= Emerging. More than 5 years before clinical availability. (29.54%)
= Expected to be clinically available in 1 to 4 years. (38.82%)
= Clinically available now. (31.65%)
MSACL 2020 US : Motorykin

MSACL 2020 US Abstract

Topic: Proteomics

Podium Presentation in Room 5 on Wednesday at 14:20 (Chair: Andrew Hoofnagle)

Innovation and Discovery in Operations: Isotopic Peak Index, Relative Retention Time and Tandem MS for Detection of 15 IGF-1 Variants in Clinical Analysis

Ievgen Motorykin (Presenter)
Quest Diagnostics

Presenter Bio(s): 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 South California. Favorite hobby: hiking and camping.

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

Abstract

INTRODUCTION

In the clinical laboratory, identification and quantification of intact proteins by high-resolution, accurate-mass (HRAM) mass spectrometry is a highly specific and robust technique. However, protein variants with mass-to-charge ratios (m/z) differing from that of the wild type (WT) are not reported.

In the case of insulin-like growth factor-1 (IGF-1), all intact variants produce wide isotopic envelopes of up to 14 detectable peaks, most of which are not unique for any variants at the level of the instrument’s resolution. Knowing the width of isotopic envelopes and their overlap can help identify different variants; failure to account for width can results in false positives.

OBJECTIVES

In this study, we further develop a system to differentiate IGF-1 variants by mass spectrometry (MS), using Isotopic Peak Index (IPi), relative retention time (rRT) and tandem MS.

METHODS

Previously, we developed a novel naming convention for isotopic peaks, called the IPi, wherein peaks within any isotopic envelope are designated by a subscript. The monoisotopic peak is designated as IP0, and heavier isotopic peaks are identified as IP1 through IP13. To make the detection more efficient, we selected as few m/z as possible to detect all the variants. This resulted in 4 variant groups (VGs), each monitored at a single m/z. The presence of a peak at a VG m/z indicates the presence of a variant from that group.

For variants within VGs, we have 3 ways to distinguish between them. The first is using IPi concept, which can distinguish variants within each VG, as long as they have different IPis. The second way is using the relative retention time (rRT), which is defined as the difference between the RT of a variant and that of the WT. This value can help distinguish variants from the same VG that have the same IPi, such as A38V and A67V; R55K and R36Q. The third way is tandem MS, which can distinguish between the most abundant pair of variants, A67T and A70T, owing to specific y ions generated during their fragmentation. We recently developed such a method.

RESULTS

Of the 307,269 samples we analyzed, 1,266 (0.4%) variants were identified. The following variants (and their count) were identified: R50W/T4M/A67T/A70T (1,210), A67V (23), A38V (9), P66A (6), R36Q/R50Q (5), V17M/V44M (5), A67S (4), R55K/R56K (3), T29I/T41I (1), S34N (1), S33P (1), Y31H (1).

For 2 samples, IPi and rRT did not match WT or known variants; follow-up by DNA sequencing identified new variants: S33P and Y31H. In addition, IPi and rRT results matched known variants, but follow-up DNA sequencing identified 3 new point mutations: R50Q, R56K, and T41I. We tested 15 samples in the R50W/T4M/A67T/A70T group using tandem MS, and were able to distinguish 9 A67T variants and 6 A70T variants. The MS/MS technique will be applied to this patient set.

CONCLUSIONS

HRAM, IPi, rRT, and tandem MS can be used together to identify and differentiate IGF-1 variants in routine clinical analysis.


Financial Disclosure

DescriptionY/NSource
Grantsno
SalaryyesQuest Diagnostics
Board Memberno
Stockyes Quest Diagnostics
ExpensesyesQuest Diagnostics
IP Royaltyno

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

yes