Biology subjects

Morin, G. B.

Publications and source records attributed to Morin, G. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Dependency Map correlation analysis reveals WDR89 as a genome maintenance factor

The Cancer Dependency map project (DepMap) has created an unprecedented resource of siRNA and CRISPR genetic screens across more than 1000 cell line models. Numerous computational tools have been developed to analyze DepMap outputs and have revealed new genetic dependencies relevant to cancer. Here we extracted correlation information from the DepMap using curated gene sets of biological pathways to determine if this approach could reveal new high confidence candidates. Beginning with a published set of curated DNA repair proteins we determined correlations of query gene knockout fitness across all DepMap cell lines. As expected, this approach extracted many known genome stability genes and also suggested several additional candidates not previously linked to genome maintenance. To validate our analysis we used siRNA depletion for candidate genes and identified several factors whose depletion increases DNA damage or delay repair. Among these was WDR89. WDR89 relocalizes to the nucleolus in an ATM dependent manner upon DNA damage and physically interacts with a number of nucleolar proteins. Depletion of WDR89 also abrogates nuclear p53 accumulation upon irradiation, suggesting a role in p53 signaling. These data establish WDR89 as a new genome stability regulator and highlight the value of DepMap for predicting previously unknown biology.

cell biology↗

bioRxiv2026

Hepatocyte growth factor activator inhibitor-2 rapidly inactivates airway-expressed human Type II Transmembrane Serine Proteases

Human Type II Transmembrane Serine Proteases (TTSPs) are essential entry factors for various influenza A and coronaviruses, and drive cancer metastasis when they are overexpressed by tumor cells. However, the natural inhibition mechanisms that regulate these proteases are not well understood. One natural transmembrane protease inhibitor, hepatocyte growth factor activator inhibitor-2 (HAI-2), has been shown to block TMPRSS2 activity and can prevent SARS-CoV-2 infection and reduce TMPRSS2-driven prostate cancer metastasis when overexpressed. In this study, we present biochemical and biophysical evidence showing that HAI-2 effectively inactivates TMPRSS2 and other TTSPs only after they have undergone zymogen activation. Through mutagenesis and ligand binding assays, we demonstrate that Kunitz Domain 1 (KD1) and KD2 can form stable ternary complexes with TMPRSS2 and other TTSPs, but do not employ the typical Laskowski inhibitor mechanism found for other macromolecular serine protease inhibitors. We also show that HAI-2 proteins do not inhibit the coagulation protease thrombin and that multivalent human IgG-tagged HAI-2 proteins are highly potent TMPRSS2 inhibitors. Our findings provide a mechanistic understanding of how TTSP activity is regulated in human airway cells and offer a foundation for developing engineered soluble HAI-2 proteins as anti-TTSP antivirals and anti-cancer therapeutics.

biochemistry↗

bioRxiv2025

Prognostically favorable immune responses to ovarian cancer are distinguished by self-reactive intra-epithelial plasma cells

Tumor-infiltrating B cells (TIL-Bs) are strongly associated with patient survival; however, the underlying mechanisms are poorly understood. Using integrated single-cell and spatial biology approaches, we defined at clonal resolution the molecular phenotypes, tumor reactivity patterns, and microenvironmental locations of TIL-Bs in high-grade serous ovarian cancer (HGSC). Prognostic benefit was associated with a TIL-B-rich tumor microenvironment with marked infiltration of malignant epithelium by plasma cells (PCs). PCs spanned five molecular phenotypes; exhibited high rates of somatic hypermutation and clonal expansion; and expressed predominantly IgG1 antibodies recognizing broadly expressed nuclear, cytoplasmic, and cell surface self-antigens. Many PC-derived antibodies were polyreactive. Self- and poly-reactive TIL-Bs penetrated tumor epithelium and stroma and expressed interferon-stimulated genes, indicating strong in situ activation. The self- and poly-reactive nature of TIL-B responses, reminiscent of autoimmune disease, may provide a means for the immune system to combat tumor heterogeneity and could potentially be harnessed for more effective immunotherapy.

cancer biology↗

bioRxiv2024

Spatial single cell transcriptomic analysis of a novel DICER1 Syndrome GEMM informs the cellular origin and developmental hierarchy of associated sarcomas

DICER1 syndrome predisposes children and young adults to tumor development across various organs. Many of these cancers are sarcomas, which uniquely express the RNase IIIb domain-deficient form of DICER1 and exhibit consistent histological and molecular similarities regardless of their anatomical origins. To uncover their cellular origin and developmental hierarchy, we established a lineage-traceable genetically engineered mouse model that allows for precise activation of Dicer1 mutations in Hic1+ mesenchymal stromal cells. This model resulted in the development of renal tumors closely mirroring human DICER1 sarcoma histologically and molecularly. Single-cell transcriptomics coupled with targeted spatial gene expression analysis revealed a Hic1+ progenitor population marked by Pdgfra, Dpt, and Mfap4, corresponding to universal fibroblasts of steady-state kidneys. These fibroblastic progenitors exhibit the capacity to undergo rhabdomyoblastic differentiation or transition to highly proliferative anaplastic sarcoma. Investigation of patient samples identified analogous cell states. This study uncovers a fibroblastic origin for DICER1 sarcoma and provides a faithful model for mechanistic investigation and therapeutic development for tumors within the rhabdomyosarcoma spectrum.

cancer biology↗

bioRxiv2024

Neutrophil Extracellular Trap gene expression signatures identify prognostic and targetable signaling axes for inhibiting metastasis of pancreatic tumours

Tumour associated neutrophils (TANs) can promote metastasis through the interaction of Neutrophil Extracellular Traps (NETs) with tumour cells. Here, we examined the contribution of NETs in the progression of Pancreatic Ductal Adenocarcinoma (PDAC), which is characterized by high metastatic propensity. We carried out consensus clustering and pathway enrichment analysis of NET-related genes in an integrated cohort of 369 resectable and metastatic PDAC patient tumour samples, and compiled two gene expression signatures comprising of either, integrin-actin cytoskeleton, and Epithelial to Mesenchymal Transition (EMT) signaling, or cell death signaling, which identified patients with very poor to better overall survival, respectively. Tumour Infiltrating neutrophils and NETs associate with ITGB1, CCDC25 and ILK, within clinical and experimental PDAC tumours. Functionally, exposure of PDAC cells to NETs identified a cytoskeletal dynamic-associated CCDC25/ITGB1/ILK signaling complex which stimulates EMT and migration/ invasion. Furthermore, NETosis-driven experimental metastasis of PDAC cells is significantly inhibited by ILK knock down. Our data identify novel NET-related gene expression signatures for PDAC patient stratification, and reveal targetable signaling axes to prevent and treat disease progression and metastasis.

cancer biology↗

bioRxiv2024

Structural basis of TMPRSS11D specificity and autocleavage activation

Transmembrane Protease, Serine-2 (TMPRSS2) and TMPRSS11D are human proteases that enable SARS-CoV-2 and Influenza A/B virus infections, but their biochemical mechanisms for facilitating viral cell entry remain unclear. We demonstrate these proteases can spontaneously and efficiently cleave their own zymogen activation motifs, thereby activating their wider protease activity on other cellular substrates. We determined TMPRSS11D co-crystal structures in complexes with a native TMPRSS11D zymogen activation motif and with an engineered activation motif, providing insights into TMPRSS11D autocleavage activation and revealing unique regions of its substrate binding cleft. We further show that a protease inhibitor that underwent clinical trials for TMPRSS2-targeted COVID-19 therapy, nafamostat mesylate, was rapidly cleaved by TMPRSS11D and converted to low activity derivatives. These insights into human protease viral tropism and into liabilities with existing human serine protease inhibition strategies will guide future drug discovery campaigns for these targets.

biochemistry↗

bioRxiv2024

IMMUNOTAR - Integrative prioritization of cell surface targets for cancer immunotherapy

Cancer remains a leading cause of mortality globally. Recent improvements in survival have been facilitated by the development of less toxic immunotherapies; however, identifying targets for immunotherapies remains a challenge in the field. To address this challenge, we developed IMMUNOTAR, a computational tool that systematically prioritizes and identifies candidate immunotherapeutic targets. IMMUNOTAR integrates user-provided RNA-sequencing or proteomics data with quantitative features extracted from publicly available databases based on predefined optimal immunotherapeutic target criteria and quantitatively prioritizes potential surface protein targets. We demonstrate the utility and flexibility of IMMUNOTAR using three distinct datasets, validating its effectiveness in identifying both known and new potential immunotherapeutic targets within the analyzed cancer phenotypes. Overall, IMMUNOTAR enables the compilation of data from multiple sources into a unified platform, allowing users to simultaneously evaluate surface proteins across diverse criteria. By streamlining target identification, IMMUNOTAR empowers researchers to efficiently allocate resources and accelerate immunotherapy development.

bioinformatics↗

bioRxiv2023

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97

The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCPs potential as a novel therapeutic target.

cancer biology↗

bioRxiv2022

Loss of FBXO11 function establishes a stem cell program in acute myeloid leukemia through dysregulation of the mitochondrial protease LONP1

Acute myeloid leukemia (AML) is an aggressive cancer with very poor outcomes. To identify additional drivers of leukemogenesis, we analyzed sequence data from 1,727 unique individual AML patients, which revealed mutations in ubiquitin ligase family genes in 11.2% of adult AML samples with mutual exclusivity. The Skp1/Cul1/Fbox (SCF) E3 ubiquitin ligase complex gene FBXO11 was the most significantly downregulated gene of the SCF complex in AML. FBXO11 catalyzes K63-linked ubiquitination of a novel target, LONP1, which promotes entry into mitochondria, thereby enhancing mitochondrial respiration. Reduced mitochondrial respiration secondary to FBXO11 depletion imparts myeloid-biased stem cell properties in primary CD34+ hematopoietic stem progenitor cells (HSPC). In a human xenograft model, depletion of FBXO11 cooperated with AML1-ETO and mutant KRASG12D to generate serially transplantable AML enriched for primitive cells. Our findings suggest that reduced FBXO11 primes HSPC for myeloid-biased self-renewal through attenuation of LONP1-mediated regulation of mitochondrial respiration.

cancer biology↗

bioRxiv2021

A novel small molecule that induces cytotoxicity in lung cancer cells inhibits disulfide reductases GSR and TXNRD1

High-throughput phenotype-based screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Here we show that a compound termed lung cancer screen 3 (LCS3), previously selected for its ability to impair the growth of human lung adenocarcinoma (LUAD) cell lines, but not normal lung cells, induces oxidative stress and activates the NRF2 signaling pathway by generating reactive oxygen species (ROS) in sensitive LUAD cell lines. To identify the target that mediates this effect, we applied thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, we confirmed that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified the loss of NQO1 as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential utility of inhibiting disulfide reductases as a therapeutic strategy for LUAD.

cancer biology↗

bioRxiv2020

Multiomics Characterization of Potential Therapeutic Vulnerabilities in Low-grade Serous Ovarian Carcinoma

BackgroundLow-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates. As such, there is a pressing need to develop more effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use a multiomics approach to interrogate a collection of LGSOC patient-derived cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. MethodsFourteen LGSOC cell lines were interrogated using whole exome sequencing, RNA sequencing, and mass spectrometry-based proteomics. Somatic mutation, copy-number aberrations, gene and protein expression were analyzed and integrated using different computational approaches. LGSOC cell line data was compared to publicly available LGSOC tumor data (AACR GENIE cohort), and also used for predictive biomarker identification of MEK inhibitor (MEKi) efficacy. Protein interaction databases were evaluated to identify novel therapeutic targets. ResultsKRAS mutations were exclusively found in MEKi-sensitive and NRAS mutations mostly in MEKi-resistant cell lines. Analysis of COSMIC mutational signatures revealed distinct patterns of nucleotide substitution mutations in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes (chromosome 9p21) were much more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. For in-vitro MEKi efficacy prediction, proteomic data provided better discrimination than gene expression data. Condensin, MCM, and RFC protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. ConclusionsOur LGSOC cell lines are representative models of the most common molecular aberrations found in LGSOC tumors. This study highlights the importance of using proteomic data in multiomics assessment of drug prediction and identification of potential therapeutic targets. CDKN2A/B and MTAP deficiency offer an opportunity to find synthetically lethal candidates for novel treatments. Multiomics approaches are crucial to improving our understanding of the molecular aberrations in LGSOC, establishing effective drug prediction programs and identifying novel therapeutic targets in LGSOC.

cancer biology↗

bioRxiv2020

Recombinant T cell receptors specific for HLA-A*02:01-restricted neoepitopes containing KRAS codon 12 hotspot mutations

KRAS codon 12 mutations are among the most common hotspot mutations in human cancer. Using a functional screening platform we set out to identify {beta} T-cell receptors (TCRs) as potential targeting reagents for KRASG12D and/or KRASG12V neoepitopes presented by the prevalent HLA-A*02:01 allele. Here we describe isolation and characterization of three distinct CD8+ T cell clones from a pre-treated 76 year old patient with pancreatic ductal adenocarcinoma (PDAC). One clone was KRASG12V reactive and two clones were KRASG12D reactive. Tetramer staining showed high specificity of each T cell clone for its cognate HLA-A*02:01 restricted KRASG12V or KRASG12D neoepitope (>98% tetramer positive) without appreciable cross-reactivity to wild-type KRAS (<2% tetramer positive). We amplified and sequenced the full-length TCR alpha and beta chains from each of the three T cell clones and determined that these three TCRs comprised distinct combinations of two different TCR alpha chains and two distinct TCR beta chains. We resynthesized these TCR alpha and beta chain nucleotide sequences and reconstituted the original pairs in healthy donor CD8+ T cells by lentiviral transduction, substituting the human {beta} TCR constant gene segments with murine {beta} TCR constant gene segments to prevent mispairing with endogenous TCR subunits. Tetramer analysis and IFN-{gamma} ELISpot analysis confirmed the specificity of each reconstituted TCR for its cognate HLA-A*02:01 restricted KRAS neoepitope. To test cytolytic activity TCR-transduced healthy donor CD8+ T cells were co-cultured with KRASG12V, KRASG12D or KRASwt peptide-pulsed K562-HLA-A*02:01 antigen presenting cells at an effector to target cell ratio of 4:1. Under these conditions we observed neoepitope-specific killing of 16.5% to 19.0% of target cell populations. To assess in vivo activity we developed a KRASG12V/A*02:01 patient-derived xenograft (PDX) mouse model. Over a 56-day period, PDX bearing mice infused with human TCR-transduced T cells had significantly reduced tumor growth and longer survival compared to mice infused with non-transduced control T cells. In conjunction with other therapeutic approaches, immune effector cell therapies expressing these TCRs may improve outcomes for HLA-A*02:01 patients with KRASG12V and/or KRASG12D positive tumors.

immunology↗

bioRxiv2020

The pathognomonic FOXL2 C134W mutation alters DNA binding specificity

The somatic missense point mutation c.402C>G (p.C134W) in the FOXL2 transcription factor is pathognomonic for adult-type granulosa cell tumours (AGCT) and a diagnostic marker for this tumour type. However, the molecular consequences of this mutation and its contribution to the mechanisms of AGCT pathogenesis remain unclear. To explore the mechanisms driving FOXL2C134W pathogenicity we engineered V5-FOXL2WT and V5-FOXL2C134W inducible isogenic cell lines and performed ChIP-seq and transcriptome profiling. We found that FOXL2C134W associates with the majority of the FOXL2 WT DNA elements as well as a large collection of unique elements genome-wide. We confirmed an altered DNA binding specificity for FOXL2C134W in vitro and identified unique targets of FOXL2C134W including SLC35F2 whose expression increased sensitivity to YM155 in our model. Statement of SignificanceMechanistic understanding of FOXL2C134W induced regulatory state alterations drives discovery of a rationally designed therapeutic strategy.

cancer biology↗

bioRxiv2019

Arginine depletion through ADI-PEG20 to treat argininosuccinate synthase deficient ovarian cancer, including small cell carcinoma of the ovary, hypercalcemic type

PurposeMany rare ovarian cancer subtypes such as small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) have poor prognosis due to their aggressive nature and resistance to standard platinum and taxane based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes. Experimental DesignWe compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype high grade serous ovarian cancer (HGSC) to identify potential therapeutic targets. Immunohistochemistry of tissue microarrays were used as validation of ASS1 deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient derived xenograft mouse models representing SCCOHT. ResultsGlobal proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared to HGSC. Low ASS1 levels were validated through IHC in a large patient cohort. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 2/15 cases, and expressed in less than 5% of the tumor cells in 8/15 cases. ASS1 deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient derived mouse xenograft model of SCCOHT, once a week treatment of ADI-PEG20 (30mg/kg and 15mg/kg) inhibited tumor growth in vivo. ConclusionsPreclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers including SCCOHT. Translational relevanceMany rare ovarian cancers lack effective management strategies and are resistant to the standard platinum- and taxane-based chemotherapy. Thus, for a rare ovarian cancer subtype like small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) - an aggressive malignancy affecting young women in their twenties, effective targeted therapeutics are urgently needed. We used global proteomics to identify a deficiency in arginosuccinate synthase (ASS1) as a common feature among some rare ovarian cancer subtypes. Using in-vitro and in-vivo models, we demonstrated that the arginine-depriving investigational agent ADI-PEG20 effectively inhibited cell growth in ASS1 deficient ovarian cancers including SCCOHT, establishing it as a potential therapeutic agent for rare ovarian cancer subtypes deficient in ASS1. Further clinical investigation is warranted.

cancer biology↗

bioRxiv2019

Loss of macp{Psi} ribosomal RNA modification is a major feature of cancer

The ribosome is an RNA-protein complex essential for translation in all domains of life. The structural and catalytic core of the ribosome is its ribosomal RNA (rRNA). While mutations in ribosomal protein (RP) genes are known drivers of oncogenesis, oncogenic rRNA variants have remained elusive. We discovered a cancer-specific single nucleotide variation in 18S rRNA at nucleotide 1248.U in up to 45.9% of colorectal carcinoma (CRC) patients and present across >22 cancer types. This is the site of a unique hyper-modified base, 1-methyl-3--amino--carboxyl-propyl pseudouridine (m1acp3{Psi}), a >1 billion years conserved RNA modification at the ribosomes peptidyl decoding-site. A sub-set of CRC tumors we term hypo-m1acp3{Psi}, show sub-stoichiometric m1acp3{Psi}-modification unlike normal control tissues. A m1acp3{Psi} knockout model and hypo-m1acp3{Psi} patient tumors share a translational signature, characterized by highly abundant ribosomal proteins. Thus, m1acp3{Psi}-deficient rRNA forms an uncharacterized class of onco-ribosome which may serve as a chemotherapeutic target for treating cancer patients.

cancer biology↗

bioRxiv2019

Loss of CIC promotes mitotic dysregulation and chromosome segregation defects

CIC encodes a transcriptional repressor and MAPK signalling effector that is inactivated by loss-of-function mutations in several cancer types, consistent with a role as a tumour suppressor. Here, we used bioinformatic, genomic, and proteomic approaches to investigate CICs interaction networks. We observed both previously identified and novel candidate interactions between CIC and SWI/SNF complex members, and also observed novel interactions between CIC and cell cycle regulators and RNA processing factors. We found that CIC loss is associated with an increased frequency of mitotic defects in human cell lines and an in vivo mouse model and with dysregulated expression of mitotic regulators. We also observed aberrant splicing in CIC-deficient cell lines predominantly at 3 and 5 untranslated regions of genes, including genes involved in MAPK signalling, DNA repair, and cell cycle regulation. Our study thus characterises the complexity of CICs functional network and describes the effect of its loss on cell cycle regulation, mitotic integrity, and transcriptional splicing, thereby expanding our understanding of CICs potential roles in cancers. In addition, our work exemplifies how multi-omic, network-based analyses can be used to uncover novel insights into the interconnected functions of pleiotropic genes/proteins across cellular contexts. Simple summaryCapicua (CIC) is a gene that is frequently mutated in several cancer types, including stomach cancers and certain subtypes of brain tumours and sarcomas. CIC, the protein encoded by the CIC gene, has been shown to play a multitude of roles in both normal and cancer cell functions; however, most studies exploring these roles focus on a single aspect of CIC function and may therefore overlook complex interconnected activities in which CIC is involved. In this study, we have used multiple complementary approaches to obtain a broader view of CICs complex functional networks. We observed novel interactions (genetic or physical) between CIC and genes/proteins involved in various aspects of cellular function, including regulation of cell division and processing of RNA molecules. Altogether, our work characterises the complexity of CICs functional network and expands our understanding of its potential roles in cancer.

cancer biology↗

bioRxiv2018

High-flow Nano-chromatography Columns Facilitate Rapid and High-quality Proteome Analysis on Standard Nano-LC Hardware

Optimizing the acquisition of proteomics data collected from a mass spectrometer (MS) requires careful selection of processed material quantities, liquid-chromatography (LC) setup, and data acquisition parameters. The small internal diameter (ID) columns standardly used in nano-chromatography coupled MS result in long per injection overhead times that require sacrifices in design of offline-fractionation and data acquisition schemes. As cohort sizes and the numbers of samples to be analyzed continue to increase, there is a need to investigate methods for improving the efficiency and time of an acquisition (LC + MS). In this work, the ability to improve throughput in single runs or as part of an in-depth proteome analysis of a fractionated sample using standard LC hardware is investigated. Capitalizing on the increased loading capacity of nanochromatography columns with larger IDs, substantially improved throughput with no reduction in detection sensitivity is achieved in single-injection proteome analyses. An optimized 150 m ID column setup is paired with an offline fractionation-concatenation scheme to demonstrate the ability to perform in-depth proteome analysis on-par with current state-of-the-art studies, while minimizing sample loading overhead. Together, these data demonstrate an easy and effective means to improve sample analysis throughput with no reduction in data quality using an approach that is applicable to any standard nano-LC hardware.

biochemistry↗

bioRxiv2018

RawTools: Rapid and Dynamic Interrogation of Orbitrap Data Files for Mass Spectrometer System Management

Optimizing the quality of proteomics data collected from a mass spectrometer (MS) requires careful selection of acquisition parameters and proper assessment of instrument performance. Software tools capable of extracting a broad set of information from raw files, including meta, scan, quantification, and identification data are needed to provide guidance for MS system management. In this work, direct extraction and utilization of these data from Thermo Orbitrap raw files is demonstrated using the RawTools software. RawTools is a standalone tool for extracting meta and scan data directly from raw MS files generated on Thermo Orbitrap instruments. RawTools generates summarized and detailed plain text outputs after parsing individual raw files, including scan rates and durations, duty cycle characteristics, precursor and reporter ion quantification, and chromatography performance. RawTools also contains a diagnostic module that includes an optional preview database search for facilitating informed decision-making related to optimization of MS performance based on a variety of metrics. RawTools has been developed in C# and utilizes the Thermo RawFileReader library, and thus can process raw MS files with high speed and high efficiency on all major operating systems (Windows, MacOS, Linux). To demonstrate the utility of RawTools, extraction of meta and scan data from both individual and large collections of raw MS files was carried out to identify problematic characteristics of instrument performance. Taken together, the combined rich feature-set of RawTools with the capability for interrogation of MS and experiment performance makes this software a valuable tool for proteomics researchers.

bioinformatics↗