Integration of Ion Mobility MS^E after Fully Automated, Online, High-Resolution Liquid Extraction Surface Analysis Micro-Liquid Chromatography

Direct analysis by mass spectrometry (imaging) has become increasingly deployed in preclinical and clinical research due to its rapid and accurate readouts. However, when it comes to biomarker discovery or histopathological diagnostics, more sensitive and in-depth profiling from localized areas is required. We developed a comprehensive, fully automated online platform for high-resolution liquid extraction surface analysis (HR-LESA) followed by micro–liquid chromatography (LC) separation and a data-independent acquisition strategy for untargeted and low abundant analyte identification directly from tissue sections. Applied to tissue sections of rat pituitary, the platform demonstrated improved spatial resolution, allowing sample areas as small as 400 μm to be studied, a major advantage over conventional LESA. The platform integrates an online buffer exchange and washing step for removal of salts and other endogenous contamination that originates from local tissue extraction. Our carry over–free platform showed high reproducibility, with an interextraction variability below 30%. Another strength of the platform is the additional selectivity provided by a postsampling gas-phase ion mobility separation. This allowed distinguishing coeluted isobaric compounds without requiring additional separation time. Furthermore, we identified untargeted and low-abundance analytes, including neuropeptides deriving from the pro-opiomelanocortin precursor protein and localized a specific area of the pituitary gland (i.e., adenohypophysis) known to secrete neuropeptides and other small metabolites related to development, growth, and metabolism. This platform can thus be applied for the in-depth study of small samples of complex tissues with histologic features of ∼400 μm or more, including potential neuropeptide markers involved in many diseases such as neurodegenerative diseases, obesity, bulimia, and anorexia nervosa

Overlapping binding of TAX1BP1 zinc finger domains with ubiquitin and myosin VI.

<p>(<b>A</b>) Domain organisation of TAX1BP1 and designated point mutations. LIR, LC3-interaction region; ZF, zinc-finger; SKICH, SKIP carboxyl homology. (<b>B</b>) Ribbon presentation of the structures of the TAX1BP1 zinc fingers together with two molecules of ubiquitin. The residues are colored according to the extent of the chemical shift changes observed where blue represents small or no chemical shift changes observed, and red represents the maximum chemical shift changes observed for the backbone amides. The side chains of the residues in TAX1BP1 that when mutated abolish ubiquitin binding are displayed and labelled. (<b>C</b>) RPE cells were transfected with GFP empty vector, with GFP-TAX1BP1 wild-type, GFP-TAX1BP1 ZF1 mutant (Q743A/E747K), GFP-TAX1BP1 ZF2 mutant (Q770A/E774K) or the Q743A/E747K/Q770A/E774K double zinc finger mutant of GFP-TAX1BP1 followed by GFP immunoprecipitation and pull-down with K63-linked polyubiquitin (2–7). Western blot analysis was performed and immunoblotting against the indicated proteins. (<b>D</b>) Mammalian 2-hybrid assay in CHOK.1 cells with myosin VI tail as bait and TAX1BP1 full-length wild-type and mutants as prey. Results are represented as the normalised luciferase activity against a bait only control.</p

The effect of charge location in ion mobility mass spectrometry for small molecule analytes

<p>Presented at the British Mass Spectrometry Society meeting in 2014 and awarded the Bordoli Prize</p> <p> </p> <p>In travelling wave IM-MS an electric field is applied across the IMS cell and analyte collisions with the inert gas give rise to a relationship between the drift time and the molecular shape and overall charge.</p> <p>Recent findings show near baseline IMS separation (R>1) where shape and overall charge appear not to explain the IMS separation. Multiple ion mobility peaks are observed for a single m/z including the fluoroquinolone antibiotics norfloxacin and lomefloxacin, the pesticide indoxacarb and a number of steroids.</p> <p> </p> <p>The Bordoli Award was judged by Prof. Graham Cooks (Henry Bohn Hass Distinguished Professor of Chemistry at Purdue University, ISI Highly Cited Chemist with over 950 publications and an H-index of 81) and Prof. Mike Morris (‎Senior Director MS Research at Waters).</p> <p> </p> <p> </p

TAX1BP1 interacts with LC3B as well as LC3C and requires its zinc finger domains to localise to ubiquitylated Salmonella.

<p>(<b>A</b>) Mammalian 2-hybrid assay in CHOK.1 cells with LC3A, B, or C and GABARAP, GABARAPL1 or L2 as bait and TAX1BP1 full-length wild-type as prey. Results are represented as the normalised luciferase activity against a bait only control. <b>(B)</b> Mammalian 2-hybrid assay in CHOK.1 cells using LC3B or LC3C as bait and full-length TAX1BP1, NDP52, optineurin wild-type and LIR mutants as prey. Results are represented as the normalised luciferase activity against a bait only control. (C) Mammalian 2-hybrid assay in CHOK.1 cells with LC3B as bait and TAX1BP1 full-length wild-type and mutants as prey. Results are represented as the normalised luciferase activity against a bait only control. (<b>D</b>) HeLa cells transfected with GFP-TAX1BP1 wild-type or V144S LIR mutant, Q743A/E747K/Q770A/E774K double zinc finger mutant, and V144S/Q743A/E747K/Q770A/E774K LIR and double zinc finger mutant were infected with mCherry expressing Salmonella for 1 hour prior to saponin extraction and fixation. Cells were immunostained for GFP and ubiquitin and processed for confocal microscopy. Nuclei are labelled with Hoechst (blue). Scale bar, 20 μm.</p

Suppression of myosin VI expression leads to a hyper-proliferation of Salmonella and an accumulation of ubiquitylated Salmonella within LC3-positive autophagosomes.

<p>(<b>A</b>) HeLa cells following myosin VI siRNA or TAX1BP1, NDP52, OPTN (TNO) siRNA transfection were subjected to Salmonella infection for the indicated time points. Gentamicin protection assays were performed and colonies were counted at each time point. Results are represented as the fold replication from 2 hours, which represents the amount of bacterial proliferation. Results are from >3 independent experiments and error bars represent s.d. Western blot analysis was performed on whole cell lysates and immunoblotting was performed to the indicated proteins. (<b>B</b>) HeLa cells mock, myosin VI siRNA, or TNO siRNA treated were infected with mCherry expressing Salmonella for 8 hours, followed by processing for confocal microscopy. Cells were immunostained for ubiquitin (green). Nuclei were labelled with Hoechst (blue). The % of infected cells with ubiquitin (+) Salmonella was quantified. The results depict >3 independent experiments and the error bars represent the s.d. (<b>C</b>) HeLa cells transfected with myosin VI or TNO siRNA were infected with BFP-expressing Salmonella for 8 hours. Cells were processed for confocal microscopy and immunostained for LC3 (green) and ubiquitin (red). Scale bar, 20 μm. Quantitation at 4 and 8 hours post-infection, with BFP-expressing Salmonella, of the % of ubiquitin (+) Salmonella which are LC3 (+). Results represent 3 independent experiments and the error bars indicate the s.d.</p