Characterization of Clinically-Relevant Stable Isotope Labeled Recombinant Proteins For Use As Internal Standards in Quantitative MS Workflows

By: Kevin Ray, Pegah Jalili, James Walters, David Rhee, Sigma-Aldrich, St. Louis, MO

Objective

Characterization of heavy (SIL) proteins for use in quantitative MS workflows

Introduction

Methods for quantification of clinical protein biomarkers by mass spectrometry demand a high level of analytical quality. Procedures must be developed to minimize and control experimental variations in all steps of the workflow including protein extraction, fractionation, enrichment, proteolysis and analysis. To this end, we have characterized stable isotope labeled (SIL) full length proteins for utility as internal standards in quantititative MS workflows. These SIL proteins can be spiked at the initial stage of the analytical workflow to significantly minimize variations. Clinically-relevant SIL proteins expressed in E. coli, such as IGF1, and in mammalian HEK293 cells, such as Thyroglobulin and APOA1, have been characterized in this study.

SIL Incorporation of ApoA1 and Thyroglobulin

Incorporation of 13C6 15N2 labeled lysine in DYVSQFEGSALGK liberated from SIL-APOA1

Figure 1. Incorporation of 13C6 15N2 labeled lysine in DYVSQFEGSALGK liberated from SIL-APOA1 produced by Sigma R&D and another commercial source.

 

Incorporation of 13C6 15N4 labeled arginine in two surrogate peptides

Figure 2. Incorporation of 13C6 15N4 labeled arginine in two surrogate peptides liberated from SIL-Thyroglobulin.

15N-Incorporation Analysis of SIL-IGF1

Experimentally measured and predicted isotope distributions of GFYFNKPTGYGSSSR of SIL-IGF1

Figure 3. Experimentally measured and predicted isotope distributions of GFYFNKPTGYGSSSR of SIL-IGF1. The % incorporation of 15N atoms was calculated to be 99.0% using the best correlation between experimentally measured isotope distribution and predicted isotope distributions spanning 95- 100% theoretical incorporation.

Purity Analysis of SIL-IGF1

RP-LC-UV analysis of intact SIL-IGF1 at 214 nm wavelength

Figure 4. RP-LC-UV analysis of intact SIL-IGF1 at 214 nm wavelength. A significant amount of disulfide-swapped isomer, which elutes earlier in RP-LC, was observed in a commercially sourced SIL-IGF1.

Intact Mass Analysis of SIL-ApoA1

Intact spectrum of SIL-APOA1 reveals a mixture of the proprotein (+RHFWQQ) and fully mature protein

Figure 5. Intact spectrum of SIL-APOA1 reveals a mixture of the proprotein (+RHFWQQ) and fully mature protein.

LC-HRMS of Intact IGF1 in Human Serum

SIL-IGF1 spiked into human serum fortified with 5 g/mL light IGF1

Figure 6. SIL-IGF1 was spiked into human serum fortified with 5 g/mL light IGF1. Proteins were extracted using acetone precipitation and analyzed using LC-HRMS. The most abundant isotopes of the most abundant charge state were used to create extracted ion chromatograms (not shown) for quantitation.

SIL-APOA1 Digestion in Human Sera

sil-apoa1 digestion if human sera

Figure 7. SIL-APOA1 was spiked into human serum, denatured with urea, digested using FASP protocol, and sampled over a 24-hour digestion timecourse.

Summary

We have characterized stable isotope labeled (SIL) full length versions of IGF1, Thyroglobulin, and APOA1 for utility as internal standards in quantititative MS workflows. These clinically-relevant SIL proteins can be spiked at the initial stage of the analytical workflow to significantly minimize process variations and therefore represent a more suitable choice for use as an internal standard in quantitative MS-based protein assays.

Materials

     
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