Related Experiment Video
Updated: Apr 21, 2026
09:35
Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling
Published on: April 1, 2017
14.5K
Analysis of protein composition using multidimensional chromatography and mass spectrometry
Andrew J Link1, Michael P Washburn2
1Vanderbilt University School of Medicine Nashville, Tennessee.
Current Protocols in Protein Science
|November 5, 2014
Summary
Multidimensional Protein Identification Technology (MudPIT) enables extensive protein identification in complex mixtures using liquid chromatography and mass spectrometry. This advanced technique offers a powerful alternative to gel electrophoresis for analyzing whole proteomes and protein complexes.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Gel electrophoresis and in-gel digestion methods have limitations for analyzing complex protein mixtures.
- Direct analysis of proteins in solution is desirable for comprehensive proteomic studies.
- Automated database searching coupled with mass spectrometry is crucial for high-throughput protein identification.
Purpose of the Study:
- To describe and highlight the utility of Multidimensional Protein Identification Technology (MudPIT) for analyzing complex protein samples.
- To present MudPIT as an advancement over traditional gel-based proteomics methods.
- To emphasize the application of MudPIT in quantitative proteomic analysis for biological discovery.
Main Methods:
- Multidimensional liquid chromatography, combining reversed-phase and strong cation-exchange chromatography in a microcapillary column.
- Direct coupling of the multidimensional column inline between High-Performance Liquid Chromatography (HPLC) and tandem mass spectrometry.
- Automated database searching of mass spectrometry data for protein identification.
Main Results:
- Successful identification of large numbers of proteins from complex biological samples.
- Demonstration of MudPIT's capability to analyze whole proteomes and protein complexes directly in solution.
- Establishment of MudPIT as a powerful and versatile approach for complex mixture analysis.
Conclusions:
- MudPIT overcomes limitations associated with gel electrophoresis, offering a more direct and comprehensive protein identification strategy.
- This technology is highly effective for quantitative proteomic analysis, enabling the generation of novel biological insights.
- MudPIT represents a significant advancement in the field of proteomics for analyzing complex biological systems.
Keywords:
chromatographydirect analysis of large protein complexesmultidimensional protein identification technologyprotein complexesproteomicsMore Related Videos
08:04A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry
Published on: March 13, 2014
11.5K
07:49Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa
Published on: September 9, 2013
81.7K
Related Concept Videos
01:33Peptide Identification Using Tandem Mass Spectrometry
6.2K
Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
6.2K
01:21Tandem Mass Spectrometry
3.0K
Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and reduce chemical noise during analyte detection. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called...
3.0K
01:21Mass Spectrometry: Complex Analysis
2.1K
Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
2.1K
01:19MALDI-TOF Mass Spectrometry
5.7K
Mass spectrometry is a powerful characterization technique that can identify and separate a wide variety of compounds ranging from chemical to biological entities, based on their mass-to-charge ratio (m/z). The instruments that allow this detection, known as mass spectrometers, have three components: an ion source, a mass analyzer, and a detector. These spectrometers differ based on the nature of their ion source and analyzers.Matrix-assisted laser desorption ionization (MALDI) is a commonly...
5.7K
01:33Proteomics
7.4K
A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term...
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term...
7.4K