UniDec Processing Pipeline for Rapid Analysis of Biotherapeutic Mass Spectrometry Data.

Recent advances in native mass spectrometry (MS) and denatured intact protein MS have made these techniques essential for biotherapeutic characterization. As MS analysis has increased in throughput and scale, new data analysis workflows are needed to provide rapid quantitation from large datasets. Here, we describe the UniDec processing pipeline (UPP) for the analysis of batched biotherapeutic intact MS data.

Complement C1q-dependent excitatory and inhibitory synapse elimination by astrocytes and microglia in Alzheimer's disease mouse models.

Microglia and complement can mediate neurodegeneration in Alzheimer's disease (AD). By integrative multi-omics analysis, here we show that astrocytic and microglial proteins are increased in Tau synapse fractions with age and in a C1q-dependent manner. In addition to microglia, we identified that astrocytes contribute substantially to synapse elimination in Tau hippocampi.

Discovery of a caspase cleavage motif antibody reveals insights into noncanonical inflammasome function.

Inflammasomes sense a number of pathogen and host damage signals to initiate a signaling cascade that triggers inflammatory cell death, termed pyroptosis. The inflammatory caspases (1/4/5/11) are the key effectors of this process through cleavage and activation of the pore-forming protein gasdermin D. Caspase-1 also activates proinflammatory interleukins, IL-1β and IL-18, via proteolysis.

Recurrent Loss of NFE2L2 Exon 2 Is a Mechanism for Nrf2 Pathway Activation in Human Cancers.

The Nrf2 pathway is frequently activated in human cancers through mutations in Nrf2 or its negative regulator KEAP1. Using a cell-line-derived gene signature for Nrf2 pathway activation, we found that some tumors show high Nrf2 activity in the absence of known mutations in the pathway. An analysis of splice variants in oncogenes revealed that such tumors express abnormal transcript variants from the NFE2L2 gene (encoding Nrf2) that lack exon 2, or exons 2 and 3, and encode Nrf2 protein isoforms missing the KEAP1 interaction domain.

A Biologist's Field Guide to Multiplexed Quantitative Proteomics.

High-throughput genomic and proteomic studies have generated near-comprehensive catalogs of biological constituents within many model systems. Nevertheless, static catalogs are often insufficient to fully describe the dynamic processes that drive biology. Quantitative proteomic techniques address this need by providing insight into closely related biological states such as the stages of a therapeutic response or cellular differentiation.

Ubiquitination profiling identifies sensitivity factors for IAP antagonist treatment.

Evasion of cell death is one crucial capability acquired by tumour cells to ward-off anti-tumour therapies and represents a fundamental challenge to sustaining clinical efficacy for currently available agents. Inhibitor of apoptosis (IAP) proteins use their ubiquitin E3 ligase activity to promote cancer cell survival by mediating proliferative signalling and blocking cell death in response to diverse stimuli. Using immunoaffinity enrichment and MS, ubiquitination sites on thousands of proteins were profiled upon initiation of cell death by IAP antagonists in IAP antagonist-sensitive and -resistant breast cancer cell lines.

The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy.

Cells maintain healthy mitochondria by degrading damaged mitochondria through mitophagy; defective mitophagy is linked to Parkinson's disease. Here we report that USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy driven by the ubiquitin ligase parkin (also known as PARK2) and protein kinase PINK1, which are encoded by two genes associated with Parkinson's disease. Parkin ubiquitinates and tags damaged mitochondria for clearance.

Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition.

Targeted therapeutics that block signal transduction through the RAS-RAF-MEK and PI3K-AKT-mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death.

Loss of the tumor suppressor BAP1 causes myeloid transformation.

De-ubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS).

Characterizing ubiquitination sites by peptide-based immunoaffinity enrichment.

Advances in high resolution tandem mass spectrometry and peptide enrichment technologies have transformed the field of protein biochemistry by enabling analysis of end points that have traditionally been inaccessible to molecular and biochemical techniques. One field benefitting from this research has been the study of ubiquitin, a 76-amino acid protein that functions as a covalent modifier of other proteins. Seminal work performed decades ago revealed that trypsin digestion of a branched protein structure known as A24 yielded an enigmatic diglycine signature bound to a lysine residue in histone 2A.

Complementary proteomic tools for the dissection of apoptotic proteolysis events.

Proteolysis is a key regulatory event that controls intracellular and extracellular signaling through irreversible changes in a protein's structure that greatly alters its function. Here we describe a platform for profiling caspase substrates which encompasses two highly complementary proteomic techniques--the first is a differential gel based approach termed Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) and the second involves affinity enrichment of peptides containing a C-terminal aspartic acid residue. In combination, these techniques have enabled the profiling of a large cellular pool of apoptotic-mediated proteolytic events across a wide dynamic range.

Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals.

Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub.

The impact of peptide abundance and dynamic range on stable-isotope-based quantitative proteomic analyses.

Recently, mass spectrometry has been employed in many studies to provide unbiased, reproducible, and quantitative protein abundance information on a proteome-wide scale. However, how instruments' limited dynamic ranges impact the accuracy of such measurements has remained largely unexplored, especially in the context of complex mixtures. Here, we examined the distribution of peptide signal versus background noise (S/N) and its correlation with quantitative accuracy.

A quantitative atlas of mitotic phosphorylation.

The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated.

Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.

Despite the success of tyrosine kinase-based cancer therapeutics, for most solid tumors the tyrosine kinases that drive disease remain unknown, limiting our ability to identify drug targets and predict response. Here we present the first large-scale survey of tyrosine kinase activity in lung cancer. Using a phosphoproteomic approach, we characterize tyrosine kinase signaling across 41 non-small cell lung cancer (NSCLC) cell lines and over 150 NSCLC tumors.

The effects of mass accuracy, data acquisition speed, and search algorithm choice on peptide identification rates in phosphoproteomics.

Proteomic analyses via tandem mass spectrometry have been greatly enhanced by the recent development of fast, highly accurate instrumentation. However, successful application of these developments to high-throughput experiments requires careful optimization of many variables which adversely affect each other, such as mass accuracy and data collection speed. We examined the performance of three shotgun-style acquisition methods ranging in their data collection speed and use of mass accuracy in identifying proteins from yeast-derived complex peptide and phosphopeptide-enriched mixtures.

ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins.

Catch-and-release reagents for broadscale quantitative proteomics analyses.

The relative quantification of protein expression levels in different cell samples through the utilization of stable isotope dilution has become a standard method in the field of proteomics. We describe here the development of a new reductively cleavable reagent which facilitates the relative quantification of thousands of proteins from only tens of micrograms of starting protein. The ligand features a novel disulfide moiety that links biotin and a thiol-reactive entity.

Enhanced analysis of metastatic prostate cancer using stable isotopes and high mass accuracy instrumentation.

The primary goal of proteomics is to gain a better understanding of biological function at the protein expression level. As the field matures, numerous technologies are being developed to aid in the identification, quantification and characterization of protein expression and post-translational modifications on a near-global scale. Stable isotope labeling by amino acids in cell culture is one such technique that has shown broad biological applications.

Optimization and use of peptide mass measurement accuracy in shotgun proteomics.

Mass spectrometers that provide high mass accuracy such as FT-ICR instruments are increasingly used in proteomic studies. Although the importance of accurately determined molecular masses for the identification of biomolecules is generally accepted, its role in the analysis of shotgun proteomic data has not been thoroughly studied. To gain insight into this role, we used a hybrid linear quadrupole ion trap/FT-ICR (LTQ FT) mass spectrometer for LC-MS/MS analysis of a highly complex peptide mixture derived from a fraction of the yeast proteome.

Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon.

ClpXP, a bacterial AAA+ protease, controls intracellular levels of many stress-response proteins. To investigate substrate profile changes caused by a specific environmental stress, quantitative mass spectrometry (SILAC) was used to analyze proteins trapped by ClpXP(trap) before and after DNA damage. The abundance of half of the trapped proteins changed more than 3-fold after damage.

A proteomic strategy for gaining insights into protein sumoylation in yeast.

Sumoylation represents a vital post-translational modification that pervades numerous aspects of cell biology, including protein targeting, transcriptional regulation, signal transduction, and cell division. However, despite its broad reaching effects, most biological outcomes of protein sumoylation remain poorly understood. In an effort to provide further insight into this complex process, a proteomics approach was undertaken to identify the targets of sumoylation en mass.