Insulin-like Growth Factor I (IGF-I) is a 70 amino acid (7.6 kDa) peptide hormone, with 3 internal di-sulphide bonds. It plays a significant role in mediating the effects of Growth Hormone (GH), circulates at ng/mL levels, and is strongly bound to Insulin-like Growth Factor Binding Protein (IGFBP). Improper balance of GH and IGF-I can lead to conditions such as acromegaly, dwarfism, and increased risk of cancer. Thus, there is significant interest in measuring IGF-I as a biomarker.2
Historically, immunoassays have been used for quantification of IGF-I. In recent years, use of LC-MS for quantification of IGF-I has increased. Most labs using LC-MS use the surrogate peptide approach, with or without immuno-affinity extraction, followed by quantification of resulting signature peptides by analytical scale LC and a tandem quadrupole (TQ) instrument. Although widely accepted, digestion may not always be required for proteins under 10 kDa on a TQ. However, achieving relevant sensitivity levels for such intact proteins does require meticulous attention to sample preparation. The immuno-affinity extraction and the surrogate peptide workflows described in literature are complex and laborious, adding cost and complexity to the analysis.
High resolution mass spectrometry (HRMS) systems are usually the preferred platforms to perform intact protein analysis, but have rarely been used routinely for quantitative bioanalytical applications. With recent advances in MS instrumentation and software capabilities, use of HRMS for bioanalytical quantification is on the rise. Some labs have reported quantifying IGF-I using immuno-affinity extraction and followed by nano-flow LC and a HRMS system.
Here, we highlight a simplified sample preparation workflow using sample pretreatment, protein precipitation, and solid phase extraction (SPE) for the quantification of intact IGF-I from human serum using an analytical LC and a tandem quadrupole instrument. We further compare its performance characteristics to a targeted HRMS approach for quantification.
Sample Preparation: 100 µL of 0.6% Sodium Dodaceyl Sulphate (SDS) is added to 100 µL of plasma/serum and incubated at 37˚C for 45 mins. After incubation, 200 µL of Acetonitrile containing 5% Acetic Acid is added and samples are vortexed, followed by centrifugation at 18,000 G for 10 minutes. 300 µL of the supernatant is added to a 2-mL 96 well plate containing 900 µL 5% Ammonium Hydroxide for pre-treatment. Pre-treated samples are then passed through a conditioned and equilibrated Oasis MAX µElution SPE plate, washed, eluted and diluted before injecting into LC-MS system.
LC-MS analysis: Quantification of IGF-I was performed using a low dispersion LC, coupled to a TQ or a QTof HRMS system. Chromatographic separation was achieved using a sub-2 µm charged surface hybrid C18 column, (2.1 mm x 50 mm), at a flow rate of 0.4 mL/min using a linear gradient with 0.1% formic acid in water and acetonitrile.
Using the described sample preparation strategy, accurate quantification of IGF-I from 5-1000 ng/mL was achieved on a TQ. Calibration curves were linear (1/x2 weighting) with r2 values >0.99 and mean accuracies between 99-102%. QC performance was excellent with accuracy ranges between 93-108% and CV’s < 10%, well within the acceptable FDA bioanalytical guidelines. In addition, this assay was reproducible and robust across multiple days.
Similar performance characteristics were observed for an analytical LC-HRMS system. IGF-I was accurately quantified from 10-1000 ng/mL using the targeted mode of the QToF system. Calibration curves were linear (1/x2 weighting) with r2 values >0.99 and mean accuracies between 98-103%. QC performance was excellent with accuracy ranges between 91-97% and CV’s < 10%
Here, we demonstrate a simple sample preparation approach combined with analytical flow LC and tandem-quadrupole MS for the fast, direct analysis and quantification of intact IGF-I from serum/plasma. This method eliminates the need for complex and costly sample preparation like protein digestion or affinity. The analytical sensitivity of (5 ng/mL), linear dynamic range, and excellent reproducibility of the method described reliably measure low endogenous and elevated levels of IGF-I in serum.
Additionally, the work highlighted also compares the robustness, sensitivities and bioanalytical performance of the TQ and QToF systems for quantifying intact proteins like IGF-I.