Characterization of Stable Isotope Labeled APO-A1 for Use as an Internal Standard in a Quantitative MS Workflow

By: Kevin Ray, Pegah Jalili, Gordon Nicol, and James J. Walters, Sigma-Aldrich, 2909 Laclede Ave, St. Louis, MO, 63103

Objective

Characterize heavy SIL-APOA1 (SILu™ Prot APOA1 Apolipoprotein A-I) for use in quantitative MS.

Overview

The ideal internal standard for quantitative LC/MS assays would be a stable-isotope-labeled full-length protein that is equivalent to the native target protein which can be introduced at the beginning of the workflow. We have extensively characterized heavy APO-A1 expressed in HEK293 cells to show that it is sufficiently similar to native APO-A1 using peptide mapping, intact mass, and protein-antibody binding affinity assays on a Biacore. We have also studied the digestion time course to demonstrate that SIL-APO-A1 peptides are liberated at the same rate as those from the endogenous protein using optimized digestion conditions.

Results

Purity and Sequence Construct

Purity and Sequence Construct

Figure 1. Sequence of SIL-APOA1, indicating propeptide (blue), V5 (purple), and 8X HIS (green) affinity tags. SDS-PAGE of Sigma R&D batch and a commercially sourced material.

 

SIL Incorporation

SIL Incorporation

Figure 2. Incorporation of stable isotopes for peptide DYVSQFEGSALGK of SIL-APOA1 from a Sigma R&D batch and a commercially sourced material.

 

LC-MS Analysis of Intact SIL-APOA1

LC-MS Analysis of Intact SIL-APOA1

Figure 3. RP-LC-MS analysis of intact SIL-APOA1 using BIOshell 400Å C4 column. The deconvoluted mass spectrum reveals a mixture of the proprotein (+RHFWQQ) and fully mature protein.

 

Binding Kinetics of SIL-APOA1 vs Native APOA1

Binding Kinetics of SIL-APOA1 vs Native APOA1

Figure 4. Binding curves for SIL-APOA1 and APOA1 isolated from human serum with a goat polyclonal antibody.

 

SIL-APOA1 Digestion in Human Sera

SIL-APOA1 Digestion in Human Sera

Figure 5. SIL-APOA1 protein was spiked into human serum pools and sampled throughout a 24-hour digestion timecourse. Serum pool A was denatured with TFE and digested with trypsin using a standard in-solution protocol. Serum pool B was denatured with urea and digested using a FASP protocol.

 

Summary

  • We have characterized SIL-APOA1 evaluating purity, incorporation of the heavy isotope, intact mass, and binding kinetics to a capture antibody.
  • The SIL-APOA1 demonstrates sufficient similarity to native APOA1 to be useful as an internal standard for quantitative MS.
  • The use of harsher denaturation conditions was found to eliminate slight differences in peptide liberation rates observed at early digestion times.