Elevated plasma albumin and apolipoprotein A-I oxidation under suboptimal specimen storage conditions.

Chad R Borges,Boriana Nikolova,Hussein Yassine,Sally Jensen,Christian Breburda,Douglas S Rehder,Matthew R Schaab,Nisha D Sherma

doi:10.1074/mcp.m114.038455

Abstract

S-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC-electrospray ionization-MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at -80 °C, -20 °C, and room temperature and exposed to multiple freeze-thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at -20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze-thaw cycles had a minimal effect on protein oxidation. In matched collections, protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface-area-to-volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress-including acute myocardial infarction and prostate cancer-demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above -30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions.

Highlights

Human apolipoprotein A-I1 contains three methionine residues (Met86, Met112, and Met148) that can be oxidized to sulfoxides [2,3,4]
We found that a similar approach could be used to analyze apolipoprotein A-I (apoA-I) simultaneously
In previous work [36] we found that the addition of 1 mM MetSer dipeptide can delay for hours the methionine oxidation of other proteins that have been pre-isolated from serum and are present at low concentrations in a acidic solution

Summary

Introduction

Improper biospecimen handling and storage can contribute to sample measurements that do not accurately reflect biological reality in vivo [13,14,15,16] This may introduce bias in analytical results, limiting the capacity for meaningful compari-. In blood P/S-based biomarker development work, verification of sample integrity is sometimes overlooked or considered only as an afterthought Contributing to this phenomenon is that fact that there are no universally accepted, globally applicable endogenous reference markers of P/S integrity. The indication of a loss of specimen integrity lies in an apparent loss of the target protein beyond the normal human reference range Such loss is often ascribed to “degradation” and in many cases likely happens because of residual proteolytic activity that occurs at temperatures above the sample freezing point. Loss of the protein marker may be due to misfolding caused by repeated freeze–thaw cycles

Results

Discussion

Conclusion