Proximity proteomics of endothelial Weibel-Palade bodies identifies novel regulator of VWF secretion
Weibel-Palade bodies (WPB) are unique secretory organelles of endothelial cells that store factors regulating vascular haemostasis and local inflammation. Endothelial activation triggers the acute exocytosis of WPB, leading to the surface presentation of adhesion molecules relevant for leukocyte rolling (P-selectin) and platelet plug formation (von-Willebrand factor, VWF). Despite its role as an important secretory organelle, a comprehensive compilation of WPB-associated factors has not been carried out. We addressed this by a proximity proteomics approach employing the peroxidase APEX2 coupled to two known WPB-associated proteins, the RabGTPases Rab3b and Rab27a. We show that APEX2-Rab3b/27a fusion constructs are correctly targeted to WPB and that proteins in their close proximity can be biotinylated through the WPB-recruited APEX2. Mass spectrometry analysis of the biotinylated proteins identified 183 WPB-associated proteins. While some of the factors identified have been reported before to localize to WPB, the majority comprises proteins not previously associated with WPB biology. These include the SNARE-interacting protein Munc13-2, which specifically localizes to WPB and serves as a novel factor promoting histamine-evoked WPB exocytosis and VWF secretion.
Sample Processing Protocol
Cells expressing Rab3b-APEX or Rab27a_APEX fusion proteins respectively were treated with biotin-phenol and hydrogenperoxide as described by Hung et al. (2016). Following lysis and streptavidin-mediated pulldown of biotinylated proteins, these were on-bead digested, desalted and subjected to LC-MSMS anaylsis using a Q Exative mass spectrometer equipped with an Easy nano-LC 1000 system and a EasyFlex electrospray source (ThermoFisher Scientific) holding a fused silica capillary chromatography column (25 cm length, ID 75 µm; NanoSeparations) that was packed in-house with Reprosil pur C18 reversed-phase material (1.9 µm; Dr. Maisch, Ammerbuch, Germany). Peptides were separated employing a linear gradient from 2 to 30 % buffer B (80 % acetonitril, 0.1 % formic acid) in 150 min and from 30 – 95 % B in 20 min at a flow rate of 250 nl/min followed by re-equilibration at starting conditions. The mass spectrometer was operated in data-dependent mode (source voltage 2.1 kV) automatically switching between a survey scan (mass range m/z = 300-1750, resolution R = 70 K; AGC target value 3e6, maximum IT 60 ms) and MS/MS acquisition of the 10 most intense peaks by higher-energy collisional dissociation (resolution 17.5 K; AGC target 1e6; max IT 108 ms; isolation width m/z = 1.6; normalized collision energy 30 % ). Dynamic exclusion was enabled for 20.0 s and double charge and higher charges were allowed.
Data Processing Protocol
MaxQuant software (184.108.40.206) involving the built-in Andromeda search engine was used to process the raw data. The search was performed against the human uniprotKB database UP000005640_9606.fasta (version from 12/2015) supplemented with common contaminants with a 1 % FDR at the peptide and protein level. Search parameteres were: Trypsin as digesting enzyme, allowing for two missed cleavages; a minimum length of 7 amino acids; carbamidomethylation at cysteine residues was set as fixed modification, while oxidation at methionine and protein N-terminal acetylation were defined as variable modifications. Maximum allowed mass deviation was 20 ppm for MS and 0.5 Da for MS/MS scans. Peptide identiﬁcations by MS/MS were allowed to be transferred between runs after retention time alignment to minimize the number of missing values for the subsequent label free quantiﬁcation process, performed with the MaxLFQ algorithm using a minimum ratio count of 1. Perseus software (version 220.127.116.11) was used for statistical and bioinformatics analysis. Proteins that were identiﬁed in the decoy reverse database or only by a site modiﬁcation, as well as common lab contaminants were excluded from further data analysis. In additon, a minimum of 2 peptides was required for protein identification, one of which had to be unique to the proteingroup. Log2 transformed LFQ values were further ﬁltered to ensure that expression values were present in at least 3 biological replicates of at least one experimental group. Missing values were substituted by imputation (down shift = 1.8 and width = 0.3). Significant differences between NES-APEX2, Rab27a-APEX2 and Rab3B-APEX2 transfected cells were determined by an ANOVA multiple sample test using a p-value of 0.02 as truncation value.
Holthenrich A, Drexler HCA, Chehab T, Naß J, Gerke V. Proximity proteomics of endothelial Weibel-Palade bodies identifies novel regulator of von-Willebrand factor secretion. Blood. 2019 PubMed: 31262780