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, MilliporeSigma, 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 quantitative 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

SIL Incorporation of ApoA1 and Thyroglobulin

Figure 1. Incorporation of 13C6 15N2 labeled lysine in DYVSQFEGSALGK liberated from SIL-APOA1 produced by Sigma R&D and another commercial source.  SIL-APOA1 was first reduced in a solution of Tris(2-carboxyethyl)phosphine hyrdochloride (Sigma-Aldrich, Prod. No. C4706), then alkylated with Iodoacetamide (Sigma-Aldrich, Prod. No. A3221), before digestion with trypsin (Sigma-Aldrich, Prod. No. T6567).  After enzymatic digestion, SIL-APOA1 was analyzed via RP LC-MS/MS using a Waters nanoAcquity UPLC, coupled to a Thermo Fischer Q Exactive Plus QTOF equipped with a Digital PicoView Spray Source (New Objective).  A New Objective PicoFrit Column HaloPeptide C18, 75 µm I.D. x 25 was utilized, with the following LC conditions: flow rate of 250 nL/min, mobile phase A of 0.1% formic acid (Prod. No. 56302) in water, mobile phase B of acetonitrile (Prod. No. 34967), and gradient of 3-35% mobile phase B in 60 minutes, 35-90% mobile phase B in 62 minutes (held for two minutes), and 90-3% mobile phase B in 65 minutes..

 

Incorporation  of 13C615N4 labeled arginine in two surrogate peptides liberated from SIL-Thyroglobulin

Figure 2. Incorporation of 13C6 15N4 labeled arginine in two surrogate peptides liberated from SIL-Thyroglobulin.  SIL-Thyroglobulin was first reduced in a solution of Tris(2-carboxyethyl)phosphine hyrdochloride (Sigma-Aldrich, Prod. No. C4706), then alkylated with Iodoacetamide (Sigma-Aldrich, Prod. No. A3221), before digestion with trypsin (Sigma-Aldrich, Prod. No. T6567).  After enzymatic digestion, SIL-Thyroglobulin was analyzed via RP LC-MS/MS using a Waters Acquity M Class, coupled to a Waters XEVO-G2S QTOF.  Two Supelco BIOshell A160 Peptide C18 15 cm x 300 µM x 2.7 µM columns (Cat# 67093-U) were run in tandem, with the following LC conditions: flow rate of 10 µL/min, mobile phase A of 0.1% formic acid (Prod. No. 56302) in water, mobile phase B of acetonitrile (Prod. No. 34967), and gradient of 3-45% mobile phase B in 60 minutes, 45-90% mobile phase B in 61 minutes (held for two minutes), and 90-3% mobile phase B in 64 minutes.

 

 

15N-Incorporation Analysis of SIL-IGF1

15N-Incorporation Analysis 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

Purity Analysis of SIL-IGF1

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-MS, was observed in a commercially sourced SIL-IGF1.  Intact mass analysis was performed by injection onto a LC-MS system consisting of a Waters Acquity UPLC coupled to a Waters QTOF Premier.  A Tosoh TSK Gel SW3000XL, 300 x 2.0 mm, 4 µm (Prod. No. 821485) was utilized, with an isocratic gradient (30% Acetonitrile, 0.1% TFA), and flow rate of 0.1 mL/min.

 

Intact Mass Analysis of SIL-ApoA1

Intact Mass Analysis of SIL-ApoA1

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

LC-HRMS of Intact IGF1 in Human Serum

Figure 6. SIL-IGF1 was spiked into human serum fortified with 5 g/mL light IGF1. Proteins were extracted using acetone precipitation. After protein precipitation, SIL-IGF1 was analyzed via RP LC-HRMS using a Waters Acquity HPLC, coupled to a Thermo Fischer Q Exactive Plus QTOF.  The LC column used for analysis was a BIOshell A400 Protein C4 (Prod. No. 67045-U).  LC conditions were as follows: flow rate of 70 µL/min, 0.1% TFA in water for mobile phase A, 0.1% TFA in acetonitrile for mobile phase B, with a gradient of 20% mobile phase B at start, 20-60% mobile phase B in 20 minutes, 60-80% mobile phase B in 20.5 minutes, 80% mobile phase B at 22.5 minutes, 80-20% mobile phase B in 23 minutes, and 20% mobile phase B at 30 minutes.  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 in 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 time course.  SIL-APOA1 was first reduced in a solution of Tris(2-carboxyethyl)phosphine hyrdochloride (Sigma-Aldrich, Prod. No. C4706), then alkylated with Iodoacetamide (Sigma-Aldrich, Prod. No. A3221), before digestion with trypsin (Sigma-Aldrich, Prod. No. T6567).  After enzymatic digestion, stopped at the various time points listed above, SIL-APOA1 was analyzed via Waters nanoACQUITY UPLC coupled to a 5500 Qtrap.  A Supelco Ascentis Express C-18, 15 cm x 200mm, 2.7 mm (Prod. No. 54261-U) column was utilized, along with a Supelco Ascentis Express Peptide ES-C18, 5 cm x 300mm, 2.7 mm (Prod. No. 53546-U) trap. LC conditions were as follows: flow rate of 4 µL/min, mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile.  Analysis began with a gradient of 99% mobile phase A at start, 40% mobile phase B at 30 minutes, 90% mobile phase B at 31 minutes, 90% mobile phase B at 35 minutes, and 1% mobile phase B at 36 minutes.

 

Summary

We have characterized stable isotope labeled (SIL) full length versions of IGF1, Thyroglobulin, and APOA1 for utility as internal standards in quantitative 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.