Development of an UPLC/MS–MS Method for Quantification of Intact IGF-I from Human Serum

Abstract

Aim: Developing LC–MS methods for biomolecules is often challenging due to issues with molecular size and complexity, nonspecific binding, protein binding, solubility and sensitivity. As a result, complex sample preparation workflows, including immune-affinity and/or protein digestion and lengthy analysis potentially using nano-flow LC, may be needed to achieve the required sensitivity. This work aims to provide a simple, sensitive, fast and robust method for quantification of intact IGF-I from human serum using UPLC–MS/MS. Methods: IGF-I serum samples were denatured with sodium dodecyl sulfate, followed by organic protein precipitation to effectively disrupt protein binding and subsequent SPE of the resulting supernatant for sample cleanup and enrichment prior to LC–MS/MS analysis. Separation was performed on an analytical scale LC using a reversed-phase column containing <2 μm solid core particle followed by detection on a tandem quadrupole MS in multiple reaction monitoring mode. Results: Intact IGF-I was quantified from serum using the method described above at a LLOQ of 5 ng/ml with a dynamic range 5–1000 ng/ml (r2>0.99) and mean accuracy of 101.76%. Accuracies for quality control samples were between 93.9–107.7% with RSD <7%. Conclusion: The analytical sensitivity, linear dynamic range and excellent reproducibility of this method reliably measures endogenous and elevated serum IGF-I levels, demonstrating its utility in discovery, bioanalysis and clinical research.

An antibody-free LC-MS/MS method for the quantification of intact insulin-like growth factors 1 and 2 in human plasma

“…Given that the method does not rely on complex and time-consuming immunoaffinity techniques and reductive, alkylating and enzymatic digestion steps, it can be readily applied in clinical mass spectrometry laboratories as an alternative to immunoassays that suffer from poor reproducibility and interlaboratory comparability. Although different LC-MS/MS methods have been described for IGF-1, these methods mostly rely on the analysis of signature peptides following reduction, alkylation and trypsin digestion [17][18][19][20][21][22][23]. The main disadvantages of this methodology are that enzymatic digestion steps are relatively time-consuming and that it does not provide direct information regarding the intact protein.…”

“…However, these IGF immunoassays tend to suffer from intraand interlaboratory variation and poor reproducibility due to their dependence on antibodies, for which significant batchto-batch variability has been observed [10][11][12][13][14]. 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 [15][16][17][18][19][20][21][22][23][24]. 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 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 [15][16][17] and are performed at the peptide level [17][18][19][20][21][22][23]. 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.…”

Optimization, validation, and comparison of a rapid method for the quantification of insulin‐like growth factor 1 in serum using liquid chromatography–high‐resolution mass spectrometry

“…To save time and cost, we optimized rapid sample preparation method suitable for intact IGF‐I analysis to avoid complicated steps, such as trypsin digestion, 13 immunoaffinity purification, 6 vacuum evaporating, 20 and solid phase extraction 21 …”

Development and single laboratory validation of a targeted liquid chromatography-triple quadrupole mass spectrometry-based method for the determination of insulin like growth factor-1 in different types of milk samples

“…In particular, Bronsema et al (2018) developed and validated an LC-MS/MS targeted method for the detection of IGF-1 in serum in the 10–1000 ng/mL concentration range by selecting two signature peptides; an urea-based sample preparation procedure was proposed and no analyte enrichment step was required. Tanna, Lame and Wrona (2020) developed and validated a sensitive (LOQ 5 ng/mL) and reliable (mean accuracy of 101.76%) ultra-pressure (UP)LC-MS/MS targeted method for the detection of intact IGF-1 in clinical samples. Pratt, van Faassen, Remmelts, Bischoff and Kema (2021) recently published an antibody free LC-MS/MS method for the detection of intact IGF-1 and IGF-2 in human plasma with LOQ values ranging between 5.9 and 8.4 ng/mL.…”