Publications by authors named "Brett Larsen"

37 Publications

Location of Traumatic Cranial Epidural Hematoma Correlates with the Source of Hemorrhage: A 12-Year Surgical Review.

World Neurosurg 2021 May 24. Epub 2021 May 24.

Department of Surgery, Division of Neurosurgery, Creighton University, School of Medicine Phoenix, Phoenix, Arizona, USA; Valleywise Health Medical Center, Phoenix, Arizona, USA; The University of Arizona, College of Medicine Phoenix, Phoenix, Arizona, USA. Electronic address:

Background: Epidural hematoma (EDH) can result in a catastrophic outcome of traumatic brain injury. Current management guidelines do not consider the source of hemorrhage in decision making. The purpose of this study was to examine the relationship between EDH location and the source of hemorrhage.

Methods: We report retrospectively reviewed, prospectively obtained surgical data of patients with acute traumatic cranial EDH treated between 2007 and 2018. Computed tomography (CT) scans were used to categorize EDH location as lateral or medial. The source of hemorrhage was identified intraoperatively by a single surgeon.

Results: Overall, of 92 evacuated EDHs (in 87 patients), 71 (77.2%) were in the lateral location. Arterial bleeding was the cause of EDH in 63.4% of the lateral EDHs and 9.2% of the medial EDHs (P < 0.0001). In the cases where surgery was done primarily to treat EDH, 65.3% had an arterial bleed source (P < 0.0001). In those treated for primary reasons other than EDH evacuation, 75% had a venous bleed source (P = 0.002).

Conclusions: The location of EDH correlates with the source of hemorrhage. The decision to operate on EDH may be influenced by this factor.
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http://dx.doi.org/10.1016/j.wneu.2021.05.052DOI Listing
May 2021

Comprehensive interactome profiling of the human Hsp70 network highlights functional differentiation of J domains.

Mol Cell 2021 Apr 27. Epub 2021 Apr 27.

Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada. Electronic address:

Hsp70s comprise a deeply conserved chaperone family that has a central role in maintaining protein homeostasis. In humans, Hsp70 client specificity is provided by 49 different co-factors known as J domain proteins (JDPs). However, the cellular function and client specificity of JDPs have largely remained elusive. We have combined affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID) to characterize the interactome of all human JDPs and Hsp70s. The resulting network suggests specific functions for many uncharacterized JDPs, and we establish a role of conserved JDPs DNAJC9 and DNAJC27 in histone chaperoning and ciliogenesis, respectively. Unexpectedly, we find that the J domain of DNAJC27 but not of other JDPs can fully replace the function of endogenous DNAJC27, suggesting a previously unappreciated role for J domains themselves in JDP specificity. More broadly, our work expands the role of the Hsp70-regulated proteostasis network and provides a platform for further discovery of JDP-dependent functions.
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http://dx.doi.org/10.1016/j.molcel.2021.04.012DOI Listing
April 2021

Functional characterization of a PROTAC directed against BRAF mutant V600E.

Nat Chem Biol 2020 11 10;16(11):1170-1178. Epub 2020 Aug 10.

Institute for Research in Immunology and Cancer, Laboratory of Intracellular Signaling, Université de Montréal, Quebec, Montreal, Canada.

The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.
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http://dx.doi.org/10.1038/s41589-020-0609-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862923PMC
November 2020

Variability in Streptavidin-Sepharose Matrix Quality Can Significantly Affect Proximity-Dependent Biotinylation (BioID) Data.

J Proteome Res 2020 08 20;19(8):3554-3561. Epub 2020 Jul 20.

Princess Margaret Cancer Centre, University Health Network, University of Toronto, 101 College Street, Toronto, ON M5G 1L7, Canada.

Due to their ease of use and high binding affinity, streptavidin-based purification tools have become widely used for isolating biotinylated compounds from complex mixtures. We and others routinely use streptavidin-sepharose matrices to isolate biotinylated polypeptides generated in proximity-dependent biotinylation approaches, such as BioID or APEX. However, we noted sporadic, substantial variation in the quality of BioID experiments performed in the same laboratories over time, using seemingly identical protocols. Identifying the source of this problem, here, we highlight considerable variability in streptavidin contamination derived from different production lots of streptavidin-sepharose beads from the same manufacturer and demonstrate that high levels of streptavidin peptide contamination can have detrimental effects on BioID data. We also describe two simple, rapid approaches to assess the degree of streptavidin "shedding" from individual lots of the sepharose matrix before use to avoid the use of lower quality reagent.
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http://dx.doi.org/10.1021/acs.jproteome.0c00117DOI Listing
August 2020

A novel role for NUAK1 in promoting ovarian cancer metastasis through regulation of fibronectin production in spheroids.

Cancers (Basel) 2020 May 15;12(5). Epub 2020 May 15.

The Mary & John Knight Translational Ovarian Cancer Research Unit, London Regional Cancer Program, London, ON N6A 4L6, Canada.

Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where cells shed from the primary tumour, form aggregates called spheroids to evade anoikis, spread through the peritoneal cavity, and adhere to secondary sites. We previously showed that the master kinase Liver kinase B1 (LKB1) is required for EOC spheroid viability and metastasis. We have identified novel (nua) kinase 1 (NUAK1) as a top candidate LKB1 substrate in EOC cells and spheroids using a multiplex inhibitor beads-mass spectrometry approach. We confirmed that LKB1 maintains NUAK1 phosphorylation and promotes its stabilization. We next investigated NUAK1 function in EOC cells. Ectopic NUAK1-overexpressing EOC cell lines had increased adhesion, whereas the reverse was seen in OVCAR8-KO cells. In fact, cells with NUAK1 loss generate spheroids with reduced integrity, leading to increased cell death after long-term culture. Following transcriptome analysis, we identified reduced enrichment for cell interaction gene expression pathways in OVCAR8-KO spheroids. In fact, the gene, encoding fibronectin, exhibited a 745-fold decreased expression in KO spheroids. Fibronectin expression was induced during native spheroid formation, yet this was completely lost in KO spheroids. Co-incubation with soluble fibronectin restored the compact spheroid phenotype to OVCAR8-KO cells. In a xenograft model of intraperitoneal metastasis, NUAK1 loss extended survival and reduced fibronectin expression in tumours. Thus, we have identified a new mechanism controlling EOC metastasis, through which LKB1-NUAK1 activity promotes spheroid formation and secondary tumours via fibronectin production.
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http://dx.doi.org/10.3390/cancers12051250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280971PMC
May 2020

Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions.

Nat Cell Biol 2020 04 23;22(4):498-511. Epub 2020 Mar 23.

Institute of Cancer Research, Chester Beatty Laboratories, London, UK.

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.
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http://dx.doi.org/10.1038/s41556-020-0488-xDOI Listing
April 2020

Mechanisms of cannabinoid CB receptor-mediated reduction of dopamine neuronal excitability in mouse ventral tegmental area.

EBioMedicine 2019 Apr 3;42:225-237. Epub 2019 Apr 3.

Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders, Shandong Provincial Collaborative Innovation Center for Neurodegenerative Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao 266071, China; Department of Neurobiology, St. Joseph's Hospital and Medical Center, Barrow Neurological Institute, Phoenix, AZ 85013, USA; Department of Pharmacology, Shantou University Medical College, Shantou, Guangdong 210854, China; Department of Neurology, Yunfu People's Hospital, Yunfu, Guangdong 527300, China. Electronic address:

Background: We have recently reported that activation of cannabinoid type 2 receptors (CBRs) reduces dopamine (DA) neuron excitability in mouse ventral tegmental area (VTA). Here, we elucidate the underlying mechanisms.

Methods: Patch-clamp recordings were performed in mouse VTA slices and dissociated single VTA DA neurons.

Findings: Using cell-attached recording in VTA slices, bath-application of CBR agonists (JWH133 or five other CBR agonists) significantly reduced VTA DA neuron action potential (AP) firing rate. Under the patch-clamp whole-cell recording model, JWH133 (10 μM) mildly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) but not miniature inhibitory postsynaptic currents (mIPSCs). JWH133 also did not alter evoked EPSCs or IPSCs. In freshly dissociated VTA DA neurons, JWH133 reduced AP firing rate, delayed AP initiation and enhanced AP after-hyperpolarization. In voltage-clamp recordings, JWH133 (1 μM) enhanced M-type K currents and this effect was absent in CB mice and abolished by co-administration of a selective CBR antagonist (10 μM, AM630). CBR-mediated inhibition in VTA DA neuron firing can be mimicked by M-current opener (10 μM retigabine) and blocked by M-current blocker (30 μM XE991). In addition, enhancement of neuronal cAMP by forskolin (10 μM) reduced M-current and increased DA neuron firing rate. Finally, pharmacological block of synaptic transmission by NBQX (10 μM), D-APV (50 μM) and picrotoxin (100 μM) in VTA slices failed to prevent CBR-mediated inhibition, while intracellular infusion of guanosine 5'-O-2-thiodiphosphate (600 μM, GDP-β-S) through recording electrode to block postsynaptic G-protein function prevented JWH133-induced reduction in AP firing.

Interpretation: Our results suggest that CBRs modulate VTA DA neuron excitability mainly through an intrinsic mechanism, including a CBR-mediated reduction of intracellular cAMP, and in turn enhancement of M-type K currents. FUND: This research was supported by the Barrow Neuroscience Foundation, the BNI-BMS Seed Fund, and CNSF (81771437).
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http://dx.doi.org/10.1016/j.ebiom.2019.03.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6491419PMC
April 2019

Retrospective Review of Clinical and Chest X-Ray Findings in Children Admitted to a Community Hospital for Respiratory Syncytial Virus Infection.

Clin Pediatr (Phila) 2018 12 3;57(14):1686-1692. Epub 2018 Sep 3.

1 Maricopa Medical Center, Phoenix, AZ, USA.

Introduction: We performed a retrospective study to evaluate demographics, clinical course, outcome, and radiological findings of children with respiratory syncytial virus (RSV) infection.

Methods: Four hundred patients admitted between October 2013 and May 2016 were enrolled. Clinical and radiographic trends were evaluated for association with severity of RSV presentation. Severity was defined as hospitalization >2 days, pediatric intensive care unit admission, or need for mechanical ventilation.

Results: Common clinical findings included fever (78.5%), coughing (97%), rhinorrhea/congestion (93%), and hypoxia (44.8%). Hypoxia was seen in 64.7% of the severe group compared with 32.0% in the nonsevere group ( P < .001). Airspace opacification was seen in 49.2% of chest X-rays of the severe group compared with 26.4% in the nonsevere group ( P < .001).

Conclusion: Higher incidence of hypoxia or airspace opacification on chest X-ray may be predictors of poorer outcomes for patients with RSV infection.
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http://dx.doi.org/10.1177/0009922818795902DOI Listing
December 2018

Mortality is predicted by Comorbidity Polypharmacy score but not Charlson Comorbidity Index in geriatric trauma patients.

Am J Surg 2018 07 19;216(1):42-45. Epub 2017 Sep 19.

Department of Surgery, Nassau University Medical Center, East Meadow, NY, USA. Electronic address:

Background: Increased life expectancy has resulted in more older patients at trauma centers. Traditional assessments of injuries alone may not be sufficient; age, comorbidities, and medications should be considered.

Methods: 446 older trauma patients were analyzed in two groups, 45-65 years and <65, using Injury Severity Score (ISS), the Charlson Comorbidity Index (CCI), and Comorbidity-Polypharmacy Score (CPS).

Results: CCI and CPS were associated with HLOS in patients <65. In patients aged 45-65, only CPS was associated with HLOS. CPS was inversely associated with in-hospital mortality in patients <65, but not patients aged 45-65. CCI score was not associated with in-hospital mortality in either group.

Conclusion: Increased CCI and CPS were associated with increased HLOS. In patients over 65, increased CPS was associated with decreased mortality. This could be due to return toward physiologic normalcy in treated patients not seen in their peers with undiagnosed or untreated comorbidities.
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http://dx.doi.org/10.1016/j.amjsurg.2017.09.011DOI Listing
July 2018

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry.

Nat Commun 2017 08 21;8(1):291. Epub 2017 Aug 21.

Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093, Zurich, Switzerland.

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.SWATH-mass spectrometry consists of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics on the scale of thousands of proteins. Here, using data generated by eleven groups worldwide, the authors show that SWATH-MS is capable of generating highly reproducible data across different laboratories.
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http://dx.doi.org/10.1038/s41467-017-00249-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566333PMC
August 2017

Neprosin, a Selective Prolyl Endoprotease for Bottom-up Proteomics and Histone Mapping.

Mol Cell Proteomics 2017 06 12;16(6):1162-1171. Epub 2017 Apr 12.

From the ‡Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N4N1, Canada;

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.
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http://dx.doi.org/10.1074/mcp.M116.066803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461545PMC
June 2017

Data Independent Acquisition analysis in ProHits 4.0.

J Proteomics 2016 10 29;149:64-68. Epub 2016 Apr 29.

Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. Electronic address:

Affinity purification coupled with mass spectrometry (AP-MS) is a powerful technique for the identification and quantification of physical interactions. AP-MS requires careful experimental design, appropriate control selection and quantitative workflows to successfully identify bona fide interactors amongst a large background of contaminants. We previously introduced ProHits, a Laboratory Information Management System for interaction proteomics, which tracks all samples in a mass spectrometry facility, initiates database searches and provides visualization tools for spectral counting-based AP-MS approaches. More recently, we implemented Significance Analysis of INTeractome (SAINT) within ProHits to provide scoring of interactions based on spectral counts. Here, we provide an update to ProHits to support Data Independent Acquisition (DIA) with identification software (DIA-Umpire and MSPLIT-DIA), quantification tools (through DIA-Umpire, or externally via targeted extraction), and assessment of quantitative enrichment (through mapDIA) and scoring of interactions (through SAINT-intensity). With additional improvements, notably support of the iProphet pipeline, facilitated deposition into ProteomeXchange repositories and enhanced export and viewing functions, ProHits 4.0 offers a comprehensive suite of tools to facilitate affinity proteomics studies.

Significance: It remains challenging to score, annotate and analyze proteomics data in a transparent manner. ProHits was previously introduced as a LIMS to enable storing, tracking and analysis of standard AP-MS data. In this revised version, we expand ProHits to include integration with a number of identification and quantification tools based on Data-Independent Acquisition (DIA). ProHits 4.0 also facilitates data deposition into public repositories, and the transfer of data to new visualization tools.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079801PMC
http://dx.doi.org/10.1016/j.jprot.2016.04.042DOI Listing
October 2016

MSPLIT-DIA: sensitive peptide identification for data-independent acquisition.

Nat Methods 2015 Dec;12(12):1106-8

Center for Computational Mass Spectrometry, University of California, San Diego, La Jolla, California, USA.

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http://dx.doi.org/10.1038/nmeth.3655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857761PMC
December 2015

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Nat Methods 2015 Aug 29;12(8):725-31. Epub 2015 Jun 29.

Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.
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http://dx.doi.org/10.1038/nmeth.3472DOI Listing
August 2015

DIA-Umpire: comprehensive computational framework for data-independent acquisition proteomics.

Nat Methods 2015 Mar 19;12(3):258-64, 7 p following 264. Epub 2015 Jan 19.

1] Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA. [2] Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.

As a result of recent improvements in mass spectrometry (MS), there is increased interest in data-independent acquisition (DIA) strategies in which all peptides are systematically fragmented using wide mass-isolation windows ('multiplex fragmentation'). DIA-Umpire (http://diaumpire.sourceforge.net/), a comprehensive computational workflow and open-source software for DIA data, detects precursor and fragment chromatographic features and assembles them into pseudo-tandem MS spectra. These spectra can be identified with conventional database-searching and protein-inference tools, allowing sensitive, untargeted analysis of DIA data without the need for a spectral library. Quantification is done with both precursor- and fragment-ion intensities. Furthermore, DIA-Umpire enables targeted extraction of quantitative information based on peptides initially identified in only a subset of the samples, resulting in more consistent quantification across multiple samples. We demonstrated the performance of the method with control samples of varying complexity and publicly available glycoproteomics and affinity purification-MS data.
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http://dx.doi.org/10.1038/nmeth.3255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399776PMC
March 2015

Albumin decrease is associated with spontaneous preterm delivery within 48 h in women with threatened preterm labor.

J Proteome Res 2015 Jan 9;14(1):457-66. Epub 2014 Oct 9.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital , 25 Orde Street, 6-1001, Toronto, ON M5T 3H7, Canada.

Threatened preterm labor (TPTL) accounts for ∼30% of pregnancy-related hospital admissions. Maternal peripheral leukocytes can be used to monitor a variety of physiological processes occurring in the body. Two high-throughput mass spectrometry methodologies, SWATH and iTRAQ, were used to study differentially expressed peripheral blood leukocyte lysate proteins in symptomatic women admitted for TPTL who had a preterm birth within 48 h (n = 16) and those who did not (n = 24). The SWATH spectral library consisted of 783 proteins. SWATH methodology quantified 258 proteins (using ≥2 peptides) and 5 proteins (ALBU, ANXA6, HNRPK, HSP90A, and PDIA1) were differentially expressed (p < 0.05, Mann-Whitney U). iTRAQ workflow identified 765 proteins; 354 proteins were quantified and 14 proteins (MIF, UBIQ, HXK3, ALBU, HNRPD, ST1A2, RS15A, RAP1B, CAN1, IQGA2, ST1A1, COX5A, ADDA, and UBQL1) were significantly different between the two groups of women (p < 0.05, Mann-Whitney U). Albumin was the only common differentially expressed protein in both SWATH (28% decrease) and iTRAQ studies (45% decrease). This decrease in albumin was validated using ELISA (11% decrease, p < 0.05, Mann-Whitney U) in another 23 TPTL women. This work suggests that albumin is a broad indicator of leukocyte activation with impending preterm birth and provides new future work directions to understand the pathophysiology of TPTL.
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http://dx.doi.org/10.1021/pr500852pDOI Listing
January 2015

Glucagon-like peptide-1 receptor agonists increase pancreatic mass by induction of protein synthesis.

Diabetes 2015 Mar 2;64(3):1046-56. Epub 2014 Oct 2.

Department of Medicine, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

Glucagon-like peptide-1 (GLP-1) controls glucose homeostasis by regulating secretion of insulin and glucagon through a single GLP-1 receptor (GLP-1R). GLP-1R agonists also increase pancreatic weight in some preclinical studies through poorly understood mechanisms. Here we demonstrate that the increase in pancreatic weight following activation of GLP-1R signaling in mice reflects an increase in acinar cell mass, without changes in ductal compartments or β-cell mass. GLP-1R agonists did not increase pancreatic DNA content or the number of Ki67(+) cells in the exocrine compartment; however, pancreatic protein content was increased in mice treated with exendin-4 or liraglutide. The increased pancreatic mass and protein content was independent of cholecystokinin receptors, associated with a rapid increase in S6 phosphorylation, and mediated through the GLP-1R. Rapamycin abrogated the GLP-1R-dependent increase in pancreatic mass but had no effect on the robust induction of Reg3α and Reg3β gene expression. Mass spectrometry analysis identified GLP-1R-dependent upregulation of Reg family members, as well as proteins important for translation and export, including Fam129a, eIF4a1, Wars, and Dmbt1. Hence, pharmacological GLP-1R activation induces protein synthesis, leading to increased pancreatic mass, independent of changes in DNA content or cell proliferation in mice.
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http://dx.doi.org/10.2337/db14-0883DOI Listing
March 2015

A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways.

Cell 2014 Jul;158(2):434-448

Whitehead Institute for Biomedical Research, Cambridge, MA 02114, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Cambridge, MA 02139, USA. Electronic address:

Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement.
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http://dx.doi.org/10.1016/j.cell.2014.05.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104544PMC
July 2014

BDNF signaling in the VTA links the drug-dependent state to drug withdrawal aversions.

J Neurosci 2014 Jun;34(23):7899-909

Department of Molecular Genetics, Neurobiology Research Group, University of Toronto, Toronto, Ontario, M5S3E1 Canada, Department of Medical Biophysics, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, M5S3E1 Canada.

Drug administration to avoid unpleasant drug withdrawal symptoms has been hypothesized to be a crucial factor that leads to compulsive drug-taking behavior. However, the neural relationship between the aversive motivational state produced by drug withdrawal and the development of the drug-dependent state still remains elusive. It has been observed that chronic exposure to drugs of abuse increases brain-derived neurotrophic factor (BDNF) levels in ventral tegmental area (VTA) neurons. In particular, BDNF expression is dramatically increased during drug withdrawal, which would suggest a direct connection between the aversive state of withdrawal and BDNF-induced neuronal plasticity. Using lentivirus-mediated gene transfer to locally knock down the expression of the BDNF receptor tropomyosin-receptor-kinase type B in rats and mice, we observed that chronic opiate administration activates BDNF-related neuronal plasticity in the VTA that is necessary for both the establishment of an opiate-dependent state and aversive withdrawal motivation. Our findings highlight the importance of a bivalent, plastic mechanism that drives the negative reinforcement underlying addiction.
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http://dx.doi.org/10.1523/JNEUROSCI.3776-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4099491PMC
June 2014

Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition.

Nat Methods 2013 Dec 27;10(12):1239-45. Epub 2013 Oct 27.

1] Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. [2].

Characterizing changes in protein-protein interactions associated with sequence variants (e.g., disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies in which cost and time are major considerations. We have coupled AP to data-independent mass spectrometric acquisition (sequential window acquisition of all theoretical spectra, SWATH) and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. We used AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes and propose a scalable pipeline for systems biology studies.
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http://dx.doi.org/10.1038/nmeth.2702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3882083PMC
December 2013

Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions.

Proteomics 2013 Apr 12;13(8):1334-8. Epub 2013 Mar 12.

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

Gangliosides are ubiquitous components of cell membranes. Their interactions with bacterial toxins and membrane-associated proteins (e.g. receptor tyrosine kinases) have important roles in the regulation of multiple cellular functions. Currently, an effective approach for measuring ganglioside-protein interactions especially in a large-scale fashion is largely missing. To this end, we report a facile MS-based approach to explore gangliosides extracted from cells and measure their interactions with protein of interest globally. We optimized a two-step protocol for extracting total gangliosides from cells within 2 h. Easy-to-use magnetic beads conjugated with a protein of interest were used to capture interacting gangliosides. To measure ganglioside-protein interaction on a global scale, we applied a high-sensitive LC-MS system, containing hydrophilic interaction LC separation and multiple reaction monitoring-based MS for ganglioside detection. Sensitivity for ganglioside GM1 is below 100 pg, and the whole analysis can be done in 20 min with isocratic elution. To measure ganglioside interactions with soluble vascular endothelial growth factor receptor 1 (sFlt1), we extracted and readily detected 36 species of gangliosides from perivascular retinal pigment epithelium cells across eight different classes. Twenty-three ganglioside species have significant interactions with sFlt1 as compared with IgG control based on p value cutoff <0.05. These results show that the described method provides a rapid and high-sensitive approach for systematically measuring ganglioside-protein interactions.
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http://dx.doi.org/10.1002/pmic.201200410DOI Listing
April 2013

Label-free quantitative proteomics trends for protein-protein interactions.

J Proteomics 2013 Apr 12;81:91-101. Epub 2012 Nov 12.

AB Sciex, 71 Four Valley Drive, Concord, Ontario, Canada.

Understanding protein interactions within the complexity of a living cell is challenging, but techniques coupling affinity purification and mass spectrometry have enabled important progress to be made in the past 15 years. As identification of protein-protein interactions is becoming easier, the quantification of the interaction dynamics is the next frontier. Several quantitative mass spectrometric approaches have been developed to address this issue that vary in their strengths and weaknesses. While isotopic labeling approaches continue to contribute to the identification of regulated interactions, techniques that do not require labeling are becoming increasingly used in the field. Here, we describe the major types of label-free quantification used in interaction proteomics, and discuss the relative merits of data dependent and data independent acquisition approaches in label-free quantification. This article is part of a Special Issue entitled: From protein structures to clinical applications.
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http://dx.doi.org/10.1016/j.jprot.2012.10.027DOI Listing
April 2013

Using ProHits to store, annotate, and analyze affinity purification-mass spectrometry (AP-MS) data.

Curr Protoc Bioinformatics 2012 Sep;Chapter 8:Unit8.16

Centre for Systems Biology, Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario, Canada.

Affinity purification coupled with mass spectrometry (AP-MS) is a robust technique used to identify protein-protein interactions. With recent improvements in sample preparation, and dramatic advances in MS instrumentation speed and sensitivity, this technique is becoming more widely used throughout the scientific community. To meet the needs of research groups both large and small, we have developed software solutions for tracking, scoring and analyzing AP-MS data. Here, we provide details for the installation and utilization of ProHits, a Laboratory Information Management System designed specifically for AP-MS interaction proteomics. This protocol explains: (i) how to install the complete ProHits system, including modules for the management of mass spectrometry files and the analysis of interaction data, and (ii) alternative options for the use of pre-existing search results in simpler versions of ProHits, including a virtual machine implementation of our ProHits Lite software. We also describe how to use the main features of the software to analyze AP-MS data.
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http://dx.doi.org/10.1002/0471250953.bi0816s39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3669397PMC
September 2012

Novel NEDD1 phosphorylation sites regulate γ-tubulin binding and mitotic spindle assembly.

J Cell Sci 2012 Aug 17;125(Pt 16):3745-51. Epub 2012 May 17.

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.

During cell division, microtubules organize a bipolar spindle to drive accurate chromosome segregation to daughter cells. Microtubules are nucleated by the γ-TuRC, a γ-tubulin complex that acts as a template for microtubules with 13 protofilaments. Cells lacking γ-TuRC core components do nucleate microtubules; however, these polymers fail to form bipolar spindles. NEDD1 is a γ-TuRC-interacting protein whose depletion, although not affecting γ-TuRC stability, causes spindle defects similar to the inhibition of its core subunits, including γ-tubulin. Several residues of NEDD1 are phosphorylated in mitosis. However, previously identified phosphorylation sites only partially regulate NEDD1 function, as NEDD1 depletion has a much stronger phenotype than mutation of these residues. Using mass spectrometry, we have identified multiple novel phosphorylated sites in the serine (S)557-S574 region of NEDD1, close to its γ-tubulin-binding domain. Serine to alanine mutations in S565-S574 inhibit the binding of NEDD1 to γ-tubulin and perturb NEDD1 mitotic function, yielding microtubule organization defects equivalent to those observed in NEDD1-depleted cells. Interestingly, additional mutations in the S557-T560 region restore the capacity of NEDD1 to bind γ-tubulin and promote bipolar spindle assembly. All together, our data suggest that the NEDD1/γ-tubulin interaction is finely tuned by multiple phosphorylation events in the S557-S574 region and is critical for spindle assembly. We also found that CEP192, a centrosomal protein similarly required for spindle formation, associates with NEDD1 and modulates its mitotic phosphorylation. Thus CEP192 may regulate spindle assembly by modulating NEDD1 function.
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http://dx.doi.org/10.1242/jcs.105130DOI Listing
August 2012

A cost-benefit analysis of multidimensional fractionation of affinity purification-mass spectrometry samples.

Proteomics 2011 Jul 1;11(13):2603-12. Epub 2011 Jun 1.

Centre for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada.

Affinity purification coupled to mass spectrometry (AP-MS) is gaining widespread use for the identification of protein-protein interactions. It is unclear, however, whether typical AP sample complexity is limiting for the identification of all protein components using standard one-dimensional LC-MS/MS. Multidimensional sample separation is useful for reducing sample complexity prior to MS analysis and increases peptide and protein coverage of complex samples. Here, we monitored the effects of upstream protein or peptide separation techniques on typical mammalian AP-MS samples, generated by FLAG affinity purification of four baits with different biological functions and/or subcellular distribution. As a first separation step, we employed SDS-PAGE, strong cation exchange LC, or reversed-phase LC at basic pH. We also analyzed the benefits of using an instrument with a faster scan rate, the new TripleTOF 5600 mass spectrometer. While all multidimensional approaches yielded a clear increase in spectral counts, the increase in unique peptides and additional protein identification was modest and came at the cost of increased instrument and handling time. The use of a high duty-cycle instrument achieved similar benefits without these drawbacks. An increase in spectral counts is beneficial when data analysis methods relying on spectral counts, including Significance Analysis of INTeractome (SAINT), are used.
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http://dx.doi.org/10.1002/pmic.201000571DOI Listing
July 2011

Structure-function analysis of core STRIPAK Proteins: a signaling complex implicated in Golgi polarization.

J Biol Chem 2011 Jul 11;286(28):25065-75. Epub 2011 May 11.

Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.

Cerebral cavernous malformations (CCMs) are alterations in brain capillary architecture that can result in neurological deficits, seizures, or stroke. We recently demonstrated that CCM3, a protein mutated in familial CCMs, resides predominantly within the STRIPAK complex (striatin interacting phosphatase and kinase). Along with CCM3, STRIPAK contains the Ser/Thr phosphatase PP2A. The PP2A holoenzyme consists of a core catalytic subunit along with variable scaffolding and regulatory subunits. Within STRIPAK, striatin family members act as PP2A regulatory subunits. STRIPAK also contains all three members of a subfamily of Sterile 20 kinases called the GCKIII proteins (MST4, STK24, and STK25). Here, we report that striatins and CCM3 bridge the phosphatase and kinase components of STRIPAK and map the interacting regions on each protein. We show that striatins and CCM3 regulate the Golgi localization of MST4 in an opposite manner. Consistent with a previously described function for MST4 and CCM3 in Golgi positioning, depletion of CCM3 or striatins affects Golgi polarization, also in an opposite manner. We propose that STRIPAK regulates the balance between MST4 localization at the Golgi and in the cytosol to control Golgi positioning.
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http://dx.doi.org/10.1074/jbc.M110.214486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3137080PMC
July 2011

The Crumbs complex couples cell density sensing to Hippo-dependent control of the TGF-β-SMAD pathway.

Dev Cell 2010 Dec;19(6):831-44

Center for Systems Biology, Mount Sinai Hospital, Toronto, ON, Canada.

The Hippo pathway senses cell density information to control tissue growth by regulating the localization of the transcriptional regulators TAZ and YAP (TAZ/YAP). TAZ/YAP also regulate TGF-β-SMAD signaling, but whether this role is linked to cell density sensing is unknown. Here we demonstrate that TAZ/YAP dictate the localization of active SMAD complexes in response to cell density-mediated formation of polarity complexes. In high-density cell cultures, the Hippo pathway drives cytoplasmic localization of TAZ/YAP, which sequesters SMAD complexes, thereby suppressing TGF-β signaling. We show that during mouse embryogenesis, this is reflected by differences in TAZ/YAP localization, which define regions of active SMAD2/3 complexes. Interfering with TAZ/YAP phosphorylation drives nuclear accumulation of TAZ/YAP and SMAD2/3. Furthermore, we demonstrate that the Crumbs polarity complex interacts with TAZ/YAP, which relays cell density information by promoting TAZ/YAP phosphorylation, cytoplasmic retention, and suppressed TGF-β signaling. Accordingly, disruption of the Crumbs complex enhances TGF-β signaling and predisposes cells to TGF-β-mediated epithelial-to-mesenchymal transitions.
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http://dx.doi.org/10.1016/j.devcel.2010.11.012DOI Listing
December 2010

SAINT: probabilistic scoring of affinity purification-mass spectrometry data.

Nat Methods 2011 Jan 5;8(1):70-3. Epub 2010 Dec 5.

Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.

We present 'significance analysis of interactome' (SAINT), a computational tool that assigns confidence scores to protein-protein interaction data generated using affinity purification-mass spectrometry (AP-MS). The method uses label-free quantitative data and constructs separate distributions for true and false interactions to derive the probability of a bona fide protein-protein interaction. We show that SAINT is applicable to data of different scales and protein connectivity and allows transparent analysis of AP-MS data.
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http://dx.doi.org/10.1038/nmeth.1541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064265PMC
January 2011

SCFCdc4 enables mating type switching in yeast by cyclin-dependent kinase-mediated elimination of the Ash1 transcriptional repressor.

Mol Cell Biol 2011 Feb 22;31(3):584-98. Epub 2010 Nov 22.

School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, United Kingdom.

In the budding yeast Saccharomyces cerevisiae, mother cells switch mating types between a and α forms, whereas daughter cells do not. This developmental asymmetry arises because the expression of the HO endonuclease, which initiates the interconversion of a and α mating type cassettes, is extinguished by the daughter-specific Ash1 transcriptional repressor. When daughters become mothers in the subsequent cell cycle, Ash1 must be eliminated to enable a new developmental state. Here, we report that the ubiquitin ligase SCF(Cdc4) mediates the phosphorylation-dependent elimination of Ash1. The inactivation of SCF(Cdc4) stabilizes Ash1 in vivo, and consistently, Ash1 binds to and is ubiquitinated by SCF(Cdc4) in a phosphorylation-dependent manner in vitro. The mutation of a critical in vivo cyclin-dependent kinase (CDK) phosphorylation site (Thr290) on Ash1 reduces its ubiquitination and rate of degradation in vivo and decreases the frequency of mating type switching. Ash1 associates with active Cdc28 kinase in vivo and is targeted to SCF(Cdc4) in a Cdc28-dependent fashion in vivo and in vitro. Ash1 recognition by Cdc4 appears to be mediated by at least three phosphorylation sites that form two redundant diphosphorylated degrons. The phosphorylation-dependent elimination of Ash1 by the ubiquitin-proteasome system thus underpins developmental asymmetry in budding yeast.
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http://dx.doi.org/10.1128/MCB.00845-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3028614PMC
February 2011