Plrp s

The sarcomeric protein mixture extracted from VL muscle tissue was separated by reverse phase chromatography (RPC) using a home-packed reversed-phased column (PLRP-S, 200 mm length × 500 μm id, 10 μm particle size, 1,000 Å pore size, Agilent). The RPC separation was performed in a 60 min gradient with mobile phase B increases from 5% to 95% (Mobile phase A: 0.1% formic acid (FA) in water, Mobile phase B: 0.1% FA in 1:1 ethanol:acetonitrile) in a nanoACQUITY UPLC system (Waters, Milford, MA, USA). The nanoACQUITY UPLC system was coupled to an impact II quadrupole-time-of-flight (q-TOF) mass spectrometer (Bruker, Bremen, Germany) for online LC/MS analysis. Mass spectra were collected at 1 Hz over a 500–3000 m/z range.

Reverse phase chromatography was performed with a nanoACQUITY UPLC system (Waters; Milford, MA, USA). Mobile phase A (MPA) contained 0.2 % formic acid in nanopure water, and mobile phase B (MPB) contained 0.2% formic acid in 50:50 acetonitrile and isopropanol. Prior to injection, protein samples were desalted by washing through 10 kDa MWCO filters using water six times. For each injection, 5 μL of desalted protein sample (1 μg/μL) was loaded on a home-packed 200 mm × 250 μm PLRP-S (5 μm, 1000Å; Agilent Technology, Santa Clara, CA, USA) capillary column. The column was placed in a column heater set at 50 °C with a constant 6 μL/min flow rate. The RPC gradient consisted of the following concentrations of MPB: 10% MPB at 0 min, 20% at 5 min, 65% at 65 min, 90% at 70 min, held at 90% until 75 min, adjusted back to 10% at 75.1 min, and held at 10% until 80 min. Each run was 80 min long.

LC-MS/MS analysis was carried out using a Waters ACQUITY UPLC M-class system (Milford, MA, USA) coupled to a maXis II ETD Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Myofilament protein extracts from rat SOL and GAS skeletal muscle tissues were diluted using 0.1% formic acid, 2 mM TCEP in water. 5 μL of the diluted protein extracts (500 ng) were loaded on a home-packed PLRP column (PLRP-S, 250 mm long, 0.25 mm i.d., 10 μm particle size, 1000 Å pore size, Agilent). Myofilament proteins were eluted by a linear 50 minute 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 flow rate of 8 μL/min. End plate offset and capillary voltage were set at 500 and 4,500 V, respectively.
Data-dependent LC-MS/MS was performed on the rat SOL and GAS skeletal muscle myofilament 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 in 500–3000 m/z. The isolation window for online auto MS/MS CAD was 5–8 m/z. The collision DC bias was set from 18 to 45 eV for CAD with nitrogen as collision gas.

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.