Insights into the formation of ATPsynthase: A new important function of the folding helper Hsp70 NatureComms
Differentially SILAC-labelled samples were combined in a 1:1 ratio. 50 µg mitochondria were used for mitochondrial protein analysis and 100% of the elution fraction from affinity purification via mtHsp70were used for protein analysis. Proteins were precipitated by addition of acetone at a sample to acetone ratio of 1:4 [v/v] followed by incubation at −20 °C overnight. Subsequently, proteins were collected by centrifugation and washed twice with ice-cold acetone.
Analytical columns were produced in-house by the generation of spray tips from fused silica capillaries with a P-2000 laser puller . Spray tips were packed with 3 μm ReproSil-Pur AQ Cparticles . Peptides were separated with 120 min and 240 min linear gradients from 5-35% [v/v] solvent B at a flow rate of 300 nl/min. For DDA analyses, MS1 spectra were acquired from m/z 350-1,200 in the Orbitrap mass analyser .
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