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Discovering underneath a “MudPit”


Fan Liu

Fan Liu

What is referred to as “MudPit” here is not “a pit of mud” but a technique in the mass spectrometry field which stands for “multi-dimensional protein identification technology”, a very powerful approach that has been widely used since the year of its inception in 2001.

Many efficient technologies have been developed to reveal global behaviors of DNA (genome) and RNA (transcriptome), such as DNA-DNA interactions (through chromatin conformation capture on chip) and global gene expression (through transcriptome sequencing). However, the systematic study of proteins (proteome) lags behind the analytical DNA and RNA analysis.

There are mainly two reasons for this. First, even though mass spectrometry is very powerful for protein sequencing, it primarily does it one at a time as opposed to parallel sequencing of DNA and RNA, thus requiring more material or less complex samples. Second, because of the dynamic range limitations of mass spectrometry analysis, accurate measurements of very low abundant components are compromised by the present of highly abundant species. Therefore, the pre-separation of the complex proteome samples prior to entering the mass spectrometer is required.

MudPit is an approach for deep protein sequencing from complex mixtures. The technique consists of a multi-dimensional (normally two dimensional) chromatography separation, prior to mass spectrometry sequencing. The first dimension is usually based on a peptide’s unique physical properties of charge and hydrophobicity, such as strong cation exchange (SCX) chromatography. The second dimension is mostly reversed-phase (RP) chromatography, which complements SCX as it is efficient at removing salts and has the added advantage of being compatible with electrospray mass spectrometry.

The technique of MuPit was first developed by John Yates and colleagues in 2001, where they used SCX as the first dimension and RP as the second dimension of chromatographic separation1. It was applied to the proteomic study of Saccharomyces cerevisiae and yielded the largest proteome analysis at the time the paper was published: a total of 1,484 proteins were detected and identified.

Later on, more and more effective fractionation procedures were being developed. Here I just list two as examples.

MudPit technology

Fractionation procedures improve MudPit analysis

The first example is by Dr. Richard Smith’s group, where they applied a different type of two-dimensional separation2.  As oppose to SCX, they performed high pH reversed-phase liquid chromatography (RPLC) as the first dimension. As a result, they were able to obtain a significant enhancement for protein identification, including improved protein sequence coverage (1.6 fold), simplified sample processing and reduced sample losses, making this an attractive alternative to SCX chromatography in conjunction with the second dimension of low pH RPLC for two-dimensional proteomics analyses. Figure 1 illustrates the protein profile by different separation strategy nicely.

MudPit Technology - Results

3D separation system for MudPit analysis

The second example is a three-dimensional separation system developed by Dr. Neil Kelleher and colleagues, where they applied isoelectric focusing, multiplex GELFrEE and RPLC as the three dimensions (Figure 2).3  This work was published in Nature in 2011. In total, they identified 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modifications (PTMs), RNA splicing, and endogenous proteolysis.

Suffice it to say, MudPit changed the world of proteomics. Now we are approaching extremely deep proteome coverage near the transcriptome level, which was the dream of many scientists over a decade ago.


Washburn, M., Wolters, D., & Yates, J. (2001). Large-scale analysis of the yeast proteome via multidimensional protein identification technology. Nature Biotechnology, 19 (3), 242-247 DOI: 10.1038/85686

Yang, F., Shen, Y., Camp, D., & Smith, R. (2012). High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis Expert Review of Proteomics, 9 (2), 129-134 DOI: 10.1586/epr.12.15

Tran, J., Zamdborg, L., Ahlf, D., Lee, J., Catherman, A., Durbin, K., Tipton, J., Vellaichamy, A., Kellie, J., Li, M., Wu, C., Sweet, S., Early, B., Siuti, N., LeDuc, R., Compton, P., Thomas, P., & Kelleher, N. (2011). Mapping intact protein isoforms in discovery mode using top-down proteomics Nature, 480 (7376), 254-258 DOI: 10.1038/nature10575