PINK1-PARKIN pathway mitochondrial phosphoproteomics
This protocol is extracted from research article:
RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway
Sci Adv, Nov 21, 2018; DOI: 10.1126/sciadv.aav0443

To examine the phosphoproteome of mitochondria in response to mitochondrial depolarization, biological duplicate cultures of HFT-PARKINWT or S65A mutant cells, or HFT-PARKINWT;PINK1−/− cells, were depolarized for the indicated times, and mitochondria were isolated and digested as described (33). The flow-through from Kgg-enriched samples was acidified and subjected to Sep-Pak (Waters, C18) for desalting. Samples were resuspended in 80% ACN/0.1% TFA solution, and phosphopeptides were enriched using IMAC magnetic agarose beads (Ni-NTA, catalog no. 36111, Qiagen). Enriched peptides were subjected to Sep-Pak for desalting, resuspended in 100 μl of 0.1 M EPPS/20% ACN (pH 8.2), and labeled with TMT reagent for 1 hour at room temperature before quenching with 5 μl of 5% hydroxylamine. Labeled peptide was acidified to 0.1% TFA (pH ~2 to 3), and the Pierce High pH Reversed-Phase Peptide Fractionation Kit was used to collect 12 fractions and recombined for a total of 6 fractions before desalting and subsequent LC–tandem mass spectrometry (LC-MS/MS) processing.

MS data were collected on an Orbitrap Fusion Lumos mass spectrometer in line with a Proxeon NanoLC-1200 UHPLC. The 100-μm capillary column was packed with 35 cm of Accucore 150 resin (2.6 μm, 150 Å; Thermo Fisher Scientific). Samples were separated with a gradient consisting of 3 to 23% (ACN; 0.1% FA) over 150 min at ~550 nl/min. Each analysis used the MultiNotch SPS-MS3–based TMT method (26) to reduce ion interference compared to MS2 quantification. The scan sequence began with FTMS1 spectra (resolution of 120,000, mass range of 400 to 1400 m/z, AGC target of 5 × 105, and maximum injection time of 100 ms). Precursors for MS2 analysis were selected using a Top10 method. Precursors were filtered according to charge state ≥2 and ≤6. Monoisotopic peak assignment was used, and previously interrogated precursors were excluded using a dynamic window (90 s ± 7 ppm). Minimum intensity to trigger an MS2 scan was 2 × 104. MS2 precursors were isolated using the quadrupole (0.7-Th window) and analyzed in the Orbitrap (FTMS2) at 30,000 resolution at 200 Th, with an AGC target of 5 × 104 and a maximum injection time of 118 ms. Precursors were fragmented by CID at a collision energy of 35% and an activation q value of 0.25. Multistage activation was triggered for neutral loss mass of 97.9763. Following acquisition of each MS2 spectrum, an SPS-MS3 scan was collected on the top 10 most intense ions in the MS2 spectrum (26). MS3 precursors were fragmented by HCD and analyzed using the Orbitrap (NCE of 55, AGC target of 3 × 105, maximum injection time of 250 ms, and resolution of 50,000 at 200 Th).

Database searching included all entries from the Human Reference Proteome (May 2017) and an in-house list of contaminants. This database was concatenated with one composed of all protein sequences in the reversed order. Searches were performed using the Comet search engine (2014.02 rev. 2) using a 20-ppm precursor ion tolerance, and the product ion tolerance was set to 0.03 Da. Peptide’s N/C terminus was required to have Lys-C/trypsin specificity [1 1 KR], allowing up to three missed cleavages. TMT tags on lysine residues and peptide N termini (+229.163 Da) and the carbamidomethylation of cysteine residues (+57.021 Da) were set as static modifications, while the oxidation of methionine residues (+15.995 Da) was set as a variable modification. For phosphorylation analysis, deamidation (+0.984) on asparagine and glutamine and phosphorylation (+79.966) on serine and threonine were set as variable modifications. PSMs were adjusted to a 1% FDR, PSM filtering was performed using an LDA, and phosphorylation site localization was determined using the Ascore algorithm, as described previously (49). Ascore is a probability-based approach for high-throughput protein phosphorylation site localization. Specifically, a threshold of 13 corresponded to 95% confidence in site localization. For quantification, reporter ion counts were summed across all matching PSMs using an in-house software (49). Briefly, a 0.003-Th window around the theoretical m/z value of each reporter ion was scanned for ions, and the maximum intensity nearest the theoretical m/z was used. Reporter ion intensities were adjusted to correct for the isotopic impurities of the different TMT reagents according to the manufacturer’s specifications. MS3 spectra with TMT reporter summed signal-to-noise ratio of <150 or isolation purities of <50% were excluded from quantitation (26). The extracted signal-to-noise measurements of peptides were then normalized to the corresponding protein level previously measured from the total mitochondrial proteome (33). Individual ratios to HFT-PARKINWT untreated cells were calculated, and values were imported into Perseus (version 1.6.1.3) (50) for downstream statistical processing. T tests with Welch’s correction for unequal variances were performed with the following parameters (S0 = 1), and permutation-based FDR correction to 1% was applied to correct for multiple test correction. Results from the statistical analysis were then exported and plotted as volcano plots.

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