Label-free quantitative proteomic analysis of adult Drosophila heads

Summary LIR motif-containing proteins (LIRCPs) bind to LDS (LIR motif docking site) of Atg8-family proteins. In this protocol, we describe steps to identify Drosophila LIRCPs, in Atg8a LDS mutants we have created, via label-free quantitative proteomic analysis. We detail steps for extraction of proteins from adult Drosophila heads, followed by liquid chromatography–mass spectrometry (LC-MS/MS) analysis. We also describe screening steps of upregulated proteins in Atg8a LDS mutants, leading to identification of novel LIRCPs in Drosophila. For complete details on the use and execution of this protocol, please refer to Rahman et al. (2022).

Note: Initially, add all the reagents to 20 mL of water. Make the volume to 30 mL when all reagents are dissolved.
Note: UA buffer can be stored at 25 C for 3 days (Prepare fresh before beginning the experiment).
Note: ABC buffer can be stored at 25 C for 1 week.
Note: DTT and IAA stocks are stored at À20 C, for a maximum of 6 months. IAA should be weighed in the fume hood.
Note: DTT and IAA are harmful if swallowed. DTT causes skin irritation and serious eye damage. IAA may cause an allergic skin reaction. Wear eye and face protection and prevent release to the environment when you are preparing.

STEP-BY-STEP METHOD DETAILS
Quantitative proteomics analysis of adult Drosophila heads

Timing: 2 days
In this study, we use two autophagy mutants: Atg8a KG07569 (Scott et al., 2007) and Atg8a LDS (LIR motif docking site) mutant (Rahman et al., 2022). It is hypothesized that these flies have defective LIR motif binding ability and thus accumulate LIR motif containing proteins. We perform labelfree quantitative proteomic to analyze the accumulated proteins in Atg8a LDS, Atg8a mutant compare to wild type flies. The accumulated proteins are putative selective autophagy receptors.
1. Sample preparation for Label-Free-Quantification (LFQ) based mass spectrometry analysis. a. In this protocol, we use 200 mg of protein extraction for digestion. i. Calculate the required volume of 200 mg protein according to the protein concentration.
ii. Mix 200 mg of protein extraction with UA buffer in the filter tube (Millipore, UFC500396).
iii. Make sure the total volume is 400 mL. b. Centrifuge the filter units at 12, 000 g for 20 min at 4 C and discard the flow-through from the collection tube. c. Add 400 mL of UA buffer to the filter tube. d. Centrifuge at 12, 000 g for 20 min at 4 C. Discard the flow-through from the collection tube. e. Repeat steps (c-d) two times.

Reagent
Final concentration Amount f. Add 300 mL of UA buffer and 6 mL of 1 M DTT buffer to the filter tube. Mix and incubate at 37 C for 1 h. g. Add 30 mL of 1 M IAA buffer to the filter tube. Mix and incubate at 25 C for 1 h.
CRITICAL: Make sure the filter tube is cooled to 25 C before adding the IAA buffer.
Note: Incubate in dark place.
h. Centrifuge at 12, 000 g for 20 min at 4 C. Discard the flow-through from the collection tube. i. Add 400 mL of UA buffer to the filter tube. j. Centrifuge at 12, 000 g for 20 min at 4 C. Discard the flow-through from the collection tube. k. Repeat steps (i-j) two times. l. Add 400 mL of ABC buffer to the filter tube. m. Centrifuge at 12, 000 g for 20 min at 4 C. Discard the flow-through from the collection tube. n. Repeat steps (l-m) two times. o. Add 200 mL ABC buffer with trypsin (enzyme to protein ratio 1: 50), incubate at 37 C for 20 h.
Note: Change to a new collection tube before adding trypsin.
Note: When incubating, wrap the lid of the filter tube with sealing film.
p. Centrifuge the filter units at 12, 000 g for 20 min at 4 C. q. Add 200 mL ABC buffer and centrifuge the filter units at 12, 000 g for 20 min at 4 C. r. Collect the tryptic peptides from the collection tube. s. Evaporate the samples to dryness using a Labconco Speedvac (Centrivap, Labconco, USA), usually for 3-4 h. t. Store the samples at À20 C until use.
A workflow of sample preparation for Mass spectrometry is shown in Figure 2.
CRITICAL: All biological repeats are digested at the same time to improve the repeatability. If your repeats are digested in batches, make sure you use the same materials and methods.
Pause point: Samples can be stored at this point for up to 1 month at À80 C.

Mass spectrometry and statistical analysis
Timing: weeks Tryptic peptides are analyzed by mass spectrometry to identify the accumulated proteins in Atg8a KG07569 , Atg8a LDS mutant compared to wild type flies.  e. Peptides are subjected to Nano Spray ionization (NSI) source followed by tandem mass spectrometry (MS/MS) in Thermo Orbitrap Fusion coupled online to UPLC (Ultimate 3000 RSLCnano HPLC) (Dionex) or Q exactive coupled online to EASY-nLC 1000 system. f. The electrospray voltage applied is 2.1 kV. g. The m/z scan range is 375-1,575 m/z for full scan, and intact peptides are detected in the Orbitrap at a resolution of 120,000 resolution with a 2 3 10 5 ion count target. h. The maximum injection time is set to 150 ms. i. Tandem MS is performed with an isolation window at 1.2 Th using the Quadrupole. Use of wider isolation windows improves sensitivity, but noise of ions also increases. We set isolation window at 1.2 Th for Orbitrap Fusion Mass Spectrometer, 2.2 m/z for Q exactive Mass Spectrometer according to our experience. j. HCD (High-energy collisional dissociation) is significantly affected by the normalized energy applied. HCD fragmentation with normalized collision energy of 33 or 32 is recommended for Orbitrap Fusion Mass Spectrometer, 27 for Q exactive Mass Spectrometer. k. The MS 2 ion count target is set to 5 3 10 3 and maximum injection time is 200 ms. Precursors with charge state 2-7 are selected and sampled for MS 2 . l. The dynamic exclusion duration is set to 50 s with a 10 ppm tolerance around the selected precursor and its isotopes. Fixed first mass is set as 120 m/z. m. Monoisotopic precursor selection is turned on and instrument is run in top speed mode. 3. MaxQuant analysis of Samples. All acquired raw data are searched against Drosophila melanogaster database and the common contaminant database by MaxQuant software (v1.6.5.0) (Cox et al., 2011;Tyanova et al., 2016). Please see Figure 3 for workflow on MaxQuant detailed parameters for analysis of these data. Unless explicitly stated, parameters in MaxQuant have not been changed from their standard values. a. Along the top of MaxQuant are six tabs. Select Raw data, then click ''Load'' to load all raw data (step 1). b. Write template (step 2), then a txt named ''experimentalDesignTemplate'' will automatically appear in the folder. Assign the experiment number (step 3) and then read the ''experimental-DesignTemplate'' from file (step 4). c. In the Group-specific parameters tab, choose type as ''Standard'' and the ''Multiplicity'' is 1 for label-free quantification (step 5). Choose ''Multiplicity'' 2 if you have light and heavy labels, and 3 if you have light, medium, and heavy. d. Precursor mass tolerance is 4.5 ppm and product ions are searched at 15 ppm tolerances (steps 6 and 10). e. Peptides are generated from a tryptic digestion with up to two missed cleavages (step 7), carbamidomethylation of cysteines as fixed modifications, and oxidation of methionine as variable modifications. f. We use LFQ with minimum ratio count of 2 to perform label-free quantification (step 8). g. On the Global parameters tab, click ''sequence'' and add a FASTA file of Drosophila proteome downloaded from Uniport (step 9). h. Minimum peptide length is set at 7, while the estimated false discovery rate (FDR) threshold for peptide and protein are specified at maximum 1% (step 11).

LC parameter settings
CRITICAL: Perform three or four replicates for Atg8a, Atg8a-LDS mutant and wild type in order to improve the accuracy of results.

Identify the accumulate proteins.
a. All result files appear in the folder ''.\combined\txt'' as tab-delimited text files. Data processing and annotation are performed by manual operation. b. Open ''ProteinGroups'' with Microsoft Excel, and remove the reverse and contaminant hits (as defined in MaxQuant) from the MaxQuant output files. c. Only protein groups identified with at least one unique peptide are used for the analysis. d. Calculate the average LFQ intensity of each protein group in each type of sample. e. The ratio is calculated by dividing the average value of LFQ intensity of Atg8a, Atg8a LDS mutant sample by average value of LFQ intensity of wild type sample (Atg8a/ wild type, Atg8a LDS/ wild type). f. Protein groups are assigned a probability value (p-value) using a two-sample Student's T-Test. g. Proteins are considered significant if the p-value < 0.05 and had a more than two-fold change in protein expression.

EXPECTED OUTCOMES
Mass spectrometry-based comparative proteomic study helps efficiently to identify the accumulated proteins in Atg8a and Atg8a LDS mutant fly heads compared to wild type. Thus, a successful MSbased assay can provide high quality data. Figure 4 shows a representative chromatogram of wild type fly heads. X axis represents the retention time, and y axis represents the relative abundance.
In this study, we identified 3036, 2342, 2468 proteins from wild type, Atg8a KG07569 and Atg8a-LDS mutants, respectively. All the accumulated proteins with a difference of more than 2-fold between mutant and wild type flies are included in the below table. Figure 5 shows the all identified

LIMITATIONS
This protocol enables identification of accumulated proteins in Atg8a KG07569 and Atg8a-LDS mutant Drosophila heads compared to wild type. In this protocol, we use the fly head because the brain is relatively small and difficult to dissect out to extract enough proteins for proteomics.

TROUBLESHOOTING Problem 1
Our project is to identify accumulated proteins in autophagy mutants. In our pre-experiments, we used methanol precipitation technique to extract proteins and in-solution digestion method. Although Ref (2)p is a well-studied LIRCP, it was not identified in the pre-experiments (step for sample collection for quantitative proteomic profiling).

Potential solution
We thought methanol precipitation and in-solution digestion techniques may not have extracted some proteins. Then, we used RIPA buffer to extract proteins and Filter Aided Sample Preparation (FASP) method to digest proteins. More proteins were identified.

Problem 2
For the second pre-experiments, we collected whole flies as our samples. The number of identified proteins was very low (step for sample collection for quantitative proteomic profiling).

Potential solution
We speculated that protein composition is very diverse in whole flies. The intensity of high-abundant proteins may inhibit the low-abundant proteins. We collected fly heads to perform quantitative proteomic analysis. In addition, we increased nano-LC gradient time from 120 min to 180 min. We increased the protein number from 1843 to 2528 proteins.

Problem 3
User may find that the chromatogram peaks are discontinuous and have low relative intensity during MS analysis (step 3).

Potential solution
Check the spray needle (nano-bore emitters), and use a new one if necessary. Increase of sample loading can increase the intensity.

Problem 4
In the pre-experiments, the repeatability of proteomics was poor (step 4).

Potential solution
In the pre-experiments, we collected both male and female fly heads. To improve the repeatability, we only used the male virgin flies in the experiments. In addition, all repeat samples are digested at the same time. Perform the mass spectrometry for all repeat samples within one or two days.

Problem 5
There are many accumulated proteins identified in Atg8a KG07569 and Atg8a LDS mutant compared to wild type (step 4).

Potential solution
Select the accumulated proteins in both Atg8a KG07569 and Atg8a LDS mutants by using the cut-off p-value as less than 0.05 together with a difference of more than two-fold between mutant and wildtype flies. Choose the proteins which have putative LIR motifs by using the iLIR software (Jacomin et al., 2016).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ioannis P. Nezis (I.Nezis@warwick.ac.uk).

Materials availability
This study did not generate any new unique reagents and/or materials.

Data and code availability
This study did not generate new unique datasets or codes.