The peptide hormone insulin-like growth factor 1 (IGF1) is involved in a wide spectrum of biological processes.1 It is present in nanomolar amounts in blood. IGF-binding proteins modulate IGF1. Because the hormone is a marker for multiple disorders including osteoporosis, diabetes, obesity, neuromuscular disorders, growth hormone resistance and insulin resistance, accurate measurement of IGF1 levels is important. However, measuring  IGF-1 is complicated by its very low abundance, the presence of IGF1 binding proteins and the limited dynamic range of existing assays.

Neiderkofler et al.2 report on the development and validation of a mass spectrometry-based method for measuring IGF-1 from human serum and plasma. This study used recombinant human IGF1 variant, LR3-IGF1, as an internal standard added to the samples at the beginning of the process. LR3-IGF1 differs from IGF1 as it has one amino acid difference and an extra 13 amino acids at the N-terminus. Key steps are summarized below.

Step 1. Dissociating IGF1 from the IGF binding proteins using sodium dodecyl sulphate

Most methods dissociate IGF1 from binding proteins using acid conditions, followed by ethanol precipitation. This method, adapted from one that was previously published, used an optimized concentration of sodium dodecyl sulphate. The concentration (0.3%) dissolved the binding complex, but did not interfere with the antibody binding in the next step. The team performed dissociation reactions in a micro plate.

Step 2: Immunoaffinity retrieval of IGF1 and LR3-IGF1

These researchers included an immunoaffinity capture step acquire low-abundance IGF1 targets without interfering proteins. They used mass spectrometric immunoassay tips derivatized with polyclonal rabbit anti-human IGF1 to extract the IGF1 and LR3-IGF1 from the micro plate samples. After repeated wash cycles, the proteins were eluted into another micro plate, dried down and resuspended in appropriate buffer. Using an automated liquid handler, the team was able to perform this step in less than 45 minutes for all 96 samples.

Step 3: Trypsin digestion

Although trypsin digestion is not necessary, the researchers included this step because the triple quadrupole mass spectrometer is more sensitive at a lower peptide range.

Step 4: Quantitation using LC-MS/MS

The investigators injected all 96 samples onto a C18 column with an auto-sampler. They then shot the column using a TSQ vantage triple quadrupole mass spectrometer in selected reaction monitoring mode. They quantified targeted peptides using Pinpoint software and measured IGF1 in 289 human serum samples.

The scientists checked the assay’s performance to ensure linearity as well as intra- and inter-assay precision. The limit of detection (1ng/mL) was lower than both a commercial IGF1 assay and another recently published LC MS/MS IGF1 assay.

They conclude that this new high-throughput assay is sensitive, accurate, automated—making it suitable for clinical laboratory use. The team is now working to optimize the assay for high levels of IGF1 and to calibrate the assay against the latest IGF1 international standard. They will also optimize the assay by removing the trypsin digestion step so long as they can remove it without compromising assay sensitivity.

  1. Le Roith, D. Insulin-Like Growth Factors. N Engl J Med 1997; 336:633-640 February 27, 1997 DOI:10.1056/NEJM199702273360907
  2. Niederkofler EE, Phillips DA, Krastins B, et al. Targeted selected reaction monitoring mass spectrometric immunoassay for insulin-like growth factor 1. PLoS One. 2013 Nov 21;8(11):e81125. doi: 10.1371/journal.pone.0081125. eCollection 2013. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836743/pdf/pone.0081125.pdf