proteomics (mitochondria)

1. Proteins extracted from mitochondria are reduced using a final concentration of 5 mM of TCEP (tris(2-carboxyethyl)phosphine) at room temperature for 10 min.
2. The proteins are further alkylated using a final concentration of 20 mM of CAA (chloro-acetamide) at room temperature for 15 min.
3. A 25 ul aliquot of the lysate is acidified using 2.5 ul of 12% phosphoric acid. Using a pH test paper to make sure solution pH< 2.
4. Combine 165 ul of the S-trap buffer (90% MeOH with 100 mM triethylammonium bicarbonate) with the acidified lysate.
5. Transfer the combined colloid into the S-trap micro (Protifi, NY).
6. Centrifuge the S-trap micro at 4000 g for 1 min at room temperature.
7. Using 150 ul of the S-trap buffer to wash the S-trap and centrifuge at 4000 g for 1 min. Repeat the step two more times.
8. Add 20 ul of 100 mM triethylammonium bicarbonate containing 20 ug of Trypsin into S-trap micro. Allow the incubation stay overnight (14-16 hr) at 37 C.
9. After overnight incubation, directly adding 40 ul of the 50 mM triethylammonium bicarbonate and centrifuge at 4000 g for 1 min.
10. Add 40 ul of 2% formic acid in water to the S-trap, and centrifuge at 4000 g for 1 min.
11. Add 40 ul of 50% acetonitrile in water to the S-trap, and centrifuge at 4000 g for 1 min.
12. Speedvac the sample.
13. The sample is reconstituted in 20 ul of 2% acetonitrile and 0.2% formic acid in water and 500 ug of the peptide is used for LC-MS/MS analysis.

  An aliquot of sample containing 500 ug of the

peptides is subjected to the LC-MS/MS analysis. The sample is separated on an Aurora UHPLC Column (25 cm × 75 μm, 1.6 μm C18, AUR2-25075C18A, Ion Opticks) using an Easy-nLC 1200 liquid chromatography system. The gradient settings follows Table 1 .| A | B | C | D | | --- | --- | --- | --- | | Time | Duration | Flow (nl/min) | %B | | 0:00 | 0:00 | 350 | 3 | | 1:00 | 1:00 | 350 | 3 | | 73:00 | 72:00 | 350 | 19 | | 101:00 | 28:00 | 350 | 29 | | 121:00 | 20:00 | 350 | 41 | | 124:00 | 3 | 350 | 95 | | 131:00 | 7 | 350 | 98 |

Table 1. LC gradient for the sample Mobile Phase A: 0.2% formic acid, 2% acetonitrile, and 97.8%

water.

Mobile Phase B: 0.2% formic acid, 80% acetonitrile, and

19.8% water.

1. The sample is analyzed on a Thermo Q-Exactive HF mass spectrometer using a data-dependent acquisition method. Detailed parameters of the scans are listed in Table 2 . | A | B | | --- | --- | | Global settings | | | Ion source type | NSI | | Spray voltage | 2000 V | | Ion transfer tube temperature | 300 C | | Polarity | Positive | | MS1 scan settings | | | Resolution | 60000 | | AGC target | 3e6 | | Maximum IT | 15 ms | | Scan range | 375-1500 m/z | | MS2 scan settings | | | Resolution | 30000 | | AGC target | 1e5 | | Maximum IT | 45 ms | | Loop count | 12 | | Isolation window | 1.2 m/z | | NCE | 28 | | Spectrum data type | Centroid | | Fixed first mass | 100 m/z |

Table 2. MS settings for the run.

  The raw data generated by mass spectrometer is analyzed using Proteome Discoverer 2.5. The data is searched using the mouse proteome achieved from UniprotKB on 10/26/2020 (swissprot + trembl). The processing and consensus workflow are set according to  **Figure 1** and  **2** . The parameters are listed below in  **Table 3** . All the parameters that are not mentioned are left defaulted.<img src="https://protocols-files.s3.amazonaws.com/files/ragrbuvrx.png?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAJYFAX46LHRVQMGOA%2F20240822%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20240822T013556Z&X-Amz-Expires=604800&X-Amz-SignedHeaders=host&X-Amz-Signature=f0581a5ee8c44c37cd9b6849957b56ad24dd886aea4702bbf92030ee829f310b" alt="Figure1. Processing workflow of the PD analysis." loading="lazy" title="Figure1. Processing workflow of the PD analysis."/>

A	B
SequestHT settings
Enzyme name	Trypsin (Full)
Max. missed cleavage	2
Min. peptide length	6
Max. peptide length	144
Precursor mass tolerance	10 ppm
Fragment mass tolerance	0.02 Da
Max. equal modification	3
Dynamic modification	Oxidation/ +15.995 Da (M)
Dynamic modification (protein terminus)	Acetyl/ + 42.011 Da (N-Terminal)
Dynamic modification (protein terminus)	Met-loss/ - 131.040 Da (M)
Dynamic modification (protein terminus)	Met-loss+Acetyl/ - 89.030 Da (M)
Static modification	Carbamidomethyl/ + 57.021 Da (C)
Percolator
Target/Decoy selection	Concatenated
Validation based on	q-Value
Target FDR (Strict)	0.01
Target FDR (Relaxed)	0.05
Feature Mapper settings
Maximum RT shift (min)	2
Mass tolerance	5 ppm

Table 3. Parameters for PD searching

1. The protein list exported from PD results are further analyzed using R scripts based on the TidyProteomics package (jeffsocal.github.io/tidyproteomics/articles/overview.html).