Targeted proteomics of serum IGF-I, -II, IGFBP-2, -3, -4, -5, -6 and ALS

Jakob Albrethsen; Lylia Drici; Lea Marie Slot Vilmann; Stine A. Holmboe; Charlotte Ehlers Thomsen; Veronica Lykke Rogaczewska Groendahl; Maud Eline Ottenheijm; Annelaura Bach Nielsen; Christina Christoffersen; Lise Aksglaede; Casper P. Hagen; Nicolai J. Wewer Albrechtsen; Anders Juul

doi:10.1515/cclm-2024-1428

Article

Targeted proteomics of serum IGF-I, -II, IGFBP-2, -3, -4, -5, -6 and ALS

Jakob Albrethsen , Lylia Drici , Lea Marie Slot Vilmann , Stine A. Holmboe , Charlotte Ehlers Thomsen , Veronica Lykke Rogaczewska Groendahl , Maud Eline Ottenheijm , Annelaura Bach Nielsen , Christina Christoffersen , Lise Aksglaede , Casper P. Hagen , Nicolai J. Wewer Albrechtsen and Anders Juul

Published/Copyright: March 24, 2025

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 63 Issue 8

Abstract

Objectives

The insulin-like growth factors (IGFs) regulate growth in humans. IGF-I and IGF binding protein (IGFBP)-3 are biomarkers in children with growth disorders. We investigate a targeted proteomics method for absolute quantitation of eight IGF protein family members in human serum, including the peptide hormones IGF-I and -II, and the six binding proteins IGFBP-2, -3, -4, -5, -6 and acid labile subunit (ALS).

Methods

Serum preparation was optimized for targeted proteomics of IGF related proteins on a clinical LC-MS/MS platform (UHPLC coupled with Triple-Q MS). We created quality controls, standards and internal standards and 289 serum samples from healthy children and adolescents were measured in ten batches over two months. The method was compared to WHO reference standards, clinical and research immunoassays, and relative proteomics profiling.

Results

The sensitivity and reproducibility were sufficient for most but not all IGF protein family members. Targeted proteomics correlated well with clinical immunoassays for IGF-I (R²=0.88) and for IGFBP-3 (R²=0.46), (p<0.001). The correlation between targeted proteomics and non-clinical immunoassays for IGF-II, IGFBP-2, -4, -5, -6 and ALS varied between proteins.

Conclusions

We present a method for parallel quantification of IGF-I, IGFBP-3, 5 and ALS for clinical verification studies, whereas targeted proteomics of the five remaining IGF related proteins (IGF-II, IGFBP-2, -4, and -6) require further examination. The sensitivity of our new IGF-I method suggests a possible diagnostic role for targeted proteomics of IGF-I in the management of children with extremely low levels of circulating IGF-I.

Keywords: insulin-like growth factor (IGF); LC-MS/MS; proteomics; serum

Corresponding author: Jakob Albrethsen, Senior Scientist, PhD, Department of Growth and Reproduction, Copenhagen University Hospital – Rigshospitalet, Section 5064, Blegdamsvej 9, 2100 Copenhagen, Denmark; and International Centre for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet and University of Copenhagen, Copenhagen, Denmark, E-mail: jakob.christian.albrethsen@regionh.dk

Funding source: TømmerhandlerVilhelm Bangs Foundation

Award Identifier / Grant number: 12298

Funding source: Danmarks Frie Forskningsfond

Award Identifier / Grant number: 1052-00003B

Funding source: Kirsten and the Freddy Johansen Foundation

Award Identifier / Grant number: n.a.

Funding source: Miljøstyrelsen

Award Identifier / Grant number: MST-611-00012

Funding source: Novo Nordisk Fonden

Award Identifier / Grant number: NNF14CC0001

Award Identifier / Grant number: NNF17OC0027594

Award Identifier / Grant number: NNF19OC0055001

Award Identifier / Grant number: NNF24OC0088402

Acknowledgments

We thank all participants in the cohort studies, which provided serum samples and the staff involved in the projects including the recruitment procedures, physical examinations, collection, and analysis of samples. We thank Christine Rasmussen (Department of Clinical Biochemistry, Copenhagen University Hospital, Bispebjerg) for her time planning, preparing and performing the proteomic analysis and we also acknowledge the Clinical Proteomic Group at the NNF Center for Protein Research, University of Copenhagen, in particular Matthias Mann.

Research ethics: The Copenhagen Puberty Study III was approved by the Regional Committee on Health Research Ethics (H-19087825) and the Danish Data Protection Agency (RH-2018-24/I-Suite nr.: 6179).
Informed consent: All parents gave written informed consent prior to participation in the study.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission. Jakob Albrethsen: project idea, planning, writing and targeted proteomics analysis. Lylia Drici, Maud Ottenheijm, Annelaura Bach Nielsen: Proteomics profiling, planning and writing. Lea Marie Slot Vilmann, Stine A. Holmboe, Charlotte Ehlers Thomsen, Veronica Lykke Rogaczewska Groendahl: Sample collection, planning and writing. Christina Christoffersen, Lise Aksglaede, Casper P. Hagen, Nicolai J. Wewer Albrechtsen, Anders Juul: project idea, planning and writing.
Use of Large Language Models, AI and Machine Learning Tools: None declared.
Conflict of interest: The authors state no conflict of interest.
Research funding: This project was financially supported by: the Centre on Endocrine Disruptors, the Danish Environmental Protection Agency (MST-611-00012), Kirsten and the Freddy Johansen Foundation. Tømmerhandler Vilhelm Bangs Foundation (J.nr. 12298). Nicolai J. Wewer Albrechtsen is supported by a NNF Excellence Emerging Investigator Grant – Endocrinology and Metabolism (Application No. NNF19OC0055001), EFSD Future Leader Award (NNF21SA0072746), NNF Ascending Investigator Grant (NNF24OC0088402) and DFF Sapere Aude (1052-00003B). NNF Center for Protein Research is supported financially by the NNF (grant agreements NNF14CC0001 and NNF17OC0027594).
Data availability: Not applicable.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2024-1428).

Received: 2024-12-08

Accepted: 2025-03-13

Published Online: 2025-03-24

Published in Print: 2025-07-28

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https://doi.org/10.1515/cclm-2024-1428

Keywords for this article

insulin-like growth factor (IGF); LC-MS/MS; proteomics; serum