Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.

Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, "rewiring" subnetworks within each cell's interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell's phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.

Integrated Proteogenomic Characterization across Major Histological Types of Pediatric Brain Cancer.

We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses. Proteomics data further reveal functional effects of somatic mutations and copy number variations (CNVs) not evident in transcriptomics data. Kinase-substrate association and co-expression network analysis identify important biological mechanisms of tumorigenesis. This is the first large-scale proteogenomics analysis across traditional histological boundaries to uncover foundational pediatric brain tumor biology and inform rational treatment selection.

TKO6: A Peptide Standard To Assess Interference for Unit-Resolved Isobaric Labeling Platforms.

Protein abundance profiling using isobaric labeling is a well-established quantitative mass spectrometry technique. However, ratio distortion resulting from coisolated and cofragmented ions, commonly referred to as interference, remains a drawback of this strategy. Tribrid mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple mass analyzer configuration, facilitate methods (namely, SPS-MS3) that can help alleviate interference. However, few standards are available to measure interference and thereby aid in method development. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) mass resolution. We use TKO6 to compare interference in MS2- versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion-trap-based tandem mass tag reporter ion analysis (IT-MS3) with conventional Orbitrap-based analysis (OT-MS3). We show that the TKO6 standard is a valuable tool for assessing quantification accuracy in isobaric-tag-based analyses.

Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu.

Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from Pseudomonas putida (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant, and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from Bacillus anthracis (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC-SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC-SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in B. anthracis.

Growth factor and ultrasound-assisted bioreactor synergism for human mesenchymal stem cell chondrogenesis.

Ultrasound at 5.0 MHz was noted to be chondro-inductive, with improved SOX-9 gene and COL2A1 protein expression in constructs that allowed for cell-to-cell contact. To achieve tissue-engineered cartilage using macroporous scaffolds, it is hypothesized that a combination of ultrasound at 5.0 MHz and transforming growth factor-ß3 induces human mesenchymal stem cell differentiation to chondrocytes. Expression of miR-145 was used as a metric to qualitatively assess the efficacy of human mesenchymal stem cell conversion. Our results suggest that in group 1 (no transforming growth factor-ß3, no ultrasound), as anticipated, human mesenchymal stem cells were not efficiently differentiated into chondrocytes, judging by the lack of decrease in the level of miR-145 expression. Human mesenchymal stem cells differentiated into chondrocytes in group 2 (transforming growth factor-ß3, no ultrasound) and group 3 (transforming growth factor-ß3, ultrasound) with group 3 having a 2-fold lower miR-145 when compared to group 2 at day 7, indicating a higher conversion to chondrocytes. Transforming growth factor-ß3-induced chondrogenesis with and without ultrasound stimulation for 14 days in the ultrasound-assisted bioreactor was compared and followed by additional culture in the absence of growth factors. The combination of growth factor and ultrasound stimulation (group 3) resulted in enhanced COL2A1, SOX-9, and ACAN protein expression when compared to growth factor alone (group 2). No COL10A1 protein expression was noted. Enhanced cell proliferation and glycosaminoglycan deposition was noted with the combination of growth factor and ultrasound stimulation. These results suggest that ultrasound at 5.0 MHz could be used to induce chondrogenic differentiation of mesenchymal stem cells for cartilage tissue engineering.

Enhanced depth-independent chondrocyte proliferation and phenotype maintenance in an ultrasound bioreactor and an assessment of ultrasound dampening in the scaffold.

Chondrocyte-seeded scaffolds were cultured in an ultrasound (US)-assisted bioreactor, which supplied the cells with acoustic energy around resonance frequencies (~5.0 MHz). Polyurethane-polycarbonate (BM), chitosan (CS) and chitosan-n-butanol (CSB) based scaffolds with varying porosities were chosen and the following US regimen was employed: 15 kPa and 60 kPa, 5 min per application and 6 applications per day for 21 days. Non-stimulated scaffolds served as control. For BM scaffolds, US stimulation significantly impacted cell proliferation and depth-independent cell population density compared to controls. The highest COL2A1/COL1A1 ratios and ACAN mRNA were noted on US-treated BM scaffolds compared to controls. A similar trend was noted on US-treated cell-seeded CS and CSB scaffolds, though COL2A1/COL1A1 ratios were significantly lower compared to BM scaffolds. Expression of Sox-9 was also elevated under US and paralleled the COL2A1/COL1A1 ratio. As an original contribution, a simplified mathematical model based on Biot theory was developed to understand the propagation of the incident US wave through the scaffolds and the model analysis was connected to cellular responses. Scaffold architecture influenced the distribution of US field, with the US field being the least attenuated in BM scaffolds, thus coupling more mechanical energy into cells, and leading to increased cellular activity.

Ultrasonic bioreactor as a platform for studying cellular response.

The need for tissue-engineered constructs as replacement tissue continues to grow as the average age of the world's population increases. However, additional research is required before the efficient production of laboratory-created tissue can be realized. The multitude of parameters that affect cell growth and proliferation is particularly daunting considering that optimized conditions are likely to change as a function of growth. Thus, a generalized research platform is needed in order for quantitative studies to be conducted. In this article, an ultrasonic bioreactor is described for use in studying the response of cells to ultrasonic stimulation. The work is focused on chondrocytes with a long-term view of generating tissue-engineered articular cartilage. Aspects of ultrasound (US) that would negatively affect cells, including temperature and cavitation, are shown to be insignificant for the US protocols used and which cover a wide range of frequencies and pressure amplitudes. The bioreactor is shown to have a positive influence on several factors, including cell proliferation, viability, and gene expression of select chondrocytic markers. Most importantly, we show that a total of 138 unique proteins are differentially expressed on exposure to ultrasonic stimulation, using mass-spectroscopy coupled proteomic analyses. We anticipate that this work will serve as the basis for additional research which will elucidate many of the mechanisms associated with cell response to ultrasonic stimulation.

Adherence of platelets to in situ albumin-binding surfaces under flow conditions: role of surface-adsorbed albumin.

Surfaces that preferentially bind human serum albumin (HSA) were generated by grafting albumin-binding linear peptide (LP1) onto silicon surfaces. The research aim was to evaluate the adsorption pattern of proteins and the adhesion of platelets from platelet-poor plasma and platelet-rich plasma, respectively, by albumin-binding surfaces under physiological shear rate (96 and 319 s(-1)) conditions. Bound proteins were quantified using enzyme-linked immunosorbent assays (ELISAs) and two-dimensional gel electrophoresis. A ratio of ∼1000:100:1 of adsorbed HSA, human immunoglobulin (HIgG) and human fibrinogen (HFib) was noted, respectively, on LP1-functionalized surfaces, and a ratio of ∼5:2:1 of the same was noted on control surfaces, as confirmed by ELISAs. The surface-adsorbed von Willebrand factor was undetectable by sensitive ELISAs. The amount of adhered platelets correlated with the ratio of adsorbed HSA/HFib. Platelet morphology was more rounded on LP1-functionalized surfaces when compared to control surfaces. The platelet adhesion response on albumin-binding surfaces can be explained by the reduction in the co-adsorption of other plasma proteins in a surface environment where there is an excess of albumin molecules, coupled with restrictions in the conformational transitions of other surface-adsorbed proteins into hemostatically active forms.

Investigation of platelet responses and clotting characteristics of in situ albumin binding surfaces.

The response of biomaterial surfaces when exposed to blood is in part dependent upon the nature and composition of the adsorbed layer of proteins. Surfaces passivated with albumin have been shown to reduce platelet adhesion and activation. In an attempt to develop surfaces that can selectively and specifically bind albumin, silicon-based surfaces were functionalized with linear peptides and chemical ligands that displayed an affinity for albumin. Peptide functionalized surfaces were observed to preferentially bind albumin when compared to human immunoglobulin and human fibrinogen, which possess low densities of surface adsorbed platelets. The platelet morphology was noted to be discoid on the peptide modified surface. Both the unmodified control and SCL functionalized surfaces had high densities of surface adhered platelets with spread out morphology. The peptide and SCL functionalized surfaces were noted to have no impact on PTT and PT clotting times, indicating that the extrinsic and intrinsic pathways were unperturbed by the surfaces generated.

The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.

Both pulsed- and square-wave, low-intensity ultrasound (US) signals have been reported to impact chondrocyte function and biosynthetic activity. In this study, a low-intensity diffuse ultrasound (LIDUS) signal at 5.0 MHz (0.14 mW/cm(2)) was employed to stimulate bovine chondrocytes seeded in three-dimensional (3D) chitosan-based matrices. While the duration of application was constant at 51 s, US was applied once, twice, four times and eight times/day, and the impacts of US on the biosynthetic activity of chondrocytes and the expression of chondrocyte-specific genes were evaluated. When stimulated with continuous US for predetermined time intervals, chondrocytes had higher levels of type II collagen, aggrecan, L-Sox5 and Sox9 mRNA expression when compared to controls; however, under the same conditions, the expression of MMP-3 was downregulated. Interestingly, both Sox5 and Sox9 genes coordinately responded to changes in US stimulation and generally mirrored the response of collagen type II transcript to changes in US stimulation. RT-PCR analysis revealed that US stimulation increased the gene expression of cell-surface integrins α5 and ß1. The expression of integrins α2 was downregulated by US treatment, suggesting that multiple integrin subunits may be involved in the regulation of chondrocytic function in response to US stimuli. The enhancement in the abundance of the mRNA transcripts upon US stimulation was observed to correlate with the protein expression of collagen type I, collagen type II, and integrins α5 and ß1. In conclusion, the US stimulation regimen employed was shown to modulate the proliferative capacity, biosynthetic activity and integrin mRNA expression of articular chondrocytes maintained in 3D matrices.

Evaluation of in situ albumin binding surfaces: a study of protein adsorption and platelet adhesion.

Surface modification strategies that take advantage of the passivation effects of albumin are important in the development of biomaterial surfaces. In this study, linear peptides (LP1, LP2) and a small chemical ligand (SCL) with albumin binding affinities were grafted onto silane functionalized silicon substrates. Surfaces were characterized with contact angle and ellipsometric measurements, and densities of immobilized ligands were assessed spectroscopically. Ellipsometrically measured thickness correlated with the predicted molecular lengths of grafted moieties. Contact angle analysis indicated that the LP1 and LP2 functionalized surfaces were hydrophilic compared to SCL functionalized and control surfaces. Adsorption of albumin from human serum was evaluated and quantified via specific enzyme-linked immunosorbent assays and 2D gel electrophoresis. The following trend was noted for surface adsorbed albumin: LP1 > LP2 > SCL > C, with LP1 derivatized surfaces having ~2.450 µg/cm(2) of bound albumin. LP1 derivatized surfaces possessed the least number of adsorbed platelets with rounded platelet morphology when compared to control surface.