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GEN Protocols

OMICS

OMICS

Protocol

IsoCode Single-Cell Adaptive Immune: Human CAR-T Protocol Using AF647-Conjugated Antibodies to CD4 and CD8

Overview of ProtocolDay 1: Cryopreserved CAR-T cells are thawed and cultured overnight in the presence of IL-2. Cryopreserved target cell lines are thawed, cultured, and passaged several days prior to beginning the experiment.Day 2: Enrichment and Antigen-Stimulation of CD8 + and/or CD4 + CAR-T cell... More


Protocol

Rapid and sensitive label-free proteome quantification on the timsTOF Pro 2

Shotgun proteomics capacity to deliver in-depth information on the identification, the relative amount, and the modification patterns of ever larger fraction of the proteins present in a sample has made it an essential approach in most biological research areas. The recent introduction of Parallel A... More


Protocol

Ultra-high sensitivity proteomics on the timsTOF SCP

The timsTOF Pro platform, introduced in 2017, already featured a sensitivity boost from the Parallel Accumulation SErial Fragmentation (PASEF) technology, which provides time and space focusing of the ions in the Trapped Ion Mobility Spectrometry (TIMS) tunnel. The timsTOF SCP platform further enhan... More


Protocol

MRMS powered single cell metabolomics – Quantification of picogram amounts of a biocatalytic product from few living cells

Probing the metabolic state of living microbial cells via their secreted metabolic products is highly challenging as it requires measures to maintain the cellular integrity and to collect the analytes. This application note demonstrates how direct infusion magnetic resonance mass spectrometry (DI-MR... More


Protocol

prm-PASEF®: enabling high-throughput, high sensitivity targeted proteomics

In comparison with standard selected and parallel reaction monitoring (SRM and PRM), the prm-PASEF increases the number of peptides that can be targeted in a single acquisition method, without compromising the selectivity or the sensitivity. Using a prototype acquisition software, we targeted 201 is... More


Protocol

Incorporating CCS values to enable 4-dimensional annotation of metabolic features

Reliable annotation of metabolites for LC-MS/MS based data requires the adept combination of many parameters such as retention time and accurate mass. To extend our parameter portfolio, we analyzed the high reproducible measured CCS values using trapped ion mobility spectrometry (TIMS) to provide ad... More


Protocol

Studying protein-drug complexes under native conditions in the low nM range using MRMS

Protein ligand complexes need to be preserved through the electrospray and detection process. This work describes high resolution native mass spectrometric measurements of the enzyme carbonic anhydrase II (Figure 1) and the non-covalent complex with the substrate acetazolamide (Figure 2) using a sci... More


Protocol

Proteomic Interrogation of Primary Insulin-secreting Pancreatic β-cells

In this work we leveraged the analytical performance of the timsTOF Pro to identify expression of low-level enzymes for the first time in primary insulinsecreting beta cells (β-cells). We found that these proteins are critical, functional components of key metabolic pathways which can support β-cell... More


Protocol

Magnetic Resonance Mass Spectrometry (MRMS) discriminates yeast mutants through metabolomics

A eukaryote model of Saccharomyces cerevisiae was used to study the methylglyoxal pathway by Magnetic resonance mass spectrometry (MRMS). It was discovered that glutathione plays a major role in driving metabolomic differences between different strains. It is expected that metabolomics-based discrim... More


Protocol

Elucidation of metabolic changes in HFD-ApoE–/– model by SP6 peptide: A flow injection analysis magnetic resonance mass spectrometry (FIA-MRMS) study

The employment of natural peptides in the treatment of cardiovascular diseases is an attractive option to pharmacological therapy. In this study, a novel antihypertensive peptide from Spirulina Platensis (SP6) was employed to evaluate its potential anti-atherosclerotic effect in high fat diet ApoE–/... More


Protocol

Proteomic Interrogation of Primary Insulin-secreting Pancreatic β-cells

In this work we leveraged the analytical performance of the timsTOF Pro to identify expression of low-level enzymes for the first time in primary insulinsecreting beta cells (β-cells). We found that these proteins are critical, functional components of key metabolic pathways which can support β-cell... More


Protocol

Magnetic Resonance Mass Spectrometry (MRMS) discriminates yeast mutants through metabolomics

A eukaryote model of Saccharomyces cerevisiae was used to study the methylglyoxal pathway by Magnetic resonance mass spectrometry (MRMS). It was discovered that glutathione plays a major role in driving metabolomic differences between different strains. It is expected that metabolomics-based discrim... More


Protocol

Elucidation of metabolic changes in HFD-ApoE–/– model by SP6 peptide: A flow injection analysis magnetic resonance mass spectrometry (FIA-MRMS) study

The employment of natural peptides in the treatment of cardiovascular diseases is an attractive option to pharmacological therapy. In this study, a novel antihypertensive peptide from Spirulina Platensis (SP6) was employed to evaluate its potential anti-atherosclerotic effect in high fat diet ApoE–/... More


Protocol

High throughput 4D-Proteomics – Application of dia-PASEF® and the Evosep One for short gradients

In this application note, we demonstrate the benefits of dia-PASEF technology on the timsTOF Pro platform coupled to an Evosep One for high-throughput in-depth proteome analysis of up to 300 samples per day (SPD). We quantify about 5200 protein groups in only 21-minutes run time. In 4.8-minutes ultr... More


Protocol

How to Determine Glycan Profiles of Biopharmaceuticals from Peptide Mapping Data

Glycosylation is a common critical quality attribute (CQA) of therapeutic proteins and needs to be characterized during product development. Typically, this analysis is conducted after enzymatic release and tagging of the glycans followed by fluorescence and MS detection. However, that approach lose... More


Protocol

Achieving Reproducible and Automated Region-Specific Lipid Annotations using CCS-aware SpatialOMx®

The CCS-aware SpatialOMx® workflow opens new dimensions by combining the molecular and spatial information measured by MALDI-TIMS Imaging with highly confident 4D-Omics annotations. MetaboScape® 2021b and SCiLS™ Lab 2021a provide the interface to match data from both ionization techniques and enable... More


Protocol

Next-generation MALDI top-down sequencing of protein biotherapeutics – expanding the scope of timsTOF technology

This new method takes advantage of Trapped Ion Mobility Spectrometry (TIMS), to further enhance MALDI-TDS by adding another dimension of separation in the gas phase.High confidence N- and C-terminal sequencing specifically are enhanced by combining high resolution (~ 60,000 RP) and 2 ppm mass accura... More


Protocol

Investigating the increased lifespan in C. elegans daf-2 mutants by 4D-Lipidomics™

The small nematode Caenorhabditis elegans is one of the premier biomedical model organisms and employed in many aspects of basic and applied science.... More


Protocol

IsoCode Single-Cell Adaptive Immune: Human Bispecific Protocol

Overview of ProtocolDay 1: Cryopreserved T cells are thawed and cultured overnight in the presence of IL-2.Day 2: Enrichment, Staining and Antigen Stimulation of CD4+ and/or CD8+ T cells for 36-48 hours.Day3: Loading of T cells onto IsoCode chip.... More


Protocol

Single-Cell Intracellular Proteome: HeLa Cell Protocol

Overview of Protocol:Day 1-3: Cryopreserved HeLa cells are thawed and cultured for 72 hours.Day 4: Staining and stimulating of HeLa cells followed by immediate loading onto IsoCode Chip... More


Application

Access the Breadth of Sample Preparation Protocols to Awaken True Functional Immune Biology

Precedent Protocols and Processes ensure that enrichment, stimulation and labeling will result in maximizing the correlative data achieved, while minimizing non-specific impact on the cells themselves. Highlights: • Published enrichment, stimulation, and staining protocols throughout immunotherapy ... More


Primer

Source and Elimination of Common Mass Spectrometry Contaminants

Sensitivity is a key attribute of mass spectrometers to allow quantification of minute amounts of analytes. Yet this sensitivity can be a conundrum. Myriad sources can contribute contaminants that can impact background and confound interpretation of results. An increased background signal can make i... More


Primer

Selecting a Functional Oligonucleotide from a Random Mix

SELEX (Systematic Evolution of Ligands by EXponential enrichment) was first described and used in 1990 to isolate a functional oligonucleotide from a random pool of differently-shaped DNA or RNA molecules.1-2SELEX-derived oligonucleotides (RNA or single-stranded DNA) known as aptamers, bind with hig... More


Primer

Single-Cell RNA Sequencing Upgrades Sensitivity

A new method for DNA amplification and sequencing called TAS-Seq improves the accuracy of single-cell RNA sequencing (scRNA-seq). TAS-Seq uses a terminal transferase enzyme that amplifies and sequences DNA complementary to mRNA (cDNA) in solid phase on a nanowell/magnetic bead-based cell isolation p... More


Primer

Cyclic Microchip Assay Enhances Throughput of Protein Detection in Single Cells

Most existing single-cell protein analysis methods can analyze about a dozen proteins at a time when the execution of nearly every conceivable cellular function depends on a complex and diverse proteome.A new microchip assay called CycMIST1 (single-cell cyclic multiplex in situ tagging) can measure ... More


Primer

Solvent Precipitation Method Increases Protein Recovery for Proteomics

Study designs in proteomics aim for inclusivity—to characterize every protein in a sample. However, extracting proteins from complex biological samples involves the use of harsh buffers containing detergents and salts that are incompatible with the proteomics workflow. More proteins are lost in atte... More


Mixer

Mapping Tissue Carbohydrates Using Mass Spectrometry

This mixer will be relevant for all interested in methods used in glycoproteomics research that aims to understand the pivotal role of carbohydrate motifs that modify the proteome.Glycoproteomics, a dynamic area of frontier research, provides a systems biology approach to understanding the biologica... More


Mixer

Spatial Biology Tools and Techniques

Single-cell spatial phenotyping is transforming biomedical research in fields ranging from oncology to neuroscience. This GEN Protocols Mixer focuses on technological developments and protocols that have enabled spatial approaches in biology. We will explore novel applications of the technology in d... More


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