MudPIT

Introduction
Before Multidimensional Protein Identification Technology (MudPIT) came about, Liquid Chromatography (LC) and Mass Spectrometry (MS) are used separately to fractionate and then identify protein composition from biological sample.

Disadvantage of using Liquid Chromatography
- Loss of material that commonly occurs in chromatographic processes

Disadvantage of using Mass Spectrometry (gel-based)
Although gel-based methods are widely used when it comes to the identification of protein, this method has several drawbacks:
- Problem identifying hydrophobic proteins
- Difficulty detecting low-level proteins (dye staining is not sensitive)
- Long experiment duration
- Inability to be automated
- Biological sample need to undergo solubilization

These problem decrease the sensitivity of the protein identification process and MudPIT seeks to address these problems by improving the separation and identification of proteins.


So what is MudPIT?
Multidimensional Protein Identification Technology, or MudPIT is a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm.

Advantage of MudPIT
- Eliminate the problems of gel-based approach when it comes to MS
- More sensitive and thus able to detect low abundance proteins
- two-dimensional chromatography technique reduces sample loss


Workflow of MudPIT
1. Preparation of protein sample
2. Digest protein sample to peptides
3. Peptides are then seperated into two liquid column chromatography steps:
-strong cationic exchange
-reversed-phase high performance liquid chromatography (HPLC)
3. Acquire tandem mass spectra of peptide
4. Search mass spectra against a protein sequence database
5. Identification of protein in the sample using SEQUEST

Some projects using MudPIT:
1) Protein pathway and complex clustering of correlated mRNA and protein expression analyses in Saccharomyces cerevisiae

2)Food Standards Agency
This site is researching on the feasibility of using MudPIT as an alternative to gel-based approach for the rigorous safety assessment of GM plants.

3)Chloroplast proteomics: potentials and challenges
This is a site on botany research, this research is about the analysis of chloroplast proteome.

source:

Technologies and Strategies for Reseach and Development

DRUG Discovery & Development

http://www.hupo.org/educational/past_congresses/2007_seoul/3_MacCoss_color.pdf

Nature Publishing Group

posted by Alvin