Nanoacquity

Pellet samples were excised as whole lanes from gels, reduced with TCEP, alkylated with iodoacetamide, and digested with trypsin. Tryptic digests were desalted by loading onto a MonoCap C18 Trap Column (GL Sciences), flushed for 5 min at 6 μL/min using 100% Buffer A (H₂0, 0.1% formic acid), then analyzed via LC (Waters NanoAcquity) gradient using Buffer A and Buffer B (acetonitrile, 0.1% formic acid) (initial 5% B; 75 min 30% B; 80 min 80% B; 90.5–105 min 5% B) on the Thermo Q Exactive HF mass spectrometer. Data were acquired in data-dependent mode. Analysis was performed with the following settings: MS1 60K resolution, AGC target 3e6, max inject time 50 ms; MS2 Top N = 20 15K resolution, AGC target 5e4, max inject time 50 ms, isolation window = 1.5 m/z, normalized collision energy 28%. LC-MS/MS data were searched with full tryptic specificity against the UniProt human database using MaxQuant 1.6.8.0. MS data were also searched for common protein N-terminal acetylation and methionine oxidation. Protein and peptide false discovery rate was set at 1%. LFQ intensity was calculated using the MaxLFQ algorithm (Cox et al., 2014 (link)). Fold enrichment was calculated as LFQ intensity from the ΔCLPB pellet divided by the LFQ intensity from the wild-type pellet. High confidence hits were quantified as minimum absolute fold change of 2 and p-value<0.05.

Samples were resuspended in 0.1% FA and transferred into a full recovery autosampler vial (Waters). Chromatographic separation was achieved on a UPLC system (nanoAcquity, Waters) with a two buffer system (buffer A: 0.1% FA in water, buffer B: 0.1% FA in ACN). Attached to the UPLC was a C18 trap column (Symmetry C18 Trap Column, 100 Å, 5 μm, 180 μm × 20 mm, Waters) for online desalting and sample purification followed by an C18 separation column (BEH130 C18 column, 75 μm × 25 cm, 130 Å pore size, 1.7 μm particle size, Waters). Peptides were separated using a 60 min gradient with increasing ACN concentration from 2 to 30% ACN. The eluting peptides were analyzed on a quadrupole orbitrap mass spectrometer (QExactive, Thermo Fisher Scientific) in data dependent acquisition mode.
For LC-MS/MS analysis on the QExactive, the 15 most intense ions per precursor scan (1 × 10⁶ ions, 70,000 Resolution, 100 ms fill time) were analyzed by MS/MS (HCD at 25 normalized collision energy, 2 × 10⁵ ions, 17,500 Resolution, 50 ms fill time) in a range of 400 to 1200 m/z. A dynamic precursor exclusion of 20 s was used.