Fig 1.
The cyclic and aldehyde-containing linear forms of the reducing end of a polysaccharide (e.g. dextran) are in equilibrium. The linear form is able to undergo a Maillard reaction with protein amine groups during prolonged storage or upon the addition of heat forming a glycated protein (Schiff base or Amadori Product). Reduction of the polysaccharide aldehyde to an alcohol prevents the Maillard reaction during long term storage of proteins with the polysaccharide.
Fig 2.
High molecular weight smearing appears in vitrified samples.
Human serum after 16 weeks of storage after isothermal vitrification using dextran-based xeroprotectant matrix show high molecular weight smearing. Vitrified samples were either stored at room temperature (RT) or 37°C and reconstituted in PBS. Frozen samples were kept at -20°C. Gel electrophoresis was carried out using (Panel A) non-denaturing conditions, (Panel B) denaturing conditions, or (Panel C) denatured & reducing conditions. High molecular weight smears were present in all vitrified samples stored for 16 weeks and were more pronounced in samples stored at the higher temperature. Smearing did not disappear upon denaturation or reduction.
Fig 3.
High molecular weight smearing worsens over time and at elevated temperature storage, and stains positive for glycosylation.
Serum samples were vitrified in dextran-based xeroprotectant matrix and stored for 1 or 6 months at either room temperature (RT) or 37°C. Samples were run on SDS-PAGE under denaturing/reducing conditions and stained (Panel A) for total protein or (Panel B) for glycoprotein. Smearing worsened with higher temperature storage and with increased storage time. Glycoprotein stain indicates high-molecular weight smears are glycosylated; suggesting covalent attachment of dextran to proteins.
Fig 4.
Dextranol-based xeroprotectant matrix preserves proteins and prevents smearing.
Serum samples were either fresh, frozen, or vitrified in either a dextran-based or dextranol-based xeroprotectant matrix. Serum was analyzed (Panel A and C) immediately after vitrification (Day 1) or (Panel B and D) after storage for 140 days at 37°C. Vitrified samples were reconstituted in PBS. Gel electrophoresis was carried out under (Panel A and B) native conditions, and (Panel C and D) denaturing/reducing conditions. After two weeks, smearing was visible in samples preserved in dextran-based matrix, but not in dextranol-based matrix. Gel images obtained on intermediate time points can be found in S4 Fig.
Fig 5.
Dextranol-based xeroprotectant matrix protects human serum proteins and BSA when stored at 45°C.
Coomassie stained gel showing results of high temperature storage of (Panel A) BSA (Bovine serum albumin) and (Panel B) human serum. Immediately after vitrification, samples preserved in dextran and dextranol-based xeroprotectant matrices looked similar to frozen sample (left). After 7 days (mid), and 14 days (right) BSA stored in dextran-based matrix was mostly in a high-molecular weight smear. BSA stored in dextranol-based matrix still resembled the frozen sample. Note that it is common to see higher molecular bands in SDS-PAGE of BSA due to irreversible multimer formation [53, 54].
Table 1.
Biomarker preservation after 14 days storage of vitrified samples at 45°Ca.
Table 2.
High-molecular-weight adduct formation in lyophilized IgG.