End-to-End Throughput Chemical Proteomics for Photoaffinity Labeling Target Engagement and Deconvolution.

Photoaffinity labeling (PAL) methodologies have proven to be instrumental for the unbiased deconvolution of protein-ligand binding events in physiologically relevant systems. However, like other chemical proteomic workflows, they are limited in many ways by time-intensive sample manipulations and data acquisition techniques. Here, we describe an approach to address this challenge through the innovation of a carboxylate bead-based protein cleanup procedure to remove excess small-molecule contaminants and couple it to plate-based, proteomic sample processing as a semiautomated solution.

CRISPR-Cas9 knockout screen informs efficient reduction of the Komagataella phaffii secretome.

Background: The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K.

Method development workflow for quantifying protein biomarkers by hybrid LC-MS/MS.

Industry-standard guidance on method development and validation of hybrid LC-MS/MS assays for protein biomarkers, particularly on evaluation of parallelism, is lacking. Using a protein endogenous to humans and mice as a model analyte, a quantitative hybrid LC-MS/MS workflow was developed using a surrogate matrix approach with a recombinant form of the protein as the calibrant. The developed workflow identified a surrogate matrix, established parallelism between the surrogate and authentic matrices and assessed parallelism between the recombinant and authentic forms of the protein.

Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow.

Objective: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage.

Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes.

We present the development, optimization, and application of constructs, cell lines, covalent cross-linking methods, and immunoprecipitation strategies that enable robust and accurate determination of collagen interactomes via mass spectrometry-based proteomics. Using collagen type-I as an example, protocols for working with large, repetitive, and GC-rich collagen genes are described, followed by strategies for engineering cells that stably and inducibly express antibody epitope-tagged collagen-I. Detailed steps to optimize collagen interactome cross-linking and perform immunoprecipitations are then presented.

Exploration of CTCF post-translation modifications uncovers Serine-224 phosphorylation by PLK1 at pericentric regions during the G2/M transition.

The zinc finger CCCTC-binding protein (CTCF) carries out many functions in the cell. Although previous studies sought to explain CTCF multivalency based on sequence composition of binding sites, few examined how CTCF post-translational modification (PTM) could contribute to function. Here, we performed CTCF mass spectrometry, identified a novel phosphorylation site at Serine 224 (Ser-P), and demonstrate that phosphorylation is carried out by Polo-like kinase 1 (PLK1).

Genetic Intersection of Tsix and Hedgehog Signaling during the Initiation of X-Chromosome Inactivation.

X-chromosome inactivation (XCI) silences one X chromosome in the female mammal and is essential to peri-implantation development. XCI is thought to be cell autonomous, with all factors required being produced within each cell. Nevertheless, external cues may exist.

Direct evidence for cancer-cell-autonomous extracellular protein catabolism in pancreatic tumors.

Mammalian tissues rely on a variety of nutrients to support their physiological functions. It is known that altered metabolism is involved in the pathogenesis of cancer, but which nutrients support the inappropriate growth of intact malignant tumors is incompletely understood. Amino acids are essential nutrients for many cancer cells that can be obtained through the scavenging and catabolism of extracellular protein via macropinocytosis.

Mapping and Exploring the Collagen-I Proteostasis Network.

Collagen-I is the most abundant protein in the human body, yet our understanding of how the endoplasmic reticulum regulates collagen-I proteostasis (folding, quality control, and secretion) remains immature. Of particular importance, interactomic studies to map the collagen-I proteostasis network have never been performed. Such studies would provide insight into mechanisms of collagen-I folding and misfolding in cells, an area that is particularly important owing to the prominence of the collagen misfolding-related diseases.

Engineered bromodomains to explore the acetylproteome.

MS-based analysis of the acetylproteome has highlighted a role for acetylation in a wide array of biological processes including gene regulation, metabolism, and cellular signaling. To date, anti-acetyllysine antibodies have been used as the predominant affinity reagent for enrichment of acetyllysine-containing peptides and proteins; however, these reagents suffer from high nonspecific binding and lot-to-lot variability. Bromodomains represent potential affinity reagents for acetylated proteins and peptides, given their natural role in recognition of acetylated sequence motifs in vivo.

SirT1 is required in the male germ cell for differentiation and fecundity in mice.

Sirtuins are NAD(+)-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control.

Molecular characterization of EGFR and EGFRvIII signaling networks in human glioblastoma tumor xenografts.

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ≈ 50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2-7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors.

Phosphotyrosine signaling analysis of site-specific mutations on EGFRvIII identifies determinants governing glioblastoma cell growth.

To evaluate the role of individual EGFR phosphorylation sites in activating components of the cellular signaling network we have performed a mass spectrometry-based analysis of the phosphotyrosine network downstream of site-specific EGFRvIII mutants, enabling quantification of network-level effects of site-specific point mutations. Mutation at Y845, Y1068 or Y1148 resulted in diminished receptor phosphorylation, while mutation at Y1173 led to increased phosphorylation on multiple EGFRvIII residues. Altered phosphorylation at the receptor was recapitulated in downstream signaling network activation levels, with Y1173F mutation leading to increased phosphorylation throughout the network.

Quantifying oncogenic phosphotyrosine signaling networks through systems biology.

Pathways linking oncogenic mutations to increased proliferative or migratory capacity are poorly characterized, yet provide potential targets for therapeutic intervention. As tyrosine phosphorylation signaling networks are known to mediate proliferation and migration, and frequently go awry in cancers, a comprehensive understanding of these networks in normal and diseased states is warranted. To this end, recent advances in mass spectrometry, protein microarrays, and computational algorithms provide insight into various aspects of the network including phosphotyrosine identification, analysis of kinase/phosphatase substrates, and phosphorylation-mediated protein-protein interactions.