Opti-nQL: An Optimized, Versatile and Sensitive Nano-LC Method for MS-Based Lipidomics Analysis

Abstract

Lipidomics is the comprehensive analysis of lipids in a given biological system. This investigation is often limited by the low amount and high complexity of biological samples, therefore highly sensitive lipidomics methods are required. Nanoflow-LC/MS offers extremely high sensitivity; however, it is challenging as a more demanding maintenance is often needed compared to conventional microflow-LC approaches. Here, we developed a sensitive and reproducible lipidomics LC method, termed Opti-nQL, which can be applied to any biological system. Opti-nQL has been validated with cellular lipid extracts of human and mouse origin and with different lipid extraction methods. Among the resulting 4000 detected features, 700 and even more unique lipid molecular species have been identified covering 16 lipid sub-classes, while 400 lipids were uniquely structure defined by MS/MS. These results were obtained by analyzing an amount of lipids extract equivalent to 40 ng of proteins, being highly suitable for low abundant samples. MS analysis showed that theOpti-nQL method increases the number of identified lipids, which is evidenced by injecting 20 times less material than in microflow based chromatography, being more reproducible and accurate thus enhancing robustness of lipidomics analysis.

Acetyl-CoA carboxylase 1 controls a lipid droplet–peroxisome axis and is a vulnerability of endocrine-resistant ER + breast cancer

“…To address the potential metabolic differences related to lipid metabolism in endocrine therapy resistance, we subjected to lipidomic analysis an isogenic model in which parental ER + MCF7 cells were compared with LTED derivatives. Cell lysis was performed on three independent replicates, and total lipids were extracted and subjected to mass spectrometry analysis [nano-liquid chromatography–electrospray ionization–tandem mass spectrometry (nLC-MS/MS)] ( 21 ). Lipidomic analysis identified 2109 lipid species (data file S2).…”

Clonal cooperation through soluble metabolite exchange facilitates metastatic outgrowth by modulating Allee effect

“…The cell pellet was washed twice with 1× DPBS before being resuspended in 200 μl of cold MilliQ H2O. Lipids were extracted and processed using a single-step extraction protocol with methanol and chloroform as described previously ( 63 ). In brief, cells were lysed by passing them repeatedly through a 26-gauge needle.…”

FADS1/2-mediated lipid metabolic reprogramming drives ferroptosis sensitivity in triple-negative breast cancer

“…Lipids were extracted using a single-step extraction protocol with methanol and chloroform as described previously (Cattaneo et al, 2021). Briefly, cell pellets (equivalent to 1-3 × 10 3 cells) were resuspended in 200 µL of MilliQ water and mechanically disrupted by passing Sciex).…”

“…To explore the potential lipidic alterations induced by ferroptosis execution in sensitive and resistant cells, we subjected 67NR (resistant) and 4T1 (sensitive) cells, either untreated or exposed to a short-acute RSL3 treatment, to lipid extraction and subsequent lipidomic analysis (Cattaneo et al, 2021). Partial Least Square Discriminant Analysis (PLS-DA) of 1209 unambiguously identified lipids showed a clear separation between 4T1 cells and 4T1 exposed to RSL3 (Figure 4a and Supplementary Table 1).…”