VEGF Study in Primary Human Endothelial Cells
The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial post-wound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage prior to progression of wound healing toward tissue homeostasis. Due to its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 hours of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment.
Sample Processing Protocol
Phosphotyrosine mass spectrometry. Sample processing followed protocols for chemical reduction, alkylation, trypsin digestion, and fractionation as previously described. Briefly, groups of eight samples were labeled with eight unique isobaric iTRAQ reagents (AB SCIEX, Framingham, MA) for 2 hours at room temperature. Following labeling, samples were combined and concentrated before immunoprecipitation (IP) with a mixture of anti-phosphotyrosine antibodies (4G10 (Millipore, Billerica, MA), pTyr100 (Cell Signaling), and PT-66 (Sigma), 12 μg of each) immobilized onto protein G agarose beads (Calbiochem) in iTRAQ IP buffer [100 mM Tris, 100 mM NaCl, 0.3% NP-40, ph 7.4] (Sigma-Aldrich) overnight at 4°C. Beads were washed four times in rinse buffer (IP buffer without NP-40) and phosphotyrosine-containing peptides were eluted with glycine buffer (100 mM, pH=2) at room temperature for 30 minutes on a rotator. Phosphopeptides were further enriched by immobilized metal affinity chromatography (IMAC) on a custom-packed 200μm x 10cm column with Poros MS 20μm beads (Life Technologies). Phosphopeptides were eluted with phosphate buffer (250 mM, pH 8.0) directly onto a custom 100μm x 10cm precolumn packed with 10 μm YMC-Gel ODS-A beads (Waters Corporation, Milford, MA). The precolumn was immediately washed and brought inline with a custom 50μm x 10cm analytical column packed with 5μm YMC-Gel ODS-AQ beads with 120 angstrom pores (Waters Corporation), and with an integrated sub-μm nanospray tip pulled with a laser puller (Sutter, Novato, CA). Each tip was examined by microscopy with a calibrated objective and tested extensively with single femtomolar quantities of peptide standards before use. Analysis and quantification of eluted peptides were conducted on an LTQ-Orbitrap Elite mass spectrometer via nano-ESI LC/MS/MS (Thermo Fisher Scientific) with a tandem MS top ten data-dependent acquisition method. Peptides were eluted with a binary gradient with an aqueous solvent of 0.2M acetic acid in ultrapure water and an organic solvent of 0.2M acetic acid in 90% acetonitrile and ultrapure water. The gradient was 0-13% organic from 0 to 10 min, 13% to 42% from 10 to 105 min, 42% to 60% from 105 to 115 min, 60% to 100% from 115 to 120 min, held at 100% organic from 120 to 128 min, and washed indefinitely with aqueous solvent until peptides stopped eluting. The flow rate was initially set at 20 nL/min and then dropped to 5-10 nL/min once peptides began eluting.
Data Processing Protocol
Raw files were processed with MSQuant v2.0b7 software and DTASupercharge (43), followed by peptide sequence and protein identification with Mascot v2.1.03 (Matrix Science, Boston, MA). Mascot searches were run in MS/MS ion search mode with the following parameters. Fixed modifications included iTRAQ8plex (K, N-term), and Carbamidomethyl (C), while variable modifications were Oxidation (M), Phospho Serine/Threonine (ST), and Phospho Tyrosine (Y). Monoisotopic mass values were selected with no restrictions on protein mass. A tolerance of 10 ppm was selected for peptide mass and 0.8 Da for fragment mass tolerance, with a maximum of 2 missed trypsin cleavages permitted. The instrument type was specified as an LTQ-Oribitrap. Since the culture system of HDMVECs was human in origin, the database was limited to the 2009 NCBI human proteome database in all searches (H. sapiens), with 37,743 total entries. The top 1000 peptide hits were reported, neglecting protein significance score cutoffs, due to the extensive phosphotyrosine enrichment performed. Due to the challenges associated with using a collagen gel culture system, an initial MASCOT peptide score threshold of 20 was implemented prior to initial data processing and normalization to protein supernatant values. In the cases of multiple matches, the query with the highest score and smallest expect value from Mascot was selected. For search results file outputs (both phosphotyrosine enriched immunoprecipitates and IP supernatants), please see Supplemental Data. Manual validation of mass spectra fragments. Because our work involved specific phosphorylation of amino acid residues, each tyrosine phosphorylation site was subjected to manual validation. Phosphopeptide scores and signals were thresholded for significant hits (score>20) and values were normalized to a master lysate (50 ng/mL VEGF treatment for 15 minutes) and normalized to total protein values obtained from an LC/MS/MS analysis of iTRAQ channel intensities of the IP supernatant. Initial methods to normalize to a master control condition with no stimulation (no VEGF) yielded a response with high noise to signal amplification due to the absence or minimal signal present in this condition. Each nonzero normalized mass spectra output (where iTRAQ for at least 5 of 8 tags were nonzero) was manually validated by hand following previously described guidelines to ensure correct sequence identification and phosphosite assignment. Peaks were compared with theoretical values for each of the peptides assigned by Mascot using the MS-Product component of Protein Prospector v5.10.9 (Baker, P.R. and Clauser, K.R. http://prospector.ucsf.edu). The processed, validated mass spectra data are also included in the Supplemental Data, as well as scans that were rejected due to precursor contamination, as indicated in the added header on each scan plot. Amino acid residue locations of phosphorylations on peptides were verified using PTMScout in addition to the aforementioned manual validation.
Nathan Tedford, Biological Engineering
Hang TC, Tedford NC, Reddy RJ, Rimchala T, Wells A, White FM, Kamm RD, Lauffenburger DA. Vascular endothelial growth factor (VEGF) and platelet (PF-4) factor 4 inputs modulate human microvascular endothelial signaling in a three-dimensional matrix migration context. Mol Cell Proteomics. 2013 Dec;12(12):3704-18 PubMed: 24023389