Context
1.     Materials and methods. 2
1.1       Protein extraction and digestion.. 2
1.2       LC-MS/MS analysis. 3
1.3       Identification and quantitation of proteins. 3
1.4       Bioinformatic analysis. 5  

1. Materials and methods
   1. Protein extraction and digestion

SDT(4%SDS，100mM Tris-HCl，pH7.6) buffer was used for sample lysis and protein extraction. The amount of protein was quantified with the BCA Protein Assay Kit (Bio-Rad, USA). 20 µg of protein for each sample were mixed with 5X loading buffer respectively and boiled for 5 min. The proteins were separated on 4%-20% SDS-PAGE gel (constant voltage 180V, 45 min). Protein bands were visualized by Coomassie Blue R-250 staining. Protein digestion by trypsin was performed according to filter-aided sample preparation (FASP) procedure described by Matthias Mann. The digest peptides of each sample were desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma), concentrated by vacuum centrifugation and reconstituted in 40 µl of 0.1% (v/v) formic acid.
Filter-aided sample preparation (FASP Digestion) procedure：The detergent DTT (with the final concentration of 10 mM) was added to each sample respectively and mixed at 600 rpm for 1.5 h (37℃).After the samples cooled to room temperature, IAA was added with the final concentration of 20 mM into the mixture to block reduced cysteine residues and the samples were incubated for 30 min in darkness. Next, the samples were transferred to the filters respectively. The filters were washed with 100 μl UA buffer three times and then 100 μl 25mM NH4HCO3 buffer twice. Finally, trypsin was added to the samples(the trypsin : protein (wt/wt) ratio was 1:50) and incubated at 37℃ for 15-18 h (overnight), and the resulting peptides were collected as a filtrate. The peptides of each sample were desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma), concentrated by vacuum centrifugation and reconstituted in 40 µl of 0.1% (v/v) formic acid. The peptide content was estimated by UV light spectral density at 280 nm using an extinctions coefficient of 1.1 of 0.1% (g/l) solution that was calculated on the basis of the frequency of tryptophan and tyrosine in vertebrate proteins.

1. 1. LC-MS/MS analysis

LC-MS/MS analysis was performed on a timsTOF Pro mass spectrometry (Bruker) that was coupled to Nanoelute (Bruker). The peptides were loaded onto a C18-reversed phase analytical column (Thermo Scientific Easy Column, 25 cm long, 75 μm inner diameter, 1.9μm resin) in 95% buffer A (0.1% Formic acid in water) and separated with a linear gradient of buffer B (99.9% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nl/min. The mass spectrometer was operated in positive ion mode. The electrospray voltage applied was 1.5 kV. Precursors and fragments were analyzed at the TOF detector over a mass range of m/z 100-1700. The timsTOF Pro was operated in parallel accumulation serial fragmentation (PASEF) mode, PASEF mode data collection was performed based on the following parameters: Ion mobility coefficient (1/K0) value was set from 0.6 to 1.6 Vs cm2; 1 MS and 10 MS/MS PASEF scans. Active exclusion was enabled with a release time of 24 s.

1. 1. Identification and quantitation of proteins

The MS raw data for each sample were combined and searched using the MaxQuant 1.6.14 software for identification and quantitation analysis. Related parameters and instructions are as follows:
Table Maxquant identification and quantitation indexes

Item	Value
Enzyme	Trypsin
Max Missed Cleavages	2
Fixed modifications	Carbamidomethyl (C),
Variable modifications	Oxidation (M) ,
Main search	6 ppm
First search	20ppm
MS/MS Tolerance	20ppm
Database	xxxx for example, uniprot_mouse_76417 represents: “uniprot”, public database (http://www.uniprot.org/); “mouse”, organism species; “76417”, the number of sequences
Database pattern	Reverse
Include contaminants	True
protein FDR	≤0.01
Peptide FDR	≤0.01
Peptides used for protein quantification	Use razor and unique peptides
Time window (match between runs)	2min
protein quantification	LFQ
min. ratio count	1

Notes: Intensity-based absolute quantification (iBAQ ) and LFQ are two different methods for protein quantification provided by Maxquant software.
iBAQ Intensity reveals the level of protein expression in the sample X based on iBAQ algorithm，which is approximation to the absolute concentration of the protein in the sample.
LFQ Intensity reveals the level of protein expression in the sample X based on LFQ algorithm，which is often used in the comparison between groups.

1. 1.  Bioinformatic analysis
      1. Cluster analysis of phosphorylated peptides

Cluster 3.0 (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) and Java Treeview software (http://jtreeview.sourceforge.net) were used to performing hierarchical clustering analysis. Euclidean distance algorithm for similarity measure and average linkage clustering algorithm (clustering uses the centroids of the observations) for clustering were selected when performing hierarchical clustering. A heat map was often presented as a visual aid in addition to the dendrogram.

1. 1. 1. Motif analysis

The motifs were analyzed by MeMe (http://meme-suite.org/index.htm). We extracted the amino acid sequences contain the modified site and six upstream/downstream amino acids from the modified site (13 amino acid sites in total). These sequences were used to predicted motifs in this study (parameters: width：13, occurrences:20, background: species).

1. 1. 1. Subcellular localization

CELLO (http://cello.life.nctu.edu.tw/) which is a multi-class SVM classification system, was used to predict protein subcellular localization.

1. 1. 1. Domain annotation

Protein sequences are searched using the InterProScan software to identify protein domain signatures from the InterPro member database Pfam.

1. 1. 1. GO annotation

The protein sequences of the selected differentially expressed proteins were locally searched using the NCBI BLAST+ client software (ncbi-blast-2.2.28+-win32.exe) and InterProScan to find homologue sequences, then gene ontology (GO) terms were mapped and sequences were annotated using the software program Blast2GO. The GO annotation results were plotted by R scripts.

1. 1. 1. KEGG annotation

Following annotation steps, the studied proteins were blasted against the online Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://geneontology.org/) to retrieve their KEGG orthology identifications and were subsequently mapped to pathways in KEGG.

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注：1) 材料与方法仅供参考，不得用于正式文章发表，文章书写需自行修改。