, these IGF immunoassays tend to suffer from intra- and interlaboratory variation and poor reproducibility due to their dependence on antibodies, for which significant batch-to-batch variability has been observed. Quantitative analysis of both IGF-1 and IGF-2 is traditionally performed using different ligand-binding assays (LBAs). dwarfism, gigantism or acromegaly), whereas in some cases, IGF-2 is produced in excess in islet cell tumours and non-islet hypoglycaemic cell tumours, making it an interesting biomarker in these situations. In laboratory medicine, IGF-1 has various applications, including the diagnosis and monitoring of growth hormone–related disorders (e.g. Though in contrast to IGF-1, which plays an important role in childhood growth and continues to have anabolic effects in adults, the major physiological role of IGF-2 is as a growth-promoting hormone during gestation. Despite the fact that they do not originate from the same genetic locus, both IGFs exert their main physiological effects by binding to the IGF-1 receptor. Physiologically they act as the main mediators of growth hormone (GH)-stimulated cell and tissue growth. Insulin-like growth factor 1 (IGF-1, MW 7649 Da) and insulin-like growth factor 2 (IGF-2, MW 7470 Da) are polypeptide hormones, similar in molecular structure to insulin. The method can be readily implemented in clinical mass spectrometry laboratories and has the potential to be adapted for the analysis of different similarly sized peptide hormones. The LC-MS/MS method described here allows for reliable and simultaneous quantification of IGF-1 and IGF-2 in plasma, without the need for enzymatic digestion. Comparison with the IDS-iSYS IGF-1 immunoassay showed good correlation ( R 2 > 0.97), although a significant bias was observed with the immunoassay giving substantially higher concentrations. An excellent correlation was found between nominal and measured concentrations of the WHO reference standard for IGF-1. Intra-assay imprecision was below 4.5% and inter-assay imprecision was below 5.8% for both analytes.
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Lower limits of quantification were 5.9 ng/mL for IGF-1 and 8.4 ng/mL for IGF-2. Detection of intact IGF-1 and IGF-2 is performed by means of a Waters XEVO TQ-S triple quadrupole mass spectrometer in positive electrospray ionisation (ESI+) mode. It features trifluoroethanol (TFE)-based IGF/IGF-binding protein complex dissociation and a two-step selective protein precipitation workflow, using 5% acetic acid in 80/20 acetone/acetonitrile (precipitation 1) and ice-cold ethanol (precipitation 2). The method requires 50 μL of plasma and uses fully 15N-labelled IGF-1 as internal standard. We present a simple and fast antibody-free LC-MS/MS method for the quantification of intact IGF-1 and IGF-2 in human plasma. Quantitative analysis is performed using various ligand-binding assays or enzymatic digestion LC-MS/MS methods, whose widespread adoption is hampered by time-consuming sample preparation procedures. Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) are important biomarkers in research and diagnosis of growth disorders.