Biology subjects

Jenkins, C.

Publications and source records attributed to Jenkins, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2020

Heparin MicroIslands to Promote Enhanced Diabetic Wound Healing Outcomes

A powerful tool to improve tissue integration with biomaterial scaffolds for the regeneration of damaged tissues is to promote cell migration using chemotactic gradients of growth factors. This approach has been realized by the exogenous delivery of growth factors, which unfortunately also limits the scaffolds ability to meet each wounds unique spatial and temporal regenerative needs. To address this limitation, we present a new approach to gradient generation by incorporating heparin microislands, which are spatially isolated heparin-containing microparticles that create chemotactic microgradients through reorganization of endogenous local growth factors. We incorporated heparin microislands within microporous annealed particle (MAP) scaffolds, which allows us to tune their incorporation ratiometrically to create a heterogenous microenvironment. In this manuscript, we demonstrate the ability of heparin microislands to organize uniform growth factors into spontaneous microgradients and control downstream cell migration in vitro. Further, we present their ability to significantly improve wound healing outcomes (epidermal regeneration and vascularization) in a diabetic wound model relative to two clinically relevant controls.

bioengineering↗

bioRxiv2020

Global population structure and genotyping framework for genomic surveillance of the major dysentery pathogen, Shigella sonnei

Shigella sonnei is the most common agent of shigellosis in high-income countries, and causes a significant disease burden in low- and middle-income countries. Antimicrobial resistance is increasingly common in all settings. Whole genome sequencing (WGS) is increasingly utilised for S. sonnei outbreak investigation and surveillance, but comparison of data between studies and labs is challenging. Here, we present a genomic framework and genotyping scheme for S. sonnei to efficiently identify genotype and resistance determinants from WGS data. The scheme is implemented in the software package Mykrobe and tested on thousands of genomes. Applying this approach to analyse >4,000 S. sonnei isolates sequenced in public health labs in three countries identified several common genotypes associated with increased rates of ciprofloxacin resistance and azithromycin resistance, confirming intercontinental spread of highly-resistant S. sonnei clones and demonstrating the genomic framework can facilitate monitoring of the emergence and spread of resistant clones at local and global scales.

microbiology↗

bioRxiv2020

Accessory genome dynamics and structural variation of Shigella from persistent infections

Shigellosis is a diarrhoeal disease caused mainly by Shigella flexneri and Shigella sonnei. Infection from Shigella is thought to be largely self-limiting, with short- to medium- term and serotype-specific immunity provided following clearance. However, cases of men who have sex with men (MSM) associated shigellosis have been reported where Shigella of the same serotype were serially sampled from individuals between 1 to 1862 days apart, possibly due to persistent carriage or reinfection with the same serotype. Here, we investigate the accessory genome dynamics of MSM associated S. flexneri and S. sonnei isolates serially sampled from individual patients at various days apart. We find that pairs likely associated with persistent carriage infection and with smaller single nucleotide polymorphism (SNP) distance, demonstrated significantly less variation in accessory genome content than pairs likely associated with reinfection and with greater SNP-distance. We also observed evidence of antimicrobial resistance (AMR) acquisition during persistent Shigella infection, specifically the gain of extended spectrum beta-lactamase genes in two pairs associated with persistent carriage. Finally, we explored chromosomal structural variations and rearrangements in seven (5 chronic and 2 reinfection associated) pairs of S. flexneri 3a isolates from a MSM-associated epidemic sublineage, which revealed variations at several common regions across pairs. These variations were mediated by insertion sequence (IS) elements which facilitated plasticity of genetic material with a distinct predicted functional profile. This study provides insight on the variation of accessory genome dynamics and large structural genomic changes in Shigella during persistent infection. ImportanceShigella spp are Gram-negative bacteria that are the etiological agent of shigellosis, the second most common cause of diarrhoeal illness globally, particularly among children under the age of 5 in low-income countries. In high-income countries, an alternative transmission pathway of sexually transmissible disease among men who have sex with men (MSM) is emerging as the dominant presentation of the disease. Within MSM we have captured prolonged infection and/or recurrent infection with shigellae of the same serotype, challenging the belief that Shigella infection is short-lived, and confers homologous serotypic immunity. Using this recently-emerged transmission scenario we comprehensively characterise the genomic changes that occur over the course of individual infection with Shigella and uncover a distinct functional profile of variable genome regions in these globally important pathogens.

microbiology↗

bioRxiv2020

In silico approach to accelerate the development of mass spectrometry-based proteomics methods for detection of viral proteins: Application to COVID-19

We describe a method for rapid in silico selection of diagnostic peptides from newly described viral pathogens and applied this approach to SARS-CoV-2/COVID-19. This approach is multi-tiered, beginning with compiling the theoretical protein sequences from genomic derived data. In the case of SARS-CoV-2 we begin with 496 peptides that would be produced by proteolytic digestion of the viral proteins. To eliminate peptides that would cause cross-reactivity and false positives we remove peptides from consideration that have sequence homology or similar chemical characteristics using a progressively larger database of background peptides. Using this pipeline, we can remove 47 peptides from consideration as diagnostic due to the presence of peptides derived from the human proteome. To address the complexity of the human microbiome, we describe a method to create a database of all proteins of relevant abundance in the saliva microbiome. By utilizing a protein-based approach to the microbiome we can more accurately identify peptides that will be problematic in COVID-19 studies which removes 12 peptides from consideration. To identify diagnostic peptides, another 7 peptides are flagged for removal following comparison to the proteome backgrounds of viral and bacterial pathogens of similar clinical presentation. By aligning the protein sequences of SARS-CoV-2 field isolates deposited to date we can identify peptides for removal due to their presence in highly variable regions that may lead to false negatives as the pathogen evolves. We provide maps of these regions and highlight 3 peptides that should be avoided as potential diagnostic or vaccine targets. Finally, we leverage publicly deposited proteomics data from human cells infected with SARS-CoV-2, as well as a second study with the closely related MERS-CoV to identify the two proteins of highest abundance in human infections. The resulting final list contains the 24 peptides most unique and diagnostic of SARS-CoV-2 infections. These peptides represent the best targets for the development of antibodies are clinical diagnostics. To demonstrate one application of this we model peptide fragmentation using a deep learning tool to rapidly generate targeted LCMS assays and data processing method for detecting CoVID-19 infected patient samples. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=156 HEIGHT=200 SRC="FIGDIR/small/980383v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1d7fd4borg.highwire.dtl.DTLVardef@136563borg.highwire.dtl.DTLVardef@57641dorg.highwire.dtl.DTLVardef@16de9a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Standard Flow Multiplexed Proteomics (SFloMPro). An Accessible and Cost-Effective Alternative to NanoLC Workflows

Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid "standard flow" HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 hours. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 g of labeled peptides per fraction with SFloMPro, compared to 1 g per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample. Data are available via ProteomeXchange with identifier PXD016704. O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/964379v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1c9f848org.highwire.dtl.DTLVardef@cb4711org.highwire.dtl.DTLVardef@1a6b7c1org.highwire.dtl.DTLVardef@69f751_HPS_FORMAT_FIGEXP M_FIG Abstract Graphic C_FIG

biochemistry↗

bioRxiv2019

BoxCar Assisted MS Fragmentation (BAMF)

A recent study described the utilization of complex quadrupole isolation schemes to reduce high abundance ion saturation in Orbitrap systems. The BoxCar technique effectively democratizes MS1 scans by restricting high abundance ions from consuming as much space in the trap. This restriction allows lower abundance ions more acquisition time and can increase the signal to noise by a full order of magnitude. While effective at the MS1 level, BoxCar does not show an improvement in MS/MS acquisition as ions selected for fragmentation must come from an additional MS1 full scan in the method. In this study we describe BoxCar Assisted MS Fragmentation (BAMF), wherein ions for fragmentation are selected directly from the BoxCar scans. When utilizing BAMF, we observe the identification of ions by MS/MS that are not at all detectable in the MS1 scans of identical concentrations of peptides analyzed by standard data dependent acquisition experiments. Abstract Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2019

Comparison of Shiga toxin-encoding bacteriophages in highly pathogenic strains of Shiga toxin-producing Escherichia coli O157:H7 in the UK.

2.Over the last 35 years in the UK the burden of Shiga toxin-producing Escherichia coli (STEC) O157:H7 infection has, during different periods of time, been associated with five different sub-lineages (1983-1995: Ia, I/IIa and I/IIb, 1996-2014: Ic and 2015-2018: IIb). The acquisition of a stx2a-encoding bacteriophage by these five sub-lineages appears to have coincided with their respective emergences. Oxford Nanopore Technology (ONT) was used to sequence, characterise and compare the stx-encoding prophage harboured by each sub-lineage to investigate the integration of this key virulence factor. The stx2a-encoding prophage from each of the lineages causing clinical disease in the UK were all different, including the two UK sub-lineages (Ia and I/IIa) circulating concurrently and causing severe disease in the early 1980s. Comparisons between the stx2a-encoding prophage in sub-lineages I/IIb and IIb revealed similarity to the prophage commonly found to encode stx2c, and the same site of bacteriophage integration (sbcB) as stx2c encoding prophage. These data suggest independent acquisition of previously unobserved stx2a-encoding phage is more likely to have contributed to the emergence of STEC O157:H7 sub-lineages in the UK than intra-UK lineage to lineage phage transmission. In contrast, the stx2c-encoding prophage showed a high level of similarity across lineage and time, consistent with the model of stx2c being present in the common ancestor to extant STEC O157:H7 and maintained by vertical inheritance in the majority of the population. Studying the nature of the stx-encoding bacteriophage contributes to our understanding of the emergence of highly pathogenic strains of STEC O157:H7. 3. Impact statementThe application of ONT technology to sequence UK epidemic strains of STEC O157:H7 revealed stx2a-encoding prophage exhibit a high level of diversity. There was little evidence of geographical or temporal patterns of relatedness, or of intra-UK transmission of stx2a-encoding prophage between indigenous strains. The stx2a-encoding prophage in the UK lineages associated with severe disease appear to be acquired independently and most likely from different geographical and/or environmental sources. These data provide supporting evidence for the existence of a dynamic environmental reservoir of stx2a-encoding prophage that pose a threat public health due to their potential for integration into competent, indigenous sub-lineages of STEC O157:H7. We also provide further evidence that stx2c-encoding prophage exhibit a high level of similarity across lineage, geographical region and time, and have likely been maintained and inherited vertically. 4. Data summaryAll FASTQ files and assemblies of samples sequenced in this project were submitted to the National Centre for Biotechnology Information (NCBI). All data can be found under BioProject: PRJNA315192 - https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA315192. Strain specific details can be found in the methods section under data deposition. Publicly available data used in this project can be found via Table 1 and data bibliography. O_TBL View this table: org.highwire.dtl.DTLVardef@178103aorg.highwire.dtl.DTLVardef@1718d1corg.highwire.dtl.DTLVardef@1e7605forg.highwire.dtl.DTLVardef@1683451org.highwire.dtl.DTLVardef@42c400_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1 -C_FLOATNO O_TABLECAPTIONSummary of the PHE archived and publicly available strains used within this study, their Strain ID, lineage, phage type, Stx profile, assembly accession numbers and NCBI BioProject. C_TABLECAPTION C_TBL

genomics↗

bioRxiv2019

Yersinia canariae sp. nov., isolated from a human yersiniosis case

A Gram-negative rod from the Yersinia genus was isolated from a clinical case of yersiniosis in the United Kingdom. Long read sequencing data from an Oxford Nanopore Technology (ONT) MinION in conjunction with Illumina HiSeq reads were used to generate a finished quality genome of this strain. Overall Genome Related Index (OGRI) of the strain was used to determine that it was a novel species within Yersinia, despite biochemical similarities to Yersinia enterocolitica. The 16S ribosomal RNA gene accessions are MN434982-MN434987 and the accession number for the complete and closed chromosome is CP043727. The type strain is CFS3336T (=NCTC 14382T/ =LMG Accession under process).

microbiology↗

bioRxiv2019

Structure of E3 ligase E6AP with a novel proteasome-binding site provided by substrate receptor hRpn10

Regulated proteolysis by the proteasome involves [~]800 enzymes for substrate modification with ubiquitin, of which [~]600 are E3 ligases. We report here that E6AP/UBE3A is distinguished from other ubiquitin E3 ligases by having a 12 nM binding site at the proteasome contributed by substrate receptor hRpn10/PSMD4/S5a. Intrinsically disordered by itself, and previously uncharacterized, this domain in hRpn10 locks into a novel well-defined helical structure to form an intermolecular 4-helix bundle with the E6AP AZUL domain, which is unique to this E3. We thus name the hRpn10 AZUL-binding domain RAZUL. We further find in human cells that loss of RAZUL by CRISPR-based gene editing leads to loss of E6AP at the proteasome, where associated ubiquitin is correspondingly reduced, suggesting that E6AP ubiquitinates substrates at or for the proteasome. Altogether, our findings indicate E6AP to be a privileged E3 for the proteasome, with a dedicated, high affinity binding site contributed by hRpn10.

molecular biology↗

bioRxiv2019

Persistent transmission of shigellosis in England is associated with a recently emerged multi-drug resistant strain of Shigella sonnei

BackgroundWhole genome sequencing has enhanced surveillance and facilitated detailed monitoring of transmission of Shigella species in England.\n\nMethodsWe undertook an epidemiological and phylogenetic analysis of isolates from all cases of shigellosis referred to Public Health England between 2015 to 2018 to explore recent strain characteristics and transmission dynamics of Shigella species.\n\nResultsOf the 4950 confirmed cases of shigellosis identified during this period, the highest proportion of isolates were S. sonnei (54.4%), followed by S. flexneri (39.2%), S. boydii (4.1%) and S. dysenteriae (2.2%). Most cases were adults (82.9%) and male (59.5%), and 34.9% cases reported recent travel outside the UK. Throughout the study period diagnoses of S. flexneri and S. sonnei were most common in men with no history of recent travel abroad. Species prevalence was not static with cases of S. flexneri in men decreasing from 2015-2016, and the number of cases of S. sonnei increasing from 2017. Phylogenetic analysis showed this recent increase in S. sonnei was attributed to a novel clade exhibiting resistance to ciprofloxacin and azithromycin, which had emerged from a Central Asia sub-lineage.\n\nConclusionsDespite changes in species prevalence, Shigella diagnoses in England are persistently the most common in adult males without reported travel history, consistent with sexual transmission amongst men who have sex with men. The trend in increasing ciprofloxacin resistance, in addition to plasmid-mediated azithromycin resistance, in S. sonnei is of significant public health concern with respect to transmission of multi-drug resistant gastrointestinal pathogens and the risk of treatment failures.\n\nSummaryShigella diagnoses in England are most prevalent amongst men who have sex with men. S. sonnei exhibiting resistance to both azithromycin and ciprofloxacin has replaced azithromycin-resistant S. flexneri as the most commonly isolated species in this setting.

genomics↗

bioRxiv2019

The First Publicly Available Annotated Genome for Cannabis plants

Recently we have seen a relaxation of the historic restrictions on the use and subsequent research on the Cannabis plants in North America. We have recently described a pipeline for the creation of annotated protein databases using a combination of genomic and proteomic data and the application of this method toward the analysis of the proteomes of Cannabis plants. In parallel with our work, we approached the National Center for Biotechnology Information (NCBI) to align and annotate publicly deposited genomic files for these plants. In September of 2019, this project was completed. The result, NCBI Cannabis sativa Annotation Release 100 is now live and publicly available. The annotated genome allows, for the first time, the use of classical genetics and proteomics tools for the interrogation of these plants. Furthermore, the presence of an annotated genome within the NCBI Genome browser now permits any researcher with a web browser to manually examine or BLAST genetic sequences, vastly reducing the resources required to create primers to amplify genes from the plants or microbial contaminants that may affect them. We describe this new resource and some points of obvious value to the scientific community as well as the integration into the Cannabis Proteome Draft Map Project.\n\nSignificance StatementUntil recently laws in North America have restricted nearly all research on Cannabis plants. Until recent research from our lab, only a few hundred genes and proteins from the plant had been annotated for putative function. The construction of a publicly available annotated genome for this plant allows, for the first time, the use of traditional genomic and proteomic tools for the investigation of these plants. We evaluate herein the first fully annotated publicly available genome for Cannabis plants and the integration of this resource into www.CannabisDraftmap.org

plant biology↗

bioRxiv2019

The Cannabis Multi-Omics Draft Map Project

Recently we have seen a relaxation of the historic restrictions on the use and subsequent research on the Cannabis plants, generally classified as Cannabis sativa and Cannabis indica. What research has been performed to date has centered on chemical analysis of plant flower products, namely cannabinoids and various terpenes that directly contribute to phenotypic characteristics of the female flowers. In addition, we have seen many groups recently completing genetic profiles of various plants of commercial value. To date, no comprehensive attempt has been made to profile the proteomes of these plants. We report herein our progress on constructing a comprehensive draft map of the Cannabis proteome. To date we have identified over 17,000 potential protein sequences. Unfortunately, no annotated genome of Cannabis plants currently exists. We present a method by which \"next generation\" DNA sequencing output and shotgun proteomics data can be combined to produce annotated FASTA files, bypassing the need for annotated genetic information altogether in traditional proteomics workflows. The resulting material represents the first comprehensive annotated FASTA for any Cannabis plant. Using this annotated database as reference we can refine our protein identifications, resulting in the confident identification of 13,000 proteins with putative function. Furthermore, we demonstrate that post-translational modifications play an important role in the proteomes of Cannabis flower, particularly lysine acetylation and protein glycosylation. To facilitate the evolution of analytical investigations into these plant materials, we have created a portal to host resources we have developed from proteomic and metabolomic analysis of Cannabis plant material as well as our results integrating these resources. All data for this project is available to view or download at www.CannabisDraftMap.Org

plant biology↗

bioRxiv2019

Direct Measurement of Synchronous Precursor Selection (SPS) Accuracy in Public Proteomics Datasets

Reporter ion quantification techniques utilizing reagents such as TMT and iTRAQ allow proteomics studies to multiplex up to 11 different samples within a single LC-MS/MS experimental run. In these experiments, peptides derived from different samples are labeled with chemical tags possessing identical mass but differing distributions of heavy isotopes through their structure. Peptides from all samples may then be physically combined prior to LC-MS/MS. Relative quantification of the peptides from each sample is obtained from the liberation of low mass reporter ions alone, as these are the only discernible factor between peptides in the entire LC-MS/MS workflow. When coeluting ions of similar mass to charge ratios are fragmented along with the ions of interest, it is not possible to determine the source of the reporter fragments and quantification is skewed, most often resulting in ratio suppression. One technique for combatting ratio suppression is the selection of MS2 fragment ions that are likely to retain the intact mass tag region by synchronous precursor selection (SPS) and the liberation of the reporter ions from this combination of ions in MS/MS/MS (MS3). In this study we utilize a new post processing tool that can directly assess the accuracy of the SPS system for picking ions for quantification that are truly derived from the peptide of interest. We then apply this tool to the re-analysis of 3 public proteomics datasets. Directly assessing SPS accuracy allows a new measurement of confidence in the quantification values obtained from these reporter ion quantification experiments.\n\nAbstract Graphic\n\nO_FIG O_LINKSMALLFIG WIDTH=185 HEIGHT=200 SRC=\"FIGDIR/small/647917v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@1186337org.highwire.dtl.DTLVardef@19c0daorg.highwire.dtl.DTLVardef@c44d31org.highwire.dtl.DTLVardef@1a7d597_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2019

Constructing a Draft Map of the Cannabis Proteome

Recently we have seen a relaxing on the historic restrictions on the use and subsequent research on the cannabis plants, generally classified as Cannabis sativa and Cannabis indica. What research has been performed to date has centered on chemical analysis of plant flower products, namely cannabinoids and various terpenes that directly contribute to phenotypic characteristics of the female flowers. In addition, we have seen many groups recently completing genetic profiles of various plants of commercial value. To date, no comprehensive attempt has been made to profile the proteomes of these plants. In this study we present our initial findings consisting of the identification of 17,269 unique proteins identified from Cannabis plant materials, as well as 6,110 post-translational modifications identified on these proteins. The results presented demonstrate the first steps toward constructing a complete draft map of the Cannabis proteome.

plant biology↗

bioRxiv2019

Comparison of single nucleotide variants identified by Illumina and Oxford Nanopore technologies in the context of a potential outbreak of Shiga Toxin Producing Escherichia coli.

BackgroundWe aimed to compare Illumina and Oxford Nanopore Technology (ONT) sequencing data from the two isolates of STEC O157:H7 to determine whether concordant single nucleotide variants were identified and whether inference of relatedness was consistent with the two technologies.\n\nResultsFor the Illumina workflow, the time from DNA extraction to availability of results, was approximately 40 hours in comparison to the ONT workflow where serotyping, Shiga toxin subtyping variant identification were available within seven hours. After optimisation of the ONT variant filtering, on average 95% of the discrepant positions between the technologies were accounted for by methylated positions found in the described 5-Methylcytosine motif sequences, CC(A/T)GG. Of the few discrepant variants (6 and 7 difference for the two isolates) identified by the two technologies, it is likely that both methodologies contain false calls.\n\nConclusionsDespite these discrepancies, Illumina and ONT sequences from the same case were placed on the same phylogenetic location against a dense reference database of STEC O157:H7 genomes sequenced using the Illumina workflow. Robust SNP typing using MinION-based variant calling is possible and we provide evidence that the two technologies can be used interchangeably to type STEC O157:H7 in a public health setting.

microbiology↗

bioRxiv2019

Application of Global Metabolomics to the Identification of Complex Counterfeit Medicinal Products

Food fraud and drug counterfeiting are of increasingly large concern to both global economics and to public health and safety. Simple medicinal products consisting of single synthesized or purified compounds can be tested for purity and authenticity rapidly with established assays such as chromatography and UV absorbance. Drugs derived from natural sources may contain hundreds or thousands of distinct chemical compounds and require correspondingly complex analytical methods. In this study we explore the use of methods developed for global metabolic profiling toward the identification of unknown complex medicinal products. By utilizing rapid solvent extraction followed by ultrahigh pressure high performance liquid chromatography (UHPLC) coupled to high resolution accurate mass spectrometry (HRAM-MS/MS), we can reliably obtain a profile of the samples molecular makeup. After profiling plant material to the depth of over 1,000 distinct molecules identified and quantified, we utilize these profiles to identify separately prepared and individually assayed blinded samples. We conclude that once a comprehensive library of small molecules has been acquired for each sample, identical preparations of products of unknown origin may be identified using simple statistical tools such as principal component analysis. We also conclude that these tools will be a valuable resource in affordably identified contaminated, adulterated and counterfeit products.\n\nAbstract Graphic\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC=\"FIGDIR/small/567339_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (31K):\norg.highwire.dtl.DTLVardef@1d985aforg.highwire.dtl.DTLVardef@1d7b68corg.highwire.dtl.DTLVardef@111b0a2org.highwire.dtl.DTLVardef@637b00_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2019

Assessing a commercial capillary electrophoresis interface (ZipChip) for shotgun proteomic applications

Capillary electrophoresis coupled electrospray ionization mass spectrometry (CE-MS) has long existed as a theoretical alternative to liquid chromatography coupled mass spectrometry (LC-MS). Until recently, however, the coupling of these technologies has occupied only a small niche within specific applications. A recent innovation in CE-MS is the ZipChip interface system from 908 devices that was pioneered by the Ramsay lab at NC State. This newly available source offers advantages over previous CE-MS interfaces including both relative ease of use and direct compatibility to thousands of mass spectrometers currently in use throughout the world with no hardware alterations. The ZipChip CE-MS has been demonstrated in recent studies to provide high resolution and rapid separations for the analysis of intact proteins, glycoproteins and glycosylated peptides, with more applications likely on the way. In this study we assess the capabilities of the ZipChip system in the context of high throughput global shotgun proteomics experiments. We find that on a high field Orbitrap system we can repeatedly identify as many as 800 unique protein groups in an experiment using a run time of 12 minutes. We find the ZipChip CE-MS system to be widely applicable for both data dependent and data independent acquisition experiments as well as targeted experiments. We conclude that the ZipChip is an attractive alternative solution to traditional nanoflow ESI-MS/MS for the analysis of the genomes of single celled organisms and for offline fractionation of eukaryotic proteomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/559591v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@6a46eforg.highwire.dtl.DTLVardef@156fc4dorg.highwire.dtl.DTLVardef@1ce562eorg.highwire.dtl.DTLVardef@17c1932_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

Bolt: A new age peptide search engine for comprehensive MS/MS sequencing through vast protein databases in minutes.

The standard platform for proteomics experiments today is mass spectrometry, particularly for samples derived from complex matrices. Recent increases in mass spectrometry sequencing speed, sensitivity and resolution now permit comprehensive coverage of even the most precious and limited samples, particularly when coupled with improvements in protein extraction techniques and chromatographic separation. However, the results obtained from laborious sample extraction and expensive instrumentation are often hindered by a sub optimal data processing pipelines. One critical data processing piece is peptide sequencing which is most commonly done through database search engines. In almost all MS/MS search engines users must limit their search space due to time constraints and q-value considerations. In nearly all experiments, the search is limited to a canonical database that typically does not reflect the individual genetic variations of the organism being studied. Searching for posttranslational modifications can exponentially increase the search space thus careful consideration must be used during the selection process. In addition, engines will nearly always assume the presence of only fully tryptic peptides. Despite these stringent parameters, proteomic data searches may take hours or even days to complete and opening even one of these criteria to more realistic biological settings will lead to detrimental increases in search time on expensive and custom data processing towers. Even on high performance servers, these search engines are computationally expensive, and most users decide to dial back their search parameters. We present Bolt, a new search engine that can search more than nine hundred thousand protein sequences (canonical, isoform, mutations, and contaminants) with 31 post translation modifications and N-terminal and C-terminal partial tryptic search in a matter of minutes on a standard configuration laptop. Along with increases in speed, Bolt provides an additional benefit of improvement in high confidence identifications, as demonstrated by manual validation of unique peptides identified by Bolt that were missed with parallel searching using standard engines. When in disagreement, 67% of peptides identified by Bolt may be manually validated by strong fragmentation patterns, compared to 14% of peptides uniquely identified by SEQUEST. Bolt represents, to the best of our knowledge, the first fully scalable, cloud based quantitative proteomic solution that can be operated within a user-friendly GUI interface. Data are available via ProteomeXchange with identifier PXD012700. Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=95 HEIGHT=200 SRC="FIGDIR/small/551622v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@8fcb0org.highwire.dtl.DTLVardef@58e02org.highwire.dtl.DTLVardef@ae5ec2org.highwire.dtl.DTLVardef@1cf5281_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗