Nuclear factor (erythroid-derived 2)-like 2 and its Caenorhabditis elegans ortholog, SKN-1, are transcription factors that have a pivotal role in the oxidative stress response, cellular homeostasis, and organismal lifespan. Similar to other defense systems, the NRF2-mediated stress response is compromised in aging and neurodegenerative diseases. Here, we report that the FDA approved drug hydralazine is a bona fide activator of the NRF2/SKN-1 signaling pathway. We demonstrate that hydralazine extends healthy lifespan (~25%) in wild type and tauopathy model C. elegans at least as effectively as other anti-aging compounds, such as curcumin and metformin. We show that hydralazine-mediated lifespan extension is SKN-1 dependent, with a mechanism most likely mimicking calorie restriction. Using both in vitro and in vivo models, we go on to demonstrate that hydralazine has neuroprotective properties against endogenous and exogenous stressors. Our data suggest that hydralazine may be a viable candidate for the treatment of age-related disorders.

Quantitation in plasma-based proteomics necessitates the reproducible removal of highly abundant proteins to enable the less abundant proteins to be visible to the mass spectrometer. We have evaluated immunodepletion (proteoprep20) and enrichment (Bio-Rad beads), as the current predominant approaches. Label-free analysis offers an opportunity to estimate the effectiveness of this approach without incorporating chemical labels. Human plasma samples were used to quantitatively assess the reproducibility of these two methods using nano-LC-data-independent acquisition MS. We have selected 18 candidate proteins and a comparison of both methodologies showed that both of the methods were reproducible and fell below 20% residual SD. With the same candidate proteins, individual inter-day variability for the samples was also processed, allowing us to monitor instrument reproducibility. Overall, a total of 131 proteins were identified by both methods with 272 proteins identified by enrichment and 200 identified by immunodepletion. Reproducibility of measurements of the amount of protein in the processed sample for individual proteins is within analytically acceptable standards for both methodologies. This enables both methods to be used for biomarker studies. However, when sample is limited, enrichment is not suitable as larger volumes (>1.0 mL) are required. In experiments where sample is not limited then a greater number of proteins can be reliably identified using enrichment.

Discovery of protein biomarkers in clinical samples necessitates significant prefractionation prior to liquid chromatography–mass spectrometry (LC–MS) analysis. Integrating traveling wave ion mobility spectrometry (TWIMS) enables in-line gas phase separation which when coupled with nanoflow liquid chromatography and data independent acquisition tandem mass spectrometry, confers significant advantages to the discovery of protein biomarkers by improving separation and inherent sensitivity. Incorporation of TWIMS leads to a packet of concentrated ions which ultimately provides a significant improvement in sensitivity. As a consequence of ion packeting, when present at high concentrations, accurate quantitation of proteins can be affected due to detector saturation effects. Human plasma was analyzed in triplicate using liquid-chromatography data independent acquisition mass spectrometry (LC-DIA-MS) and using liquid-chromatography ion-mobility data independent acquisition mass spectrometry (LC-IM-DIA-MS). The inclusion of TWIMS was assessed for the effect on sample throughput, data integrity, confidence of protein and peptide identification, and dynamic range. The number of identified proteins is significantly increased by an average of 84% while both the precursor and product mass accuracies are maintained between the modalities. Sample dynamic range is also maintained while quantitation is achieved for all but the most abundant proteins by incorporating a novel data interpretation method that allows accurate quantitation to occur. This additional separation is all achieved within a workflow with no discernible deleterious effect on throughput. Consequently, TWIMS greatly enhances proteome coverage and can be reliably used for quantification when using an alternative product ion quantification strategy. Using TWIMS in biomarker discovery in human plasma is thus recommended.