February 1, 2013 (Vol. 33, No. 3)

Next-Generation Approach Aims to Save Time and Money

Since its discovery over 50 years ago, the biological aspects of insulin-like growth factor-1 (IGF-1) have been determined to be extensive. Ranging from cell proliferation, cell differentiation and apoptosis, tissue growth, and organ-specific functions, the biological functions of IGF-1 and its measurement have been deemed significant. As a biomarker, it is used in the diagnosis and treatment of growth disorders, has been implicated in the prognosis and diagnosis of several cancers, and is used to aid in the identification of athletic enhancement from growth hormone use. Its therapeutic forms have been implicated in athletic doping as well.

On account of the significance of its measurement, immunoassays have been developed over the years in attempts to accurately and precisely measure IGF-1. Since the first IGF-1 radioimmunoassay in 1977, many methods for detecting IGF-1 have been introduced, including radioimmunoassay, ELISA, immuno-chemoluminescence, and mass spectrometry-based assays. However, these described methods are not without insufficiencies that result from the complexities in IGF-1 as a peptide.

Measurement of IGF-1 is complicated, by both the biology of IGF-1 and the recent need to differentiate the endogenous form of IGF-1, and the synthetic therapeutic forms developed to treat growth disorders.

IGF-1 is a small peptide that circulates in plasma with the majority (98%) bound to the IGF-binding proteins (IGFBP). There are six binding proteins, each with a different affinity for IGF-1. Therefore, direct measurement requires that the IGF-1/IGFBP complex be disrupted. Due to the differences in the native IGF-1/IGFBP complexes, extensive efforts have been focused on their disruption.

Over the years, methods have been refined into the most commonly used protocol today, which uses an acid/ethanol precipitation (to disrupt the IGF-1/IGFBP complex) followed by the addition of insulin-like growth factor-2 (IGF-2) to prevent the reformation of the IGF/IGFBP complex. However, this liberation methodology is not without faults.

First, in the process of disrupting the IGF-1/IGFBP complex by acid/ethanol precipitation, there is the potential IGF-1 loss, which would directly affect the measurement’s accuracy. Secondly, the post-disruption addition of IGF-2 is not a 100% prophylactic treatment in preventing IGF-1/IGFBP re-complexation. In fact, this method has little effect on preventing the reformation of the IGF-1/IGFBP complex with the smaller IGFBPs (IGFBP-1 and IGFBP-4). Hence, these contribute to the reasons for the current inconsistencies in IGF-1 measurements.

In addition to having to overcome the IGF-1/IGFBP complex issues, the IGF-1 analytical techniques have recently had to contend with the existence of therapeutic forms of IGF-1. As a result of these synthetic forms, immunoassays that are able to differentiate endogenous IGF-1 from the therapeutic forms have become highly sought after.

For conventional immunoassays, this is costly in both money and time. As these approaches require antibodies to be developed that enable this degree of differentiation. However, for mass spectrometric immunoassays (MSIA), IGF-1 is detected by the measurement of its intrinsic molecular mass and, therefore, alternate forms of IGF-1 that differ in mass can be readily differentiated.

One such MSIA, termed IGF-1 MSIA-SRM, has been developed for IGF-1 with improved protocols for disrupting the IGF-1/IGFBP complex and for monitoring the immunoassays’ efficiency from sample preparation to mass spectrometric detection.

The general workflow of the developed IGF-1 MSIA-SRM (Figure 1) begins by preparing plasma samples to efficiently disrupt the IGF-1/IGFBP complex and maintain this dissociation. For this, a dilution buffer containing 0.3% SDS (w/v) was developed. The plasma sample, along with a spiked internal reference standard (synthetic long-R3-IGF-1 (LR3)), was incubated for 30 minutes. Post incubation, the liberated IGF-1 was co-immunoaffinity extracted with the internal reference, using protein A/G MSIA pipette tips (from Thermo Fisher Scientific) loaded with antihuman IGF-1 antibody.

This antibody loading is shown in Figure 1 (step 1), in which the MSIA tips are applied using a simple iterative pipetting action (aspirating/dispensing). This flowing of an antihuman IGF-1 solution through the protein A/G MSIA tips results in antibody binding, thus creating antihuman IGF-1 MSIA tips. The antihuman IGF-1 MSIA tips are then used to extract/enrich free IGF-1 simultaneously with spiked LR3 from the analytical samples (Figure 1, step 2).

The MSIA tips are then rinsed with a series of buffer and water rinses to remove salts and detergents (Figure 1, step 3), after which captured IGF-1/LR3 are eluted from the MSIA tips by iteratively pipetting an elution solvent of 33% acetonitrile in 0.4% trifluoroacetic acid, TFA (step 4). The enriched IGF-1/LR3 eluate is dried in a speed vac to remove TFA, and then reconstituted for subsequent reduction, alkylation, and tryptic digestion.

In SRM mode, the unique tryptic peptides of IGF-1 and LR3 are monitored using a TSQ Vantage, with the resulting area of the IGF-1 peptide used to quantify the IGF-1 after its normalization to the area of the LR3 peptide.

Figure 1. IGF-1 MSIA-SRM workflow

Pipette Tip Benefits

For most mass spectrometric-based immunoassays, beads are used for the up-front purification. However, MSIA pipette tips provide benefits over the beads in the form of greater simplicity, being more conducive to high-throughput, and less nonspecific binding. Enriching targeted analyte from samples is simplified to the effortless action of pipetting.

The combination of pipetting and forcing sample volume through the micro-column in place at the distal end of a pipette tip provides enhancement of the enrichment process due to microfluidics, resulting in shorter incubation times. Furthermore, the tips may be used on a liquid handler, such as the Novus i handheld electronic pipettor or the Versette pipetting workstation, to allow for parallel processing of multiple samples, thus enabling users’ higher-throughput.

The inert chemistry of the MSIA tips provide reduced nonspecific binding, lowering background and minimizing the work load on LC systems, which contribute to increased signal-to-noise and improved assay sensitivity, as shown in Figure 2.

In the case of the developed IGF-1 MSIA-SRM, the use of the MSIA tips in comparison to beads translated into a 10-fold lower limit of detection and a 20-fold LLOQ for an extraction method requiring less than 30 minutes.

Figure 2. Comparative performance of IGF-1 MSIA-SRM using Protein A/G MSIA tips versus Protein A/G magnetic beads

The IGF-1 MSIA-SRM provides a linear dynamic range spanning 1–1,500 ng/mL (Figure 3), enabling a single assay to quantify IGF-1 over its biologically relevant concentrations and the ability to quantify as little as 5 femtomoles of IGF-1 from 40 mL human plasma. Furthermore, validation of the assay demonstrated linearity, as well as recoveries, of within 15% of expected values spanning the assay’s range, demonstrating the effectiveness of the SDS protocol to disrupt the IGF-1/IGFBP complex. Replicate analyses to determine repeatability resulted in intra- and inter-assay coefficients of variation of 4–10%, providing users with confidence in assay performance.

In summary, the developed IGF-1 MSIA-SRM proved effective and efficient in enabling the measurement of IGF-1. By monitoring peptides corresponding to each forms of IGF-1 the developed assay is capable of measuring endogenous and therapeutic forms of IGF-1, a process less feasible for nonmass spectrometry-based immunoassays. The novel use of the internal reference, LR3, spiked into the analytical samples allows for the entire workflow of the assay, sample preparation to LC-MS/MS detection, to be monitored and provides quality assurance of the assay performance.

Figure 3. IGF-1 MSIA-SRM calibration curve: Protein A/G MSIA D.A.R.T.’S with IGF1 antibody provide a wide linear dynamic range of 1–1,500 ng/mL. Replicate analyses of plasma observed %CV = 8.5%.

Eric Niederkofler, Ph.D. ([email protected]) and Urban Kiernan, Ph.D. ([email protected]), are senior scientists, both specializing in protein mass spectrometry for Thermo Fisher Scientific.

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