Nanoacquity ultra high pressure lc system

Top-down LC-MS/MS was carried out by using a NanoAcquity ultra-high pressure LC system (Waters) coupled to a high-resolution maXis II quadrupole time-of-flight mass spectrometer (Bruker Daltonics). 5 μL (400 ng) of total protein was injected onto a home-packed PLRP column (PLRP-S) (Agilent Technologies), 10-μm particle size, 500-μm inner diameter, 1,000 Å pore size) using an organic gradient of 5 to 95% mobile phase B (mobile phase A: 0.2% formic acid in water; mobile phase B: 0.2% formic acid in 50:50 acetonitrile:isoproponal) at a flow rate of 12 μL/min and temperature of 35 °C. Column pressure was maintained between 700–1200 psi. Mass spectra were taken at a scan rate of 0.5 Hz over 530–2000 m/z range. A total of three replicate runs were collected for each concentration between 250–1200 ng to establish instrument sensitivity and reproducibility. Samples were randomized during processing and LC-MS/MS analysis to correct for batch effects.³⁸ Data-dependent LC-MS/MS was performed on sarcomeric protein extracts. The three most intense ions in each mass spectrum were selected and fragmented by collision-activated dissociation (CAD) with a scan rate of 2 Hz from 200–3000 m/z. The isolation window for online AutoMS/MS CAD was 10 m/z. The collision DC bias was set from 18 to 35 eV for CAD with nitrogen as the collision gas.

LC-MS analysis was accomplished using a NanoAcquity ultra-high pressure LC system (Waters, Milford, MA, USA) coupled to a high-resolution Impact II quadrupole time-of-flight (Q-TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany). 400 ng of total sarcomeric protein was separated by reverse-phase liquid chromatography (RPLC) on a home-packed PLRP column (PLRP-S, 1000 Å pore size, 10μm particle size, 500 μm inner diameter) using an organic gradient of 5% to 95% mobile phase B (mobile phase A: 0.1% formic acid (FA) in water; mobile phase B: 0.1% FA in 50:50 acetonitrile:ethanol) at a constant flow rate of 6 μL/min to acquire intact protein spectra. Data analysis was performed using Bruker DataAnalysis (v4.3) and Mash Explorer [15 (link)]. Statistical analysis was performed using “rstatix” in R (v4.1.0). Ions used for quantification are available in Table S2, and statistical analyses are available in Table S3 and S4.

LC-MS analysis was carried out using a NanoAcquity Ultra-high Pressure LC system (Waters, Milford, MA, USA) coupled to a high-resolution Impact II quadrupole time-of- flight (Q-TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany). To evaluate the reproducibility of protein extraction and the LC-MS method, three extraction replicates at the same time point (time 0) and three injection replicates of the same protein extract were tested. The sarcomeric proteins in each sample were eluted by a gradient of 5% to 95% mobile phase B (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1 % formic acid in 50:50 acetonitrile:ethanol) at a constant flow rate of 8 μL/min. Proteins eluted were delivered to the mass spectrometer via electrospray ionization. End plate offset and capillary voltage were 450 V and 4000 V, respectively. Nebulizer pressure was set to 0.5 Bar and dry gas flow rate was 4.0 L/min. In-source collisional energy was set to 10 V. Mass spectra were collected at a scan rate of 0.5 Hz over 500–2000 m/z range.