TITLE: Proteomics workflow for whole cell lysate, endosome, and lysosome fractions 
AUTHORS: Hankum Park, Frances V Hundley, J. Wade Harper

[DESCRIPTION]
We present a protocol for sample preparation for LC-MS analysis of whole cell lysates and for lysosomal and endosomal fractions purified by Lyso-IP and Endo-IP.  Protocols for purification of lysosomes and endosomes is provided in protocol dx.doi.org/10.17504/protocols.io.byi9puh6 using cells that express endogenously tagged TMEM192-HA and stably expressing FLAG-EEA1 as descrbed in dx.doi.org/10.17504/protocols.io.byi7puhn.

[STEPS]
SECTION: Whole cell global proteomics
1. Seed each cell line in one 15cm dish (293 or 293 cells stably expressing TMEM192-HA and FLAG-EEA1).  At ~80% confluency, collect cells by scraping in DPBS
SECTION: Whole cell global proteomics
2. At ~80% confluency, harvest cells on ice by scraping in 2-3 mL DPBS and pellet at 1,000xg for 2 min at 4 ºC.  Discard supernatant.
SECTION: Whole cell global proteomics
3. Wash once with DPBS, resuspend cell pellets with 8 M urea buffer supplemented with protease and phosphatase inhibitors and lyse by sonication.
SECTION: Whole cell global proteomics
4. Centrifuge lysates at  17,000xg for 8 min at 4 ºC, and collect the supernatant.
SECTION: Whole cell global proteomics
5. Determine total protein concentration using a BCA assay, and aliquot 50 μg of the proteins.
SECTION: Whole cell global proteomics
6. Add TCEP to 5 mM final and incubate at 25 ºC for 30 min.
SECTION: Whole cell global proteomics
7. Alkylate cysteines by addition of iodoacetamide to 15 mM final and incubate at 25 ºC for 30 min protected from light.
SECTION: Whole cell global proteomics
8. Add EPPS buffer to dilute urea to1 M final concentration.
SECTION: Whole cell global proteomics
9. Precipitate protein by addition of 6.1 N TCA solution to 20% final and incubate at 4 ºC for 1h.
SECTION: Whole cell global proteomics
10. Centrifuge samples at 20,000xg for 7 min at 4 ºC, and remove supernatants.  
SECTION: Whole cell global proteomics
11. Wash pellets twice with ice-cold acetone by centrifuging at 20,000xg for 10 min at 4 ºC.
SECTION: Whole cell global proteomics
12. Briefly dry pellets in a SpeedVac.
SECTION: Whole cell global proteomics
13. Resuspend pellets in 50 µL of 8 M urea buffer, sonicate in a water bath sonicator, and dilute urea by adding of 50 µL of 200 mM EPPS, pH 8.5.
SECTION: Whole cell global proteomics
14. Digest peptides with 1 µg of LysC and incubate at 30 ºC for 4h with shaking. Further dilute urea to 1.6 M final by addition of 200 mM EPPS.
SECTION: Whole cell global proteomics
15. Further digest peptides with 1 µg trypsin and incubate at 37 ºC overnight with shaking.
SECTION: Whole cell global proteomics
16. The next day, add acetonitrile (ACN) to 30% final, and label the peptides with 10 μL of the TMT reagents (10 μg/μL in anhydrous ACN) or TMTpro reagents (12.5 μg/μL in anhydrous ACN) for 1h at 25 ºC. Quench labeling with hydroxylamine at 0.5% final followed by incubation at room temperature for 15 min.
SECTION: Whole cell global proteomics
17. Pool TMT-labeled samples with 1:1 ratio and dry.
SECTION: Whole cell global proteomics
18. Desalt the sample with C18 solid-phase extraction using Sep-Pak, and dry the eluate.
SECTION: Whole cell global proteomics
19. Resuspend samples in 110 µL of 10 mM ammonium bicarbonate, 5% ACN solution and filter through a 0.2 µm PTFE centrifugal filter.
SECTION: Whole cell global proteomics
20. Pre-fractionat samples by high-pH reverse-phase HPLC (Agilent 1260 Infinity) with an Aeris C18 column (250 mm  4.6 mm) with a gradient of mobile phase A (10 mM ammonium bicarbonate, 5% ACN) and mobile phase B (10 mM ammonium bicarbonate, 90% ACN). Collect 96 fractions between 10 min (10% mobile phase B) and 72 min (100% mobile phase B) at a flow rate of 0.6 mL/min, and concatenate into 24 fractions. Dry fractions by SpeedVac and desalt by C18 StageTip. Resuspend alternating 12 fractions out of the 24 fractions in 3% ACN, 1% FA for LC-MS analysis.
SECTION: Whole cell global proteomics
21. Perform LC-MS analysis with an Orbitrap Eclipse coupled with a Proxeon EASY-nLC1200 liquid chromatography pump. Separate peptides on a microcapillary column (100 m inner diameter) packed with ~ 35 cm of Accucore150 resin (2.6 m, 150 Å, Thermo Fisher Scientific) with 8-23% (3-73 min), 23-30% (73-80 min), 30-100% (83-86 min) gradient of ACN (0.1% of FA) at 550 nL/min flow rate. For analysis, use multi-notch MS3-based TMT method coupled with Real Time Search algorithm (REF). The scan sequence starts with MS1 spectra analyzed by Orbitrap (resolution 120,000 at 200 Th, 400-1500 m/z, automatic gain control (AGC) target 2105, maximum injection time 50 ms). Monoisotopic peaks should be assigned, use precursor fit filter (70% for a fit window of 0.5 Th), and apply dynamic exclusion window (90 s, ± 10 ppm). Analyze MS2 spectra by quadrupole-ion trap with collision-induced dissociation (Rapid scan rate, AGC 1.0104, isolation window 0.5 Th, normalized collision energy (NBE) 34, maximum injection time 80 ms). Synchronous precursor selection (SPS) API-MS3 scan should collect top 10 most intense b- or y-ions matched with the real-time search algorithm (REF). Fragment MS3 precursors by high energy collision-induced dissociation (HCD) and analyze with the Orbitrap (NCE 45, AGC 2.5105, maximum injection time 200 ms, resolution 50,000 at 200 Th). Set closeout at two peptides per protein for each fraction.
SECTION: Whole cell global proteomics
22. Convert RAW files to mzXML files. Construct the searching database from Swiss-Prot human database, which is appended with common contaminants and reversed for target-decoy false discovery rate (FDR) estimation. Perform a database searching with a 20-ppm precursor ion tolerance and 1.0005 Da product ion tolerance. Include static modifications with carbamidomethylation at cysteine (+57.021 Da), TMT labeling at lysine (+229.162 Da for TMT or +304.207 Da for TMTpro) while including variable modifications with oxidation at methionine (+15.995 Da) and TMT labeling at peptide N-termini (+229.162 Da for TMT or +304.207 Da for TMTpro). Filter peptide-spectrum matches (PSM) using a linear discriminant analysis algorithm while considering XCorr, ΔCn, missed cleavages, peptide length, charge state, and precursor mass accuracy. Control the identified peptides at 1% false discovery rate (FDR). Do a protein assembly by parsimony principle, and apply 1% FDR to the protein level. For reporter ion quantification, extract signal-to-noise (S/N) ratio for each TMT channel with an integration tolerance of 0.003 Da. Quantify proteins by summing the reporter ion counts across all matching PSMs. Adjust S/N of each channel using the isotopic impurity table of TMT reagents provided by the vendor.
SECTION: Whole cell global proteomics
23. Export protein quantitation values to csv file, and analyze by R 3.6.3. Normalize protein abundances according to the total reporter values in each channel assuming equal amount of loading. Calculate Pearson’s correlation coefficient by the basic R function, cor(). For the classification by subcellular locations, use proteins annotated as “very high” or “high” from Itzhak et al (2016).
SECTION: Lysosomal and endosomal proteomics
24. Purify lysosomal and endosomal fractions as described (dx.doi.org/10.17504/protocols.io.byi9puh6 ).  
SECTION: Lysosomal and endosomal proteomics
25. Reduce samples with TCEP at 5 mM final and incubate at 25 ºC for 30 min. 
SECTION: Lysosomal and endosomal proteomics
26. Alkylate cysteines with iodoacetamide at 15 mM final and incubate at 25 ºC for 30 min and protect from light.
SECTION: Lysosomal and endosomal proteomics
27. Dilute samples with EPPS buffer for 1 M urea final concentration.
SECTION: Lysosomal and endosomal proteomics
28. Precipitate proteins by addition of 6.1 N TCA solution to 20% final and incubate at 4 ºC for 1h.
SECTION: Lysosomal and endosomal proteomics
29. Centrifuge samples at 20,000xg for 15 min at 4 ºC, and remove supernatants.
SECTION: Lysosomal and endosomal proteomics
30. Wash samples twice with ice-cold acetone by centrifuging at 20,000xg for 10 min at 4 ºC.
SECTION: Lysosomal and endosomal proteomics
31. After final wash, briefly dry pellets in a SpeedVac.
SECTION: Lysosomal and endosomal proteomics
32. Resuspend pellets in 5 µL of 8 M urea buffer, sonicate in a water bath sonicator, and dilute urea with 5 µL of 200 mM EPPS.
SECTION: Lysosomal and endosomal proteomics
33. Digest peptides with 0.2 µg of LysC and incubate at 30 ºC for 4h with shaking. Further dilute urea to 1.6 M final by addition of 200 mM EPPS.
SECTION: Lysosomal and endosomal proteomics
34. Further digest peptides with 0.2 µg trypsin and incubate at 37 ºC overnight with shaking.
SECTION: Lysosomal and endosomal proteomics
35. The next day, add ACN to 30% final, and label the peptides with 10 μL of the TMTpro reagents (12.5 μg/μL in anhydrous ACN) for 1h at 25 ºC.
SECTION: Lysosomal and endosomal proteomics
36. Quench labeling with hydroxylamine to 0.5% final and incubate at room temperature for 15 min.
SECTION: Lysosomal and endosomal proteomics
37. Pool sample, dry by SpeedVac, and pre-fractionate using Pierce High pH Reversed-Phase Peptide Fractionation Kit following the manufacturer’s instructions.
SECTION: Lysosomal and endosomal proteomics
38. Concatenate gradient eluates to four final fractions, then desalt with C18 StageTip.
SECTION: Lysosomal and endosomal proteomics
39. Perform LC-MS with an Orbitrap Eclipse tribrid mass spectrometer coupled with a Proxeon EASY-nLC1200 liquid chromatography pump. Separate peptides on a microcapillary column (100 m inner diameter) packed with ~ 35 cm of Accucore150 resin (2.6 μm, 150 Å, Thermo Fisher Scientific) with 5-30% (3-108 min), 30-99% (108-113 min) gradient of ACN (0.1% of FA) at 550 nL/min flow rate. For analysis, use FAIMS Pro Interface, and multi-notch MS3-based TMT method coupled with Real Time Search algorithm. The scan sequence starts with MS1 spectra analyzed by Orbitrap (resolution 120,000 at 200 Th, 400-1500 m/z, automatic gain control (AGC) target 4*105, maximum injection time 50 ms). Assign monoisotopic peaks, use precursor fit filter (70% for a fit window of 0.5 Th), and apply dynamic exclusion (120 s, ± 7 ppm). Select precursor ions using a cycle type of 1.25 sec/CV with FAIMS CV of -40/60/80. Analyze MS2 spectra by quadrupole-ion trap with collision-induced dissociation (Rapid scan rate, AGC 1.0*104, isolation window 0.5 Th, normalized collision energy (NBE) 34, maximum injection time 86 ms). Use synchronous precursor selection (SPS) API-MS3 scan to collect top 10 most intense b- or y-ions matched with the real time search algorithm. Fragment MS3 precursors by high energy collision-induced dissociation (HCD) and analyze with the Orbitrap (NCE 45, AGC 2.5105, maximum injection time 200 ms, resolution 50,000 at 200 Th). Set closeout at two peptides per protein for each fraction.
SECTION: Lysosomal and endosomal proteomics
40. Convert RAW files to mzXML files. Construct the searching database from Swiss-Prot human database, which is appended with common contaminants and reversed for target-decoy false discovery rate (FDR) estimation. Perform a database searching with a 20-ppm precursor ion tolerance and 1.0005 Da product ion tolerance. Include static modifications with carbamidomethylation at cysteine (+57.021 Da), TMT labeling at lysine (+229.162 Da for TMT or +304.207 Da for TMTpro) while including variable modifications with oxidation at methionine (+15.995 Da) and TMT labeling at peptide N-termini (+229.162 Da for TMT or +304.207 Da for TMTpro). Filter peptide-spectrum matches (PSM) using a linear discriminant analysis algorithm while considering XCorr, ΔCn, missed cleavages, peptide length, charge state, and precursor mass accuracy. Control the identified peptides at 1% false discovery rate (FDR). Do a protein assembly by parsimony principle, and apply 1% FDR to the protein level. For reporter ion quantification, extract signal-to-noise (S/N) ratio for each TMT channel with an integration tolerance of 0.003 Da. Quantify proteins by summing the reporter ion counts across all matching PSMs. Adjust S/N of each channel using the isotopic impurity table of TMT reagents provided by the vendor.
SECTION: Lysosomal and endosomal proteomics
41. Export protein quantitation values to csv file, and analyze by R 3.6.3. Perform two-sided Student’s t-test by t_test() function in the rstatix package version 0.7.0. Adjust individual p-values for multiple testing correction by mt.rawp2adjp() function with two-stage Benjamini & Hochberg (2006) step-up FDR-controlling procedure in the multtest package version 2.42.0.