Recovery of Insulin-like Growth Factor 1 (IGF-1) Signature Peptides Amol Bajaj (Presenter) ARUP Laboratories

Authors: Amol O Bajaj(1), Mark M Kushnir(1), Joely A Straseski(2)
(1)ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA (2)University of Utah, Department of Pathology, Salt Lake City, Utah, USA

Summary:

We identified an issue with inconsistent recovery of an insulin-like growth factor 1 (IGF-1) specific peptide when analyzed using Q-TOF LC/MS. We hypothesized that the peptide is unstable or binds to the surfaces in the flow-path of the instrument. We ruled out non-specific binding in the LC flow-path and the analytical column. While troubleshooting the issue, we identified the cause of inconsistent recovery was adsorption by the autosampler polypropylene vials/lo-bind 96-well plates in patient samples.

Abstract Details:

1. Problem

IGF-1 signature peptide (GPETLCGAELVDALQFVCGDR, T1) was observed in freshly prepared digests of IGF-1 standard, while undetectable in patient samples.

2. Method Information

Sample preparation consisted of addition of internal standard (15N-IGF-1) to serum samples. IGF-1 was separated from binding proteins by incubation with acetic acid, followed by protein precipitation. The supernatants were transferred to a new tube, evaporated, and reconstituted with ammonium bicarbonate; disulfide bonds were reduced using dithiothreitol, alkylated with iodoacetamide, and digested using trypsin. Extraction of the IGF-1 specific peptides was performed using Strata X-A SPE (Phenomenex, CA). The final extracts were transferred to autosampler polypropylene vials/lo-bind 96-well plates.

Instrumental analysis:

1290 HPLC system (Agilent Technologies, Santa Clara, CA;

Agilent Q-TOF LC/MS 6550;

Mobile phase A: 0.1% formic acid in water;

Mobile phase B: 0.1% formic acid in acetonitrile;

5.5 min LC gradient program, 500 μL/min flow rate;

Analytical Column: Poroshell 120 EC-C8, 2.7 µm, 2.1 x 50 mm;

Column oven temperature 50°C;

Injection volume 25 μL;

Data acquisition: high resolution TOF.

3. Troubleshooting Steps

Degradation of the peptide or adsorption in the LC flow-path was suspected. Data from troubleshooting experiments ruled out sample handling and preparation as the cause of the issue. While troubleshooting the instrumental method, we passivated the analytical column along with the LC flow-path with 0.1% BSA, while the peptide was still undetectable. The issue was resolved by passivation of the autosampler polypropylene vials/lo-bind 96-well plates with 0.1% BSA; after the passivation, the peptide became detectable and was consistently observed in the analysis, while the peak intensity decreased by ~50% over 5 hours and by ~90% over 24 hours.

4. Outcome

We identified the cause of poor performance with IGF-1-specific peptide, T1, as adsorption to autosampler polypropylene vials/lo-bind 96-well plates. The method performance for the T1 peptide improved when vial passivation was incorporated in the procedure.

Problem

Method Information

Troubleshooting Steps

Outcome