AI-64310-CRFT

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Automated High-Throughput Data
Processing for Targeted Multiplexed Insulin
Analog Detection and Quantification
Scott Peterman1, Kwasi Antwi2, Bryan Krastins1, Eric E. Niederkofler2, and Mary Lopez1
1Thermo Fisher Scientific BRIMS, Cambridge, MA
2Thermo Fisher Scientific, Tempe, AZ
Key Words
Q Exactive, insulin, insulin variants, Pinpoint, HRAM, high resolution,
accurate mass, MSIA, mass spectrometric immunoassay,
automation, research
Goal
Present an automated, multiplexed, high-throughput data processing
workflow for detection and targeted quantification of insulin and its analogs
at concentration ranges of 3.75 to 960 pM in biological matrices.
Introduction
The development of robust biotechnology-based methods
to create recombinant peptide hormones with altered
peptide sequences has produced hormone variants
designed to fit specific needs such as insulin analogs. The
development of these variants requires accompanying
advances in the analytical technologies used for their
detection and quantification in research applications.
Routine, global sample preparation, data acquisition,
and data processing methods that address expected
concentration levels in biological matrices are needed. 1
Traditional global sample preparation and detection
methods tend to decrease assay selectivity and sensitivity.
Rapid, targeted quantitation of many closely related
analytes places significant demands on the software
tools used.
To address the analytical requirements of routine
detection and quantification of peptide variants in
biological matrices, a complete workflow that employs
multiplexed
as created.2
MSIA technology couples global affinity capture sample
preparation with high-resolution, accurate-mass (HRAM)
spectrometric detection.3 The
was used to collect full-scan HRAM data. 4
provided
automated qualitative and quantitative HRAM data
processing. The complementary research paper published
in Proteomics provides a detailed description of the
MSIA-HRAM method and results for targeted
quantification of intact insulin and its analogs in human
serum and plasma.5
Experimental
Reagents
Insulin analogs Humulin® S (Lilly, 100 IU/mL), Apidra®
(Sanofi Aventis, 100 U/mL), Lantus® (Sanofi Aventis,
100 U/mL), NovoRapid® (Novo Nordisk, 100 U/mL), and
Hypurin Porcine (CP Pharmaceuticals, 100 U/mL) were
provided by Dr. Stephen Morley (Sheffield Hospital, UK).
Bovine serum albumin (BSA, Calbiochem) prepared at
50 g/L in phosphate-buffered saline (10 mM phosphate,
150 mM NaCl, pH 7.4) served as the biological matrix.
Bovine insulin, TWEEN® 20, and phosphate-buffered
saline were obtained from Sigma-Aldrich®. Ultra-pure
water, trifluoroacetic acid, and ammonium acetate were
obtained from American Bioanalytical. ACTH 1-24 was
obtained as a carrier peptide from Bachem®. LC-MS grade
water, LC-MS grade acetonitrile, and formic acid were
Fisher Chemical brand
were
coupled with anti-human insulin antibody.
Application Note 619