Mass Spectrometry-based Peptidomics for Biomaker Discovery
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This document discusses the use of mass spectrometry-based peptidomics for biomarker discovery. It describes how peptidomics involves the study of low molecular weight protein fragments and peptides that can serve as biomarkers. The key steps in biomarker discovery using peptidomics are sample preparation, separation of peptides, detection and identification of peptides using mass spectrometry, quantitative evaluation of peptide biomarkers, data analysis, and biomarker validation. Mass spectrometry provides a high-throughput way to detect peptide biomarkers and determine their sequences and concentrations.
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Mass Spectrometry-based Peptidomics for Biomaker Discovery
- 1. Mass Spectrometry-based Peptidomics for Biomarker Discovery Creative Proteomics Presentation
- 2. Peptidomics • Peptidomics can be easily defined as the study of the low molecular weight fraction protein fragments from endogenous protein degradation products, or other small proteins such as cytokines and signaling peptides. • Peptide in the peptidome can be classified into two parts. One is the bioactive peptides shed from all cell types in the microenvironment, which can serve as reporters for cell-to-cell communications, such as hormones and cytokines. The other is peptide fragments cleaved by enzymes resulting from in vivo resident proteins.
- 3. Biomarkers Biomarker Forecast disease Diagnose various disorders Guide clinical therapy Monitor medicine response Biomarkers are molecules that indicate a physiological state and also the change during a disease process.
- 4. Steps Separation Sample Preparation Detection and Identification Quantitative Evaluation Data Analysis Biomakers Validation Peptidome plays an important role in biomarker discovery.
- 5. Sample Preparation Because many disease identifying biomarkers are likely to be low-abundance proteins, it is imperative to remove the high-abundance proteins or apply enrichment techniques to allow detection and better coverage of the low-abundance proteins for MS analysis. organic solvent precipitation ultrafiltration magnetic beads solid- phase extraction (SPE) affinity removal column strong cation exchange (SCX)
- 6. Separation • Before being injected into the mass spectrometer, the peptides need to be separated because of the high complexity. • For example, when using MS, the peptides with the same amino acid composition but different sequences generate the same mass spectra of molecular ions. • As a technique usually combined with MS, liquid chromatography (LC) is the most popular method for peptide separation. Capillary Electrophoresis (CE) High Performance Liquid Chromatography (HPLC)
- 7. Detection and Identification • Detection of numerous fragments necessitates the development of high-throughput, accurate, and sensitive mass spectrometer. • There are four commonly used types of mass analyzers: quadrupole, time-of-flight, ion trap, and Fourier transform ion cyclotron resonance. • In order to determine the amino acid sequence of a specific peptide, the tandem MS mode should be chosen in the operation. • The collision-induced dissociation (CID) fragmentation technique is incorporated in basically all types of tandem MS. Electron capture dissociation (ECD) and electron transfer dissociation (ETD) are developed for providing explicit fragmental spectra. Quadrupole Time of Flight Ion Trap Fourier Transform Ion Cyclotron Resonane
- 8. Quantitative Evaluation Peptide biomakers Show which peptides are present in a sample (qualitative analysis) Determine the concentration (quantitative analysis) • The non-gel quantification can be divided into two main categories: stable isotope labeling and label-free methods. • Various isotope labeling methods include ICAT, TMT, iTRAQ and so on. But it has some limitations, like the increasing cost, complexity of sample preparation, complicate data processing, etc. • Currently, label-free quantitative approaches have been developed based on the chromatographic peak area of a peptide or frequency of MS/MS spectra correlating to the peptide concentration.
- 9. Data Analysis • The mass spectrometer can produce a large amount of data, which is complex and hard to analyze. • The data is submitted to data mining algorithms to get statistical interpretation, which is divided into two categories, unsupervised methods and supervised methods. • Mass spectra are analysed using search engines. Commonly used search engines Mascot SeQuest algorithms X!Tandem Peptide sequencing information databases non-redundant NCBI Erop-Moscow Peptidome Pepbank
- 10. Biomarkers Validation • After biomarker discovery and characteristics, there is a need to validate putative biomarkers identified by MS-based analysis. • It requires to offer orthogonal analysis to rule out a false positive by MS and providing additional evidence for the biomarker candidates from the study for future potential clinical assays. • Multiple-reaction monitoring (MRM) • Antibody-based Assays Western blotting ELISA Immunochemistry
- 11. Our Services Equipped with high resolution mass spectrometer, the bioanalysts and technicians in Creative Proteomics can offer professional research assistance in your biomarker discoveries.
- 12. Thanks Please contact us for more information Web: Email: www.creative-proteomics.com info@creative-proteomics.com
Hinweis der Redaktion
- Hello, welcome to watch Creative Proteomics’ Video. Today, we are going to briefly introduce Mass Spectrometry-based Peptidomics for Biomaker Discovery
- Peptidomics can be easily defined as the study of the low molecular weight fraction protein fragments from endogenous protein degradation products, or other small proteins such as cytokines and signaling peptides. Peptide in the peptidome can be classified into two parts. One is the bioactive peptides shed from all cell types in the microenvironment, which can serve as reporters for cell-to-cell communications, such as hormones and cytokines. The other is peptide fragments cleaved by enzymes resulting from in vivo resident proteins.
- Biomarkers are molecules that indicate a physiological state and also the change during a disease process. In human bodies, peptidome biomakers can be used to forecast disease, diagnose various disorders, guide clinical therapy, and monitor medicine response.
- Peptidome plays an important role in biomarker discovery. The mass spectrometry-based peptidomics for biomaker discovery contains sample preparation, saparation, detection and identification, quantitative evaluation, data analysis, as well as Biomakers validation.
- Because many disease identifying biomarkers are likely to be low-abundance proteins, it is imperative to remove the high-abundance proteins or apply enrichment techniques to allow detection and better coverage of the lowabundance proteins for MS analysis. Many methods and technologies have been applied to extract peptides and small proteins, such as organic solvent precipitation, ultrafiltration, magnetic beads, solid-phase extraction, affinity removal column, strong cation exchange, and so on. In addition, ultrafiltration is realized by filtering the sample using a membrane of a defined molecular weight cut-off based on size exclusion mechanism
- Before being injected into the mass spectrometer, the peptides need to be separated because of the high complexity. For example, when using mass spectrometry, the peptides with the same amino acid composition but different sequences generate the same mass spectra of molecular ions. As a technique usually combined with MS, liquid chromatography is the most popular method for peptide separation. And high performance liquid chromatography has been widely used for bioactive peptide profiling. In addition, there are several other methods, including multidimensional separation technology, and capillary electrophoresis.
- A detection system for peptide biomakers has two tasks. One is to show which peptides are present in a sample (qualitative analysis). The other is to determine the concentration (quantitative analysis). With the development of the mass spectrometry, there are various techniques for gel-free quantitative analyses of peptidomics samples. The non-gel quantification can be divided into two main categories: stable isotope labeling and label-free methods. Various isotope labeling methods include ICAT, TMT, Itraq and so on. But it has some limitations, like the increasing cost, complexity of sample preparation, complicate data processing, etc. Currently, label-free quantitative approaches have been developed based on the chromatographic peak area of a peptide or frequency of MS/MS spectra correlating to the peptide concentration.
- The mass spectrometer can produce a large amount of data, which is complex and hard to analyze. The data is submitted to data mining algorithms to get statistical interpretation, which is divided into two categories, unsupervised methods and supervised methods. Mass spectra are analysed using search engines. There are some commonly used search engines, including Mascot, SeQuest algorithms, X!Tandem. Peptide sequencing information databases include non-redundant NCBI, Erop-Moscow, Peptidome, Pepbank and so on.
- Equipped with high resolution mass spectrometer, the bioanalysts and technicians in Creative Proteomics can offer professional research assistance in your biomarker discoveries.
- Thanks for watching our video. At creative proteomics, we provide the most reliable peptidomics services. If you have any questions or specific requirements. Please feel free to contact us. We are very glad to cooperate with you.