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The impact of long-term cryopreservation on serum proteomic and trace metal integrity in biobanking

ABSTRACT: Background: Long-term cryopreservation compromises biobank sample integrity, yet systematic studies on its impact on serum proteomic and trace metal profiles remain scarce, hindering reliable biomarker research. Methods: We employed inductively coupled plasma mass spectrometry (ICP-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) with data-independent acquisition (DIA) to analyze serum samples from healthy donors after short-term (1 year) versus long-term (6 years) cryopreservation. Analyses included protein integrity assessment (SDS-PAGE), quantitative proteomics, trace metal quantification (10 elements), and functional enrichment (GO, KEGG). Results: Long-term cryopreservation induced substantial molecular alterations: SDS-PAGE revealed degradation of high-molecular-weight proteins (>100 kDa) and accumulation of low-molecular-weight fragments (<35 kDa). Proteomic analysis identified 242 significantly altered proteins (176 up-regulated, 66 down-regulated), enriched in immune response, metabolic pathways, and extracellular matrix organization. Paradoxically, long-term samples exhibited superior LC-MS/MS technical performance (lower CV: 22.9% vs 26.3%; fewer missing values: 15% vs 29%) attributed to artifacts arising from sample degradation. ICP-MS revealed significant concentration changes for V, Mn, Fe, Zn, Rb, and Cs. Conclusions: Our integrated analysis demonstrates that prolonged cryopreservation compromises serum molecular integrity through protein degradation/aggregation, biological pathway dysregulation, and trace metal redistribution. Crucially, apparent improvements in LC-MS/MS data quality are degradation artifacts, challenging conventional quality metrics.

ORGANISM(S): Homo Sapiens

SUBMITTER: Wenxu Hong

PROVIDER: PXD066741 | iProX | Wed Jul 30 00:00:00 BST 2025

REPOSITORIES: iProX

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Project description:Interest in exploiting algae as a biofuel source and the role of nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can be easily manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements on the ionomes of cells would result in optimized cell growth. We characterized the ionomes of multiple wild-type Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel our measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between laboratories using the same supplement. Visual observation did not reveal defects in cell motility or mating efficiency. Ni2+-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased amount of Ni2+ supplementation due to the introduction of an EDTA buffer system in the revised medium. Equilibrium modeling of the revised supplement predicts less metal precipitation in the revised medium. Other advantages include more facile preparation of trace element stock solutions that can readily be adapted for deficiency studies, a reduction in total chemical use, a more consistent batch-to-batch formulation, and long-term stability (up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient-deficiencies. In N and S deficiency, cells accumulate TAG as well in the new medium as previously demonstrated. Fe and Zn deficiency also induced TAG accumulation as suggested by Nile Red and Bodipy staining. This ionomic approach can be used to efficiently optimize culturing conditions for other algal species to improve growth and assay cell physiology. Sampling of Chlamydomonas CC-1021 (2137) cultivated in TAP medium supplemented with a revised trace element recipe based on ionomic data.

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The impact of long-term cryopreservation on serum proteomic and trace metal integrity in biobanking

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