Nanoacquity uplc

Peptides were separated with a Waters NanoAcquity UPLC and emitted into a Thermo Q-Exactive HF tandem mass spectrometer. Pulled tip columns were created from 75 μm inner diameter fused silica capillary in-house using a laser pulling device and packed with 3 μm ReproSil-Pur C18 beads (Dr. Maisch) to 300 mm. Trap columns were created from 150 μm inner diameter fused silica capillary fritted with Kasil on one end and packed with the same C18 beads to 25 mm. Solvent A was 0.1% formic acid in water, while solvent B was 0.1% formic acid in 98% acetonitrile. For each injection, 3 μl (approximately 1 μg) was loaded and eluted using a 90-minute gradient from 5 to 35% B, followed by a 40 min washing gradient. Data were acquired using either data-dependent acquisition (DDA) or data-independent acquisition (DIA). Three DDA and DIA HeLa and yeast technical replicates were acquired by alternating between acquisition modes to minimize bias. Serum-starved HeLa acquisition was randomized within blocks to enable downstream statistical analysis.

Labeled urine peptide samples were dissolved in 0.1% FA and separated with a Waters nanoAcquity UPLC before entering a Thermo Q-Exactive Orbitrap mass spectrometer (San Jose, CA). Each sample was injected twice. Mobile phase A consisted of water with 0.1% FA, and mobile phase B was composed of ACN with 0.1% FA. Samples were loaded onto a fabricated column with an integrated emitter. The 75 μm ID column was filled to a length of 15 cm using Ethylene Bridged Hybrid C₁₈ packing material (1.7 μm, 130 Å, Waters). Peptides were trapped onto the column in 100% A and separated using a solvent gradient of 0–10% B over 0.5 min and then 10–30% B over 70 min at a flow rate of 350 nL/min. Data-dependent acquisition (DDA) parameters recorded MS scans in profile mode from m/z 380–1500 at a resolution of 35K. Automatic gain control (AGC) targets of 1 x 10⁶ and maximum injection times (IT) of 100 ms were selected. The 15 most intense precursor ions were selected for MS² higher-energy collisional dissociation (HCD) fragmentation with an isolation width of 2.0 m/z and placed on an exclusion list for 40 s. Tandem mass spectra were acquired at a resolution of 17.5K in profile mode with an AGC target of 1 x 10⁵, a maximum IT of 150 ms, a normalized collision energy (NCE) of 27, and a fixed lower mass at m/z 110.

Samples were analyzed with an LC-MS/MS system comprised of a Waters NanoAcquity UPLC and a TSQ Vantage Triple Quadripole (Thermo Scientific). The liquid chromatography separation was performed on a Waters Acquity UPLC HSS C18 column (1.0×50 mm, 1.8 μm) with mobile phase A (0.1% triethylammonium acetate in water) and mobile phase B (acetonitrile). The flow rate was 50 μL min^-1. The autosampler temperature was set at 10 °C. The injection volume was 5 μL. Mass spectra were acquired with negative electrospray ionization at the ion spray voltage of -3,000 V. The capillary temperature was 270°C.
For BAP, the LC gradient was as follows: 0–1 min, 0% B; 1–7 min, 0–100% B; 7–9 min, 00% B; 9–10.1 min, 100–0% B; 10.1–12 min, 0% B. Selective reaction monitoring followed the transition from parent ion to a metaphosphate fragment (179 to 62.9 m/z) using a collision energy of 38 eV.
For fosmidomycin, the LC gradient was as follows: 0–2 min, 0% B; 2–7 min, 0–20% B; 7–7.1 min, 20–100% B; 7.1–9 min, 100% B; 9–10.1 min, 100–0% B; 10.1–12 min, 0% B. Selective reaction monitoring followed the transition from parent ion to a metaphosphate fragment (182 to 79.0 m/z) using a collision energy of 41 eV.

Whole liver tissue was homogenized, or mt fractions were isolated as previously described (Zhang et al., 2008 (link)). Both were processed, trypsin digested, and LC-MS/MS analysis performed with a Waters nanoAcquity UPLC and a Thermo Scientific LTQ Orbitrap Velos, as previously described (Hsieh et al., 2012 (link)). The raw data from MS/MS and extended supplementary files are available at https://chorusproject.org/pages/blog.html#/351.
The topograph software program (http://proteome.gs.washington.edu/software/topograph/)(http://proteome.gs.washington.edu/software/topograph/) was developed for the measurement of peptide isotopologue abundances from LC-MS/MS chromatograms and the calculation of peptide turnover rates (Hsieh et al., 2012 (link)). It allows the measurement of the proportion of the amino acid precursor pool that is labeled, which varied over time and condition (Fig. S12A). This information allows the correct calculation of percent of new synthesis for each peptide, which when plotted for 12 biological replicates over time (four time points) generated an exponential curve following first-order kinetics (Fig. S2A). Using a logarithmic transformation, the first-order protein turnover rate (slope) was determined by linear regression (Fig. S2B). Only peptides that uniquely mapped to a single protein were used for our measurements (see supplemental methods for details).

Tissues were homogenized in cold isolation buffer (250 mm sucrose, 1 mm EGTA, 10 mm HEPES, 10 mm Tris–HCl pH 7.4). These lysates were centrifuged at 800 × g for 10 min to get rid of the debris. Whole liver and heart tissues were homogenized and trypsin‐digested, and LC‐MS/MS analysis was performed with a Waters nanoAcquity UPLC and a Thermo Scientific LTQ Orbitrap Velos, as previously described (Hsieh et al., 2012).