= Emerging. More than 5 years before clinical availability. (19.79%)
= Expected to be clinically available in 1 to 4 years. (37.97%)
= Clinically available now. (42.25%)
MSACL 2022 : Motorykin

MSACL 2022 Abstract

Self-Classified Topic Area(s): Proteomics > none > none

Podium Presentation in De Anza 2 on Thursday at 17:10 (Chair: Timothy Collier)

Detection of an Unusual IGF-2 Species Using High-resolution Mass Spectrometry During a Routine IGF-1 Clinical Assay

Ievgen Motorykin, Michael J. McPhaul, Nigel J. Clarke, Zengru Wu
Quest Diagnostics Nichols Institute, San Juan Capistrano, CA

Ievgen Motorykin (Presenter)
Quest Diagnostics

Presenter Bio: Born in Ukraine. Obtained an MS degree there and came to the US for the graduate school. Graduated from Oregon State University with a Ph.D. in Chemistry. There I met my (then future) wife, with whom I moved around the country looking for a place to settle: Pittsburgh, Ithaca and finally Southern California. Favorite hobby: hiking and camping.


Previously, we described a mass-spectrometric method for monitoring Insulin-like growth factor-1 (IGF-1) variants by using only 4 mass-to-charge ratios (m/z) comprising variant groups (VG). For each variant, the method used a concept we defined as “the isotopic peak index” (IPi), as well as relative retention time (rRT). VG, IPi, and rRT are unique values that help identify IGF-1 variants and detect novel ones. In addition, by monitoring a signature y-ion using MS/MS, we could distinguish isobaric A67T and A70T IGF-1 variants. More recently, we have used y5 ions to verify the following IGF-1 variants (corresponding amino acid sequence): P66A (AAKSA), A67V (PVKSA), and A67S (PSKSA) in specimens suspected to contain those variants (as assessed by their VG, IPi, and rRT). In several patients, a potentially recurring variant was detected ([email protected], rRT [average] = -0.19 mins) that did not match previously identified IGF-1 variants. Herein we describe how this variant was identified.

MS Analysis and Results
In the first specimen, manually analyzing m/z differences in y-ions of the MS/MS of the unknown variant, a partial sequence at the C-terminus of was identified as PAK matching the WT IGF-1 sequence, but with a shift to 214 Da higher mass indicating a change at the C-terminus (eg, amino acid substitution or modification). However, the total mass of this variant was 22 Daltons lighter than the WT IGF-1. A subsequent specimen provided a better signal for b- and y-ions for sequence information.

In the next specimen, partial sequences were deduced using b- and y-ions derived from amino acids near the C- and N-termini. The N-terminus sequence (ETL) was identified, but b-ions were 420 Da heavier that they should be if derived from the IGF-1 sequence. The C-terminal sequence identified (ATPAK) was inconsistent with IGF-1 (LKPAK) and, as in the earlier specimen, the y-ions were 214 Da heavier than predicted. A BLAST search across the human proteome using C-terminal sequence indicated a sequence consistent with human IGF-2, a 67-amino acid polypeptide. The N-terminus of IGF-2 starts with the sequence AYRPSETL, which includes the (ETL) fragment, but at much heavier m/z due to the presence of the remaining amino acids (the IGF-1 sequence is just GPETL). However, the C-terminus of the IGF-2 variant still had an additional 156 Da to be accounted for (now measured against IGF-2 sequence).

This difference in mass corresponds to a C-terminal arginine (R) extension to the mature IGF-2 sequence (1-67). An R68 immediately follows C-terminus of the mature IGF-2 polypeptide sequence and is the first amino acid of the E-domain of the prohormone. If this possibility is considered, all inconsistencies are resolved: all b- and y-ions match the correct m/z ratios, and the total m/z of the protein accurately matches the theoretical value within 0.6 ppm. The conclusion: this is a case of the IGF-2 protein with an R extension at the C-terminus (ATPAKSER).

In the IGF-2 prohormone, the E-domain (68-156), which starts with R, is cleaved off when the protein matures, leaving only the WT IGF-2 sequence. It seems that in the present case the R was retained with the IGF-2 sequence. To our knowledge, IGF-2 (1-68) has never been previously observed. It may have some relationship to so-called “big IGF-2”, which has been described as an IGF-2 protein in which incorrect cleavage results in extra amino acids from the E-domain of the pro IGF-2 sequence. Both big IGF-2 polypeptides described in the literature, 1-87 and 1-104, are biomarkers of non-islet cell tumor hypoglycemia (NICTH).

Our workflow for routine monitoring of IGF-1 variants by high-resolution mass spectrometry resulted in the detection of an unusual IGF-2 species (1-68) indicative of unexpected prohormone processing with unknown clinical significance and unknown frequency of occurrence.

Financial Disclosure

SalaryyesQuest Diagnostics
Board Memberno
Stockyes Quest Diagnostics
IP Royaltyno

Planning to mention or discuss specific products or technology of the company(ies) listed above: