2.1 Clinical samples of the platelets.
Platelet samples were taken from Shanghai Blood Center and stored under standard clinical conditions[37]. Platelet samples were used at the 1st, 3rd, 5th and 7th day of storage for procedures described below. For studies involving human subjects, approval was obtained from the Huashan Hospital Institutional Review Board (HIRB), Fudan University, Shanghai, China. Informed consent was provided in accordance with the Declaration of Helsinki.
2.2 Protein extraction from platelet and trypsin digestion.
Sample debris was removed by 20,000g centrifugation at 4°C for 10 minutes and the subsequent protein concentration was tested using the BCA Quant KIT (GE Healthcare Life Sciences™, BJ, CN.) according to the manufacturer’s instructions. For digestion, the protein solution was reduced with 10mM DL-dithiothreitol (DTT) (Sigma-Aldrich, SH, CN.) for 1 hour at 37 °C and then alkylated with 20mM 3-iodoacetamide (IAA) (Sigma-Aldrich, SH, CN.) for 45 minutes at room temperature in dark. For trypsin digestion, the protein sample was diluted by adding 100mM tetraethylammonium tetrahydroborate (TEAB) to urea concentration less than 2M. Finally, trypsin (Promega, Madison, WI, USA.) was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4-hour digestion. Approximately 100μg protein for each sample was digested with trypsin (Promega, Madison, WI, USA.) for the following experiments (Kac and Ksuss included).
2.3 TMT labeling and HPLC fractionation.
After trypsin digestion, the peptide was desalted by Strata X C18 SPE column (Phenomenex, TJ, CN.) and vacuum-dried. The peptide was reconstituted in 0.5M TEAB and processed according to the manufacturer's protocol for TMTsixplexTM Isobaric Label Reagent Set (ThermoFischer Scientific, SH, CN.). In brief, one unit of TMT reagent (defined as the amount of reagent required to label 100μg of protein) was used and reconstituted in 24μl acetonitrile (ACN) (Fisher Chemical, SH, CN.). The peptide mixtures were then incubated for 2 hours at room temperature and pooled, desalted and dried by vacuum centrifugation.
The sample was then fractionated by high pH reverse-phase HPLC using Agilent 300 Extend C18 column (5μm particles, 4.6mm ID, 250mm length). Briefly, peptides were first separated with a gradient of 2% to 60% ACN in 10mM ammonium bicarbonate pH 10 over 80min into 80 fractions. Then, the peptides were combined into 18 fractions and dried by vacuum centrifugation.
2.4 Affinity enrichment for Kac and Ksu peptides.
For Kac and Ksucc peptide enrichment, tryptic peptides were dissolved in NETN buffer (100mM NaCl, 1mM EDTA, 50mM Tris-HCl, 0.5% NP-40, pH 8.0), and then incubated with pre-washed antibody beads (PTM Biolabs, Chicago, IL, USA.) at 4°C overnight with gentle shaking. The beads were washed four times with NETN buffer and twice with ddH2O (ThermoFischer Scientific, SH, CN.). The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid (TFA) (Sigma-Aldrich, SH, CN.). The eluted fractions were combined and vacuum-dried. The resulting peptides were cleaned with C18 ZipTipÒ pipette tips (EMD Millipore, SH, CN.) according to the manufacturer’s instructions, followed by LC-MS/MS analysis.
2.5 LC-MS/MS analysis for global proteome, acetylome, and succinylome of the platelets.
Peptides were dissolved in 0.1% FA, directly loaded onto a reversed-phase pre-column, AcclaimTMPepMapTM 100 C18(ThermoFischer Scientific, SH, CN.). Peptide separation was performed using a reversed-phase analytical column, AcclaimTMPepMapTM RSLC (ThermoFischer Scientific, SH, CN.). The gradient comprised of an increase of solvent B (0.1% formic acid in 98% ACN) from 8% to 25% for the first 20 minutes, 25% to 40% for the subsequent 12 minutes,then increasing to 80% in the next 4 minutes, and maintaining at 80% for the last 4 minutes.All were performed at a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system, the resulting peptides were analyzed by QExactiveTMPlus Hybrid Quadrupole-OrbitrapTMMass Spectrometer (ThermoFisher Scientific, SH, CN.).
The peptides were subjected to NanoSpray Ionization (NSI)Source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTMPlus (ThermoFischer Scientific, SH, CN.) connected online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using normalized collision energy (NCE) setting as 30; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 5E3 in the MS survey scan with 15.0s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the Orbitrap; 5E4 ions were accumulated for the generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. The fixed first mass was set as 100 m/z.
2.6 Database search and QC validation of MS data.
The resulting MS/MS data was processed using MaxQuant[38] with an integrated Andromeda search engine (v.1.5.2.8). Tandem mass spectra were searched against the SwissProt human database concatenated with reverse decoy database. Cleavage agent Trypsin/P allowed up to 4 missing cleavages, 5 modifications per peptide and 5 charges. A mass error was set to 10 ppm for precursor ions and 0.02 Da for the fragment ions. Cysteine Carbamidomethylation was specified as fixed modification while oxidation on Methionine, acetylation on Lysine, and acetylation on protein N-terminal were specified as variable modifications. False discovery rate thresholds for protein, peptide and modification site were specified at 1%. Minimum peptide length was set at 7. TMT-6-plex was selected as the quantification method of proteins by report ion in MS/MS. All the other parameters in MaxQuant were set to default values. The site localization probability was set as > 0.75.For global proteomics, acetylome and Ksuccsearches, Cysteine Carbamidomethylation was specified as fixed modification while oxidation on Methionine, and acetylation or succinylationon Lysine, acetylationon protein N-terminal, respectively.
Validation of the MS data was done using mass error distribution of all identified peptides and peptide length distribution. Firstly, we checked the mass error of all the identified peptides. The distribution of mass error is near zero and most of them were less than 0.02 Da which indicates that the mass accuracy of the MS data meets the requirement. Secondly, the length of most peptides wasdistributed between 8 and 20, which agreed with the property of tryptic peptides, indicating that sample preparation has reached the quality standard.
2.7 Bioinformatics annotation of differential proteins.
Gene Ontology (GO) annotation[39] proteome was derived from the UniProt-GOA database (www. http://www.ebi.ac.uk/GOA/). Firstly, protein ID was converted into UniProt ID and the converted UniProtID was mappedinto GO IDs based on protein ID. If some identified proteins were not annotated by UniProt-GOA database, the InterProScan software would be used to annotated protein's GO functions based on protein sequence alignment method. Then proteins were classified by Gene Ontology annotation according to three categories: biological process, cellular component and molecular function. For each category, a two-tailed Fisher's exact test was employed to test the enrichment of the differentially expressed protein against all identified proteins. The GO with a corrected p-value < 0.05 is considered significant.
Kyoto Encyclopedia of Genes and Genomes (KEGG) database[40, 41] was used to annotate protein pathway. Firstly, KEGG online service tools, KAAS was used to annotated protein's KEGG database description. Then KEGG Mapper, a KEGG online service tool, was used to map the aforementioned annotation result. A two-tailed Fisher's exact test was applied to test the enrichment of the differentially expressed protein against all identified proteins. The pathway with a corrected p-value < 0.05 was considered significant. These pathways were classified into hierarchical categories according to the KEGG website.
2.8 Functional enrichment of differential proteins.
KEGG database was used to identify enriched pathways by a two-tailed Fisher’s exact test to test the enrichment of the differentially expressed protein against all identified proteins. Correction for multiple hypothesis testing was carried out using False Discovery Rate (FDR) control methods[42]. The pathway with a corrected p-value < 0.05 was considered significant.
2.9 Motif analysis and enrichment-based clustering of protein express profiling.
Soft motif-x[43] was applied to calculate the model of amino acid sequences in specific positions of modify-21-mers (10 amino acids upstream and downstream of the site) in all protein sequences. All the database protein sequences from Sections 2.7 and 2.8 were used as a background database parameter and other parameters were set to default.
All the substrate categories obtained after enrichment were collated along with their p-values, and then filtered for those categories which were at least enriched in one of the clusters with a lower p-value thresholdof 0.03. This filtered p-value matrix was transformed by the function x = −log10 (p-value). At last, these x values were z-transformed for each category. These z scores were then clustered by one-way hierarchical clustering (Euclidean distance, average linkage clustering) in Genesis. Cluster membership was visualized by a heat map using the “heatmap.2” function from the “gplots” R-package.