Biology subjects

Orsburn, B.

Publications and source records attributed to Orsburn, B..

At least 19 recordsLinked to original sources

bioRxiv2026

A Parallel Accumulation-Mobility Aligned Fragmentation Strategy Utilizing High-Resolution Ion Mobility for High Performance Proteomics Analysis

Here we present a novel data independent acquisition (DIA) mass spectrometry (MS) operating mode termed parallel accumulation-mobility aligned fragmentation (PAMAF) that offers enhanced speed and sensitivity of ion fragmentation analysis for nontargeted discovery workflows such as bottom-up proteomics. This mode of operation leverages high-resolution ion mobility (HRIM) separation capabilities of the structures for lossless ion manipulation (SLIM) technology to achieve HRIM-based precursor isolation in place of traditional quadrupole filtering approaches. This PAMAF mode of operation increases the number of features that can be identified per MS1/MS2 acquisition cycle by employing mobility-based time alignment to associate fragment ions with their corresponding precursor ions. By using a high-speed, lossless separation technique for precursor isolation instead of the comparatively slow and wasteful quadrupole filtering method, we can avoid ion losses up to 99% while simultaneously increasing the rate at which precursor ions are sequentially fragmented and detected. Additionally, by storing ions in a trapping region while the previous packet of ions is being analyzed, the PAMAF mode achieves [~]100% ion utilization efficiency. Benchmarking results of LC-PAMAF-MS analysis of a whole cell protein digest showed approximately 6x more protein group identifications compared to a standard data-dependent acquisition (DDA) analysis without HRIM on the same QTOF instrument, and to over 100x improvement for low-load workflows. Quantitative evaluations demonstrated that PAMAF mode could quantify low abundance peptides, including those undetectable by DDA. Additionally, since precursor isolation in PAMAF mode is size-based rather than m/z-based, many coeluting isobars and isomers can be resolved prior to fragmentation to eliminate chimeric spectra that compromise identification accuracy. In this work we also explored the benefits of combining HRIM and quadrupole isolation to achieve improved specificity. This approach, known as DIA-PAMAF mode, further reduces the frequency of chimeric fragmentation spectra, and enabled the detection of over 8,000 protein groups from a HeLa digest analysis. PAMAF mode brings a powerful new technique to the field of proteomics that has the potential to improve the sensitivity and selectivity of mass spectrometry-based proteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/704896v1_ufig1.gif" ALT="Figure 1"> View larger version (114K): org.highwire.dtl.DTLVardef@7984c3org.highwire.dtl.DTLVardef@1fb2fe3org.highwire.dtl.DTLVardef@50d35org.highwire.dtl.DTLVardef@1a62926_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

The carrier proteome limit should be reassessed for each mass analyzer architecture

A clever utilization of classic proteomics reagents now allows the effective amplification of peptide sequencing potential in shotgun proteomics. The application of this method has helped usher in the exciting new field of single cell proteomics. While it was easy to first think that the discovery of Budnik et al., was finally the answer for protein PCR, limitations were carefully described by the authors and others. A study by Cheung et al., systematically identified the consequences of higher concentration carrier proteomes and defined the "carrier proteome limit". While this work has been replicated by others, every analysis published to date has used a variation of the same mass analyzer. When the same analysis is performed on alternative instruments, these limits appear to be very different and attributable to defined characteristics of each mass analyzer. Specifically, in mass analyzers with higher relative intrascan linear dynamic range, increased carrier channels appear far less detrimental to quantitative accuracy. As such, we may be limiting the power of isobaric peptide signal "amplification" by restricting ourselves to traditional mass analyzer options.

biochemistry↗

bioRxiv2024

Single cell proteomics by mass spectrometry reveals deep epigenetic insight and new targets of a class specific histone deacetylase inhibitor

Epigenetic programming has been shown to play a role in nearly every human system and disease where anyone has thought to look. However, the levels of heterogeneity at which epigenetic or epiproteomic modifications occur at single cell resolution across a population remains elusive. While recent advances in sequencing technology have allowed between 1 and 3 histone post-translational modifications to be analyzed in each single cell, over twenty separate chemical PTMs are known to exist, allowing thousands of possible combinations. Single cell proteomics by mass spectrometry (SCP) is an emerging technology in which hundreds or thousands of proteins can be directly quantified in typical human cells. As the proteins detected and quantified by SCP are heavily biased toward proteins of highest abundance, chromatin proteins are an attractive target for analysis. To this end, I applied SCP to the analysis of cancer cells treated with mocetinostat, a class specific histone deacetylase inhibitor. I find that 16 PTMs can be confidently identified and localized with high site specificity in single cells. In addition, the high abundance of histone proteins allows higher throughput methods to be utilized for SCP than previously described. While quantitative accuracy suffers when analyzing more than 700 cells per day, 9 histone proteins can be measured in single cells analyzed at even 3,500 cells per day, a throughput 10-fold greater than any previous report. In addition, the unbiased global approach utilized herein identifies a previously uncharacterized response to this drug through the S100-A8/S100-A9 protein complex partners. This response is observed in nearly every cell of the over 1,000 analyzed in this study, regardless of the relative throughput of the method utilized. While limitations exist in the methods described herein, current technologies can easily improve upon the results presented here to allow comprehensive analysis of histone PTMs to be performed in any mass spectrometry lab. All raw and processed data described in this study has been made publicly available through the ProteomeXchange/MASSIVE repository system as MSV000093434 Abstract graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/574437v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@89811corg.highwire.dtl.DTLVardef@17b7971org.highwire.dtl.DTLVardef@16221deorg.highwire.dtl.DTLVardef@19d9968_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

An integrated method for single cell proteomics with simultaneous measurements of intracellular drug concentration implicates new mechanisms for adaptation to KRASG12D inhibitors

It is well established that a population of single human cells will often respond to the same drug treatment in a heterogeneous manner. In the context of chemotherapeutics, these diverse responses may lead to individual adaptation mechanisms and ultimately multiple distinct methods of resistance. The obvious question from a pharmacology perspective is how intracellular concentrations of active drug varies between individual cells, and what role does that variation play in drug response heterogeneity? To date, no integrated methods for rapidly measuring intracellular drug levels while simultaneously measuring drug responses have been described. This study describes a method for single cell preparation that allows proteins to be extracted and digested from single cells while maintaining conditions for small molecules to be simultaneously measured. The method as described allows up to 40 cells to be analyzed per instrument per day. When applied to a KRASG12D small molecule inhibitor I observe a wide degree of intracellular levels of the drug, and that proteomic responses largely stratify based on the concentration of drug within each single cell. Further work is in progress to develop and standardize this method and - more importantly - to normalize drug measurements against direct measurements of cell volume. However, these preliminary results appear promising for the identification of single cells with unique drug response mechanisms. All data described in this study has been made publicly available through the ProteomeXchange consortium under accession PXD046002. Abstract graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/567669v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@11e5cfborg.highwire.dtl.DTLVardef@6a7dfborg.highwire.dtl.DTLVardef@1751923org.highwire.dtl.DTLVardef@c573a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

SCP Viz - A universal graphical user interface for single protein analysis in single cell proteomics datasets

Single cell proteomics (SCP) requires the analysis of dozens to thousands of single human cells to draw biological conclusions. However, assessing of the abundance of single proteins in output data presents a considerable challenge, and no simple universal solutions currently exist. To address this, we developed SCP Viz, a statistical package with a graphical user interface that can handle small and large scale SCP output from any instrument or data processing software. In this software, the abundance of individual proteins can be plotted in a variety of ways, using either unadjusted or normalized outputs. These outputs can also be transformed or imputed within the software. SCP Viz offers a variety of plotting options which can help identify significantly altered proteins between groups, both before and after quantitative transformations. Upon the discovery of subpopulations of single cells, users can easily regroup the cells of interest using straightforward text-based filters. When used in this way, SCP Viz allows users to visualize proteomic heterogeneity at the level of individual proteins, cells, or identified subcellular populations. SCP Viz is compatible with output files from MaxQuant, FragPipe, SpectroNaut, and Proteome Discoverer, and should work equally well with other formats. SCP Viz is publicly available at https://github.com/orsburn/SCPViz. For demonstrations, users can download our test data from GitHub and use an online version that accepts user input for analysis at https://orsburnlab.shinyapps.io/SCPViz/. Abstract graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/555397v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1c9d1d5org.highwire.dtl.DTLVardef@eefcfeorg.highwire.dtl.DTLVardef@23c098org.highwire.dtl.DTLVardef@1394161_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Cocaine Regulates Antiretroviral Therapy CNS Access Through Pregnane-X Receptor-Mediated Drug Transporter and Metabolizing Enzyme Modulation at the Blood Brain Barrier

BackgroundAppropriate interactions between antiretroviral therapies (ART) and drug transporters and metabolizing enzymes at the blood brain barrier (BBB) are critical to ensure adequate dosing of the brain to achieve HIV suppression. These proteins are modulated by demographic and lifestyle factors, including substance use. While understudied, illicit substances share drug transport and metabolism pathways with ART, increasing the potential for adverse drug:drug interactions. This is particularly important when considering the brain as it is relatively undertreated compared to peripheral organs and is vulnerable to substance use-mediated damage. MethodsWe used an in vitro model of the human BBB to determine the extravasation of three first-line ART drugs, emtricitabine (FTC), tenofovir (TFV), and dolutegravir (DTG), in the presence and absence of cocaine, which served as our illicit substance model. The impact of cocaine on BBB integrity and permeability, drug transporters, metabolizing enzymes, and their master transcriptional regulators were evaluated to determine the mechanisms by which substance use impacted ART central nervous system (CNS) availability. ResultsWe determined that cocaine had a selective impact on ART extravasation, where it increased FTCs ability to cross the BBB while decreasing TFV. DTG concentrations that passed the BBB were below quantifiable limits. Interestingly, the potent neuroinflammatory modulator, lipopolysaccharide, had no effect on ART transport, suggesting a specificity for cocaine. Unexpectedly, cocaine did not breach the BBB, as permeability to albumin and tight junction proteins and adhesion molecules remained unchanged. Rather, cocaine selectively decreased the pregnane-x receptor (PXR), but not constitutive androstane receptor (CAR). Consequently, drug transporter expression and activity decreased in endothelial cells of the BBB, including p-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 4 (MRP4). Further, cytochrome P450 3A4 (CYP3A4) enzymatic activity increased following cocaine treatment that coincided with decreased expression. Finally, cocaine modulated adenylate kinases are required to facilitate biotransformation of ART prodrugs to their phosphorylated, pharmacologically active counterparts. ConclusionOur findings indicate that additional considerations are needed in CNS HIV treatment strategies for people who use cocaine, as it may limit ART efficacy through regulation of drug transport and metabolizing pathways at the BBB.

neuroscience↗

bioRxiv2023

Analysis of single cells treated with the KRASG12D inhibitor MRTX 1133 reveals new challenges for the emerging field of single cell proteomics

Mutations in KRAS are common drivers of human cancers and are often those with the poorest overall prognosis for patients. A recently developed compound, MRTX1133, has shown promise in inhibiting the activity of KRASG12D mutant proteins, one of the main drivers in pancreatic cancer. To better understand the mechanism of action of this compound I performed both proteomics and metabolomics on four KRASG12D mutant pancreatic cancer cell lines. To obtain increased granularity in the proteomic observations, single cell proteomics was successfully performed on two of these lines. Following quality filtering, a total of 1,498 single cells were analyzed. From these cells 3,140 total proteins were identified with approximately 953 proteins quantified per cell. At 48 hours of treatment, two distinct populations of cells can be observed based on the level of effectiveness of the drug in decreasing total abundance of the KRAS protein in each respective cell, results that are effectively masked in the bulk cell analysis. All mass spectrometry data and processed results are publicly available at the www.massive.ucsd.edu at accessions PXD039597, PXD039601 and PXD039600. Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC="FIGDIR/small/533981v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@87cbdorg.highwire.dtl.DTLVardef@1356aaborg.highwire.dtl.DTLVardef@1ee3a9dorg.highwire.dtl.DTLVardef@14a4fc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

The SAGA HAT module is tethered by its SWIRM domain and modulates activity of the SAGA DUB module

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a transcriptional coactivator that both acetylates and deubiquitinates histones. The histone acetyltransferase (HAT) subunit, Gcn5, is part of a subcomplex of SAGA called the HAT module. A minimal HAT module complex containing Gcn5 bound to Ada2 and Ada3 is required for full Gcn5 activity on nucleosomes. Deletion studies have suggested that the Ada2 SWIRM domain plays a role in tethering the HAT module to the remainder of SAGA. While recent cryo-EM studies have resolved the structure of the core of the SAGA complex, the HAT module subunits and molecular details of its interactions with the SAGA core could not be resolved. Here we show that the SWIRM domain is required for incorporation of the HAT module into the yeast SAGA complex, but not the ADA complex, a distinct six-protein acetyltransferase complex that includes the SAGA HAT module proteins. In the isolated Gcn5/Ada2/Ada3 HAT module, deletion of the SWIRM domain modestly increased activity but had negligible effect on nucleosome binding. Loss of the HAT module due to deletion of the SWIRM domain decreases the H2B deubiquitinating activity of SAGA, indicating a role for the HAT module in regulating SAGA DUB module activity. A model of the HAT module created with Alphafold Multimer provides insights into the structural basis for our biochemical data, as well as prior deletion studies.

biochemistry↗

bioRxiv2020

Non-targeted Screening of Commercial CBD Products in the United States Reveals Common Contamination and Adulteration

The production of hemp and products derived from these plants that contain zero to trace amounts of the psychoactive cannabinoid tetrahydrocannabidiol (THC) is a rapidly growing new market in the United States. The most common products today contain relatively high concentrations of the compound cannabidiol (CBD). Recent studies have investigated commercial CBD products using targeted assays and have found varying degrees of misrepresentation and contamination of these products. To expand on previous studies, we demonstrate the application of non-targeted screening by high resolution accurate mass spectrometry to more comprehensively identify potential adulterants and contaminants. We find evidence to support previous conclusions that CBD products are commonly misrepresented in terms of cannabinoid concentrations present. Specifically, we observe a wide variation in relative THC concentrations across the product tested, with some products containing 10-fold more relative signal than others. In addition, we find that several products appear to be purposely adulterated with over the counter drugs such as caffeine and melatonin. We also observe multiple small molecule contaminants that are typically linked to improper production or packaging methods in food or pharmaceutical production. Finally, we present high resolution accurate mass spectrometry data and tandem MS/MS fragments supporting the presence of trace amounts of fluorofentanyl in a single mail order CBD product. We conclude that the CBD industry would benefit from more robust testing regulations and that the cannabis testing industry, in general, would benefit from the use of non-targeted screening technologies.

pharmacology and toxicology↗

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

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

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↗