scholarly journals Automated high-throughput proteome and phosphoproteome analysis using paramagnetic bead technology

2019 ◽  
Author(s):  
Mario Leutert ◽  
Ricard A. Rodriguez-Mias ◽  
Noelle K. Fukuda ◽  
Judit Villén
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Large Scale ◽  
Magnetic Particles ◽  
Network Inference ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Substrate ◽  
Data Independent Acquisition ◽  
Automated Method

AbstractRecent developments in proteomics have enabled signaling studies where >10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase-substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges, we introduce rapid-robotic-phosphoproteomics (R2-P2), an end-to-end automated method that uses magnetic particles to process protein extracts to deliver mass spectrometry-ready phosphopeptides. R2-P2 is robust, versatile, high-throughput, and achieves higher sensitivity than classical protocols. To showcase the method, we applied it, in combination with data-independent acquisition mass spectrometry, to study signaling dynamics in the mitogen-activated protein kinase (MAPK) pathway in yeast. Our results reveal broad and specific signaling events along the mating, the high-osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method facilitates large-scale signaling studies involving hundreds of perturbations opening the door to systems-level studies aiming to capture signaling complexity.

scholarly journals High-Throughput Stool Metaproteomics: Method and Application to Human Specimens

mSystems ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Carlos G. Gonzalez ◽  
Hannah C. Wastyk ◽  
Madeline Topf ◽  
Christopher D. Gardner ◽  
Justin L. Sonnenburg ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Clinical Studies ◽  
Large Scale ◽  
Dietary Intervention ◽  
Negative Impact ◽  
Mass Spectrometry Analysis ◽  
Careful Study ◽  
Stool Samples ◽  
Study Participants

ABSTRACT Stool-based proteomics is capable of significantly augmenting our understanding of host-gut microbe interactions. However, compared to competing technologies, such as metagenomics and 16S rRNA sequencing, it is underutilized due to its low throughput and the negative impact sample contaminants can have on highly sensitive mass spectrometry equipment. Here, we present a new stool proteomic processing pipeline that addresses these shortcomings in a highly reproducible and quantitative manner. Using this method, 290 samples from a dietary intervention study were processed in approximately 1.5 weeks, largely done by a single researcher. These data indicated a subtle but distinct monotonic increase in the number of significantly altered proteins between study participants on fiber- or fermented food-enriched diets. Lastly, we were able to classify study participants based on their diet-altered proteomic profiles and demonstrated that classification accuracies of up to 89% could be achieved by increasing the number of subjects considered. Taken together, this study represents the first high-throughput proteomic method for processing stool samples in a technically reproducible manner and has the potential to elevate stool-based proteomics as an essential tool for profiling host-gut microbiome interactions in a clinical setting. IMPORTANCE Widely available technologies based on DNA sequencing have been used to describe the kinds of microbes that might correlate with health and disease. However, mechanistic insights might be best achieved through careful study of the dynamic proteins at the interface between the foods we eat, our microbes, and ourselves. Mass spectrometry-based proteomics has the potential to revolutionize our understanding of this complex system, but its application to clinical studies has been hampered by low-throughput and laborious experimentation pipelines. In response, we developed SHT-Pro, the first high-throughput pipeline designed to rapidly handle large stool sample sets. With it, a single researcher can process over one hundred stool samples per week for mass spectrometry analysis, conservatively approximately 10× to 100× faster than previous methods, depending on whether isobaric labeling is used or not. Since SHT-Pro is fairly simple to implement using commercially available reagents, it should be easily adaptable to large-scale clinical studies.

scholarly journals An ultrasensitive fiveplex activity assay for cellular kinases

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Christian M. Smolko ◽  
Kevin A. Janes
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Large Scale ◽  
Cell Types ◽  
Mitogen Activated Protein Kinase ◽  
Activity Measurement ◽  
Kinase Substrate ◽  
Iκb Kinase ◽  
Mitogen Activated Protein

AbstractProtein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 µg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.

scholarly journals A fully automated FAIMS-DIA proteomic pipeline for high-throughput characterization of iPSC-derived neurons

2021 ◽  
Author(s):  
Luke Reilly ◽  
Lirong Peng ◽  
Erika Lara ◽  
Daniel Ramos ◽  
Michael Fernandopulle ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Induced Pluripotent Stem Cell ◽  
Steady Increase ◽  
Parental Line ◽  
Proteomic Profiling ◽  
Data Independent Acquisition ◽  
Biological Insight ◽  
Data Browsing ◽  
Induced Pluripotent

Fully automated proteomic pipelines have the potential to achieve deep coverage of cellular proteomes with high throughput and scalability. However, it is important to evaluate performance, including both reproducibility and ability to provide meaningful levels of biological insight. Here, we present an approach combining high field asymmetric waveform ion mobility spectrometer (FAIMS) interface and data independent acquisition (DIA) proteomics approach developed as part of the induced pluripotent stem cell (iPSC) Neurodegenerative Disease Initiative (iNDI), a large-scale effort to understand how inherited diseases may manifest in neuronal cells. Our FAIMS-DIA approach identified more than 8000 proteins per mass spectrometry (MS) acquisition as well as superior total identification, reproducibility, and accuracy compared to other existing DIA methods. Next, we applied this approach to perform a longitudinal proteomic profiling of the differentiation of iPSC-derived neurons from the KOLF2.1J parental line used in iNDI. This analysis demonstrated a steady increase in expression of mature cortical neuron markers over the course of neuron differentiation. We validated the performance of our proteomics pipeline by comparing it to single cell RNA-Seq datasets obtained in parallel, confirming expression of key markers and cell type annotations. An interactive webapp of this temporal data is available for aligned-UMAP visualization and data browsing (https://share.streamlit.io/anant-droid/singlecellumap). In summary, we report an extensively optimized and validated proteomic pipeline that will be suitable for large-scale studies such as iNDI.

scholarly journals Quantification and Identification of Post-Translational Modifications Using Modern Proteomics

2021 ◽  
pp. 225-235
Author(s):  
Anja Holtz ◽  
Nathan Basisty ◽  
Birgit Schilling
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
High Throughput ◽  
High Resolution Mass Spectrometry ◽  
Label Free ◽  
Post Translational Modifications ◽  
Data Independent Acquisition ◽  
Lysine Residues ◽  
Detailed Protocol ◽  
Resolution Mass

AbstractPost-translational modifications (PTMs) occur dynamically, allowing cells to quickly respond to changes in the environment. Lysine residues can be targeted by several modifications including acylations (acetylation, succinylation, malonylation, glutarylation, and others), methylation, ubiquitination, and other modifications. One of the most efficient methods for the identification of post-translational modifications is utilizing immunoaffinity enrichment followed by high-resolution mass spectrometry. This workflow can be coupled with comprehensive data-independent acquisition (DIA) mass spectrometry to be a high-throughput, label-free PTM quantification approach. Below we describe a detailed protocol to process tissue by homogenization and proteolytically digest proteins, followed by immunoaffinity enrichment of lysine-acetylated peptides to identify and quantify relative changes of acetylation comparing different conditions.

scholarly journals Obesity Proteomics: An Update on the Strategies and Tools Employed in the Study of Human Obesity

2018 ◽  
Vol 7 (3) ◽  
pp. 27 ◽  
Author(s):  
Afshan Masood ◽  
Hicham Benabdelkamel ◽  
Assim Alfadda
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Large Scale ◽  
Protein Identification ◽  
Biochemical Characterization ◽  
Biological Fluids ◽  
Protein Composition ◽  
Cellular Protein ◽  
Human Obesity ◽  
Two Dimensional Gel Electrophoresis

Proteomics has become one of the most important disciplines for characterizing cellular protein composition, building functional linkages between protein molecules, and providing insight into the mechanisms of biological processes in a high-throughput manner. Mass spectrometry-based proteomic advances have made it possible to study human diseases, including obesity, through the identification and biochemical characterization of alterations in proteins that are associated with it and its comorbidities. A sizeable number of proteomic studies have used the combination of large-scale separation techniques, such as high-resolution two-dimensional gel electrophoresis or liquid chromatography in combination with mass spectrometry, for high-throughput protein identification. These studies have applied proteomics to comprehensive biochemical profiling and comparison studies while using different tissues and biological fluids from patients to demonstrate the physiological or pathological adaptations within their proteomes. Further investigations into these proteome-wide alterations will enable us to not only understand the disease pathophysiology, but also to determine signature proteins that can serve as biomarkers for obesity and related diseases. This review examines the different proteomic techniques used to study human obesity and discusses its successful applications along with its technical limitations.

scholarly journals Identification of an Arginase II Inhibitor via RapidFire Mass Spectrometry Combined with Hydrophilic Interaction Chromatography

2018 ◽  
Vol 24 (4) ◽  
pp. 457-465 ◽  
Author(s):  
Wataru Asano ◽  
Yu Takahashi ◽  
Motoaki Kawano ◽  
Yoshiji Hantani
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Hydrophilic Interaction Chromatography ◽  
Label Free ◽  
Hydrophilic Interaction ◽  
High Concentration ◽  
Allosteric Site ◽  
Arginase Ii

Peripheral arterial disease (PAD) is an occlusive disease that can lead to atherosclerosis. The involvement of arginase II (Arg II) in PAD progression has been proposed. However, no promising drugs targeting Arg II have been developed to date for the treatment of PAD. In this study, we established a method for detecting the activity of Arg II via high-throughput label-free RapidFire mass spectrometry using hydrophilic interaction chromatography, which enables the direct measurement of l-ornithine produced by Arg II. This approach facilitated a robust high-concentration screening of fragment compounds and the identification of a fragment that inhibits the activity of Arg II. We further confirmed binding of the fragment to the potential allosteric site of Arg II using a surface plasmon resonance assay. We concluded that the identified fragment is a promising compound that may lead to novel drugs to treat PAD, and our method for detecting the activity of Arg II can be applied to large-scale high-throughput screening to identify other structural types of Arg II inhibitors.

scholarly journals Large-scale human skin lipidomics by quantitative, high-throughput shotgun mass spectrometry

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Tomasz Sadowski ◽  
Christian Klose ◽  
Mathias J. Gerl ◽  
Anna Wójcik-Maciejewicz ◽  
Ronny Herzog ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Human Skin ◽  
High Throughput ◽  
Large Scale ◽  
Shotgun Mass Spectrometry

scholarly journals Integrated Molecular Characterisation of the MAPK Pathways in Human Cancers

2020 ◽  
Author(s):  
Musalula Sinkala ◽  
Panji Nkhoma ◽  
Nicola Mulder ◽  
Darren Patrick Martin
Keyword(s):  
Cancer Cells ◽  
Large Scale ◽  
Mapk Pathway ◽  
Gene Knockout ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Signalling Pathway ◽  
Mapk Pathways ◽  
Human Cancers ◽  
Mitogen Activated Protein ◽  
Molecular Aberrations

AbstractThe mitogen-activated protein kinase (MAPK) pathways are a crucial regulator of the cellular processes that fuel the malignant transformation of normal cells. The genetic underpinnings of molecular aberrations which lead to cancer involve mutations in and, transcription variations of, various MAPK pathway genes. Here, we use datasets of 40,848 patient-derived tumours representing 101 distinct human cancers to identify cancer-associated mutations in MAPK signalling pathway genes. We identify the subset of these genes within which mutations tend to be associated with the worst disease outcomes. Furthermore, by integrating information extracted from various large-scale molecular datasets, we expose the relationship between the fitness of cancer cells after CRISPR mediated gene knockout of MAPK pathway genes, and their dose-responses to MAPK pathway inhibitors. Besides providing new insights into MAPK pathways, we unearth vulnerabilities in specific pathway genes that are reflected in the responses of cancer cells to MAPK drug perturbations: a revelation with great potential for guiding the development of innovative therapeutic strategies.

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