No abstract

No abstract

No abstract

Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis.

Capabilities of mass spectrometry for the analysis of intact proteins can be increased through separation methods. Flow field-flow fractionation (FlFFF) is characterized by the particularly "soft" separation mechanism, which is ideally suited to maintain the native structure of intact proteins. This work describes the original on-line coupling between hollow-fiber FlFFF (HF FlFFF), the microcolumn variant of FlFFF, and electrospray ionization/time-of-flight mass spectrometry (ESI/TOFMS) for the analysis and characterization of intact proteins. The results show that the native (or pseudonative) structure of horse heart myoglobin and horseradish peroxidase is maintained. Sample desalting is also observed for horse heart myoglobin. Correlation between the molar mass values independently measured by HF FlFFF retention and ESI/TOFMS allows us to confirm the protein aggregation features of bovine serum albumin and to indicate possible changes in the quaternary structure of human hemoglobin.