Targeted Selected Reaction Monitoring Mass Spectrometric Immunoassay for Insulin-like Growth Factor 1

Abstract

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.

Targeted mass spectrometry analysis of the proteins IGF1, IGF2, IBP2, IBP3 and A2GL by blood protein precipitation

“…Their quantification is typically achieved independently by classical methods such as ELISA or RIA assays [18], necessitating blood sample volumes that range between 50 and 200 μL for each assay. Specific LC-MS/MS methods have been recently developed for plasma/ serum samples for quantification of IGF1 [25] and IGF1 and A2GL [19] by protein precipitation, and of IGF1 by specific antibody-based purification [26], necessitating 100 μL, 30 μL and 40 μL, respectively. In this manuscript, we describe a sensitive and scalable method for the quantification of all five proteins by protein precipitation and selected reaction monitoring (SRM) LC-MS/MS analysis [27], requiring a total of 7 μL of plasma, and reaching a throughput of up to 80 analyses per day.…”

Clinical assays for quantitation of insulin-like-growth-factor-1 (IGF1)

“…Several groups have reported mass spectrometric methods for IGF1 quantitation [46][47][48][49][50][51][52]. One of the first published reports described a liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method in standard samples [53,54].…”

“…As the analytical approaches for mass spectrometric quantitation have evolved, two techniques are emerging as highly amenable to implementation in high throughput reference laboratories. In this section development and performance characteristics of immunoaffinity mass spectrometric method [50] and a liquid chromatography high-resolution accurate-mass spectrometry (LC-HRAMS) method [51,52] will be discussed in detail.…”

The clinical utility of mass spectrometry based protein assays

“…One clinical LC-MS IGF1 assay employs a denaturation step of large proteins that release free IGF1 [61]. A second LC-MS assay uses an antibody to IGF1 that is not affected by IGFBP and is followed by trypsin digestion for analysis of a single free IGF1 peptide [62]. The use of trypsin to digest the sample not only cleaves the large IGF protein into more MS amenable peptides but also effectively inactivates the IGFBP.…”