Data-Independent Acquisition (DIA) has emerged as a powerful mass spectrometry (MS) strategy for comprehensive metabolomics. This study presents a novel short gradient (13 min) nanosensitive analytical method for human plasma analysis using DIA LC-MS/MS, focusing on in-depth optimization of MS parameters to maximize data quality and metabolite coverage. Key MS parameters, including scan speed, isolation window width, resolution, automatic gain control, and collision energy, were systematically tuned to balance the sensitivity and specificity while minimizing interferences. The optimized method enabled the detection of 2,907 features with 675 annotated compounds, leveraging recent progress in nano-LC-MS/MS for multiomics applications and showcasing the possibility of combining proteomics and metabolomics within a single chromatographic system. Ultimately, a comparison was performed between the data acquired through the DIA and DDA MS approaches in the context of untargeted metabolomics. This optimized analytical method yields more robust and reproducible results, thereby expanding the potential for meaningful discoveries across diverse biological fields.

Current applications of mass-spectrometry-based proteomics range from single-cell to body fluid analysis, each presenting very different demands regarding sensitivity or sample throughput. Additionally, the vast molecular complexity of proteomes and the massive dynamic range of protein concentrations in these biological systems require highly performant chromatographic separations in tandem with the high speed and sensitivity afforded by modern mass spectrometers. In this study, we focused on the chromatographic aspect and, more specifically, systematically evaluated proteome analysis performance across a wide range of chromatographic flow rates (0.3–50 μL/min) and associated column diameters using a Vanquish Neo HPLC coupled online to a Q Exactive HF-X mass spectrometer. Serial dilutions of HeLa cell line digests were used for benchmarking, and the total analysis time from injection to injection was intentionally fixed at 60 min (24 samples per day). The three key messages of the study are that (i) all chromatographic flow rates are suitable for high-quality proteome analysis, (ii) capLC (1.5 μL/min) is a very robust, sensitive, and quantitative alternative to nLC for many applications, and (iii) showcased proteome, phosphoproteome, and drug proteome data provide sound empirical guidance for laboratories in selecting appropriate chromatographic flow rates and column diameters for their specific applications.

Dopamine encoding of reward prediction errors naturally fluctuates over females’ reproductive cycles with estrogenic signaling due to reduced expression of dopamine reuptake proteins.

The Cell Dynamics and Fragmentation lab at We study how single cells move and fragment in complex environments using microfluidics. We bring tools and concep...

Solute carrier (SLC) genes encode the largest membrane transporter superfamily, with many orphan members of unknown function. In recent Cell Metabolism and Molecular Cell articles, Khan et al. and Dias et al. identify SLC25A45 as essential for mitochondrial import of methylated amino acids and subsequent carnitine synthesis.

Sharp wave-ripples from the hippocampus are shown to modulate peripheral glucose homeostasis in rats, offering insights into the mechanism that links sleep disruption and blood glucose regulation in t...

The selective analysis of isoprene-containing natural products, such as triterpenoid saponins and prenylated flavonoids, remains a major challenge in …

Cells generate purine nucleotides through de novo purine biosynthesis (DNPB) and purine salvage. Purine salvage represses DNPB to prevent excessive purine nucleotide synthesis through mechanisms that ...

Second part of Thinking like a Scientist, reflecting on how genome editing and multi-omics, while powerful, can introduce conceptual flaws that distort our understanding of gene function and biologica...

Covalent inhibitors are powerful entities in drug discovery. Now the amino acid selectivity and reactivity of a diverse electrophile library have been assessed proteome-wide using an unbiased workflow...

Lange et al. identify a lipid droplet quality control pathway in which FSP1 safeguards stored neutral lipids from lipid peroxidation, thereby preventing the induction of ferroptosis.

Phosphoinositides (PIPs), the phosphorylated derivatives of phosphatidylinositol (PI), are low-abundance yet critical components of eukaryotic membranes. They play pivotal roles in a wide array of cel...