Multidimensional protein identification

Wolters, D. A. Washburn, M. P. Yates, J. R., 3rd. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 2001, 73, 5683-5690. 

Table 5.1 lists several heart-cut and comprehensive techniques. Heart-cut 2DLC is very common and has great application for the increased resolution of one or several components from the first dimension (Augenstein and Stickler, 1990 Majors, 1980 Pasch et al., 1992 and Dixon et al., 2006). Heart-cut 2DLC for the analysis of polymers is often referred to as cross-fractionation (Balke and Patel, 1980). Protein digest analysis with MS/MS identification has been called multidimensional protein identification technology or MUDPIT. This is described in detail in Chapter 11. 

There exist essentially three categories of SCX/RP/MS/MS approaches. In one approach, SCX is run off-line followed by on-line RP/MS/MS (Fig. 11.1). In the offline SCX approach, fractions do not directly elute onto RP material but rather are collected. In one of the two in-line approaches, SCX is run in line with RP/MS/MS using different columns for SCX and RP (Fig. 11.2). In the multidimensional protein identification technology approach (MudPIT), SCX and RP are run in line in the same column, and this column serves as the ion source for a tandem mass spectrometer (Fig. 11.3). Both the in-line approaches are true SCX/RP/MS/MS approaches the first approach could be abbreviated as SCX—RP/MS/MS where... 

Washburn, M.P., Wolters, D., Yates, J.R., 3rd (2001). Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242-247. 

This multidimensional protein identification technology (MudPIT) specifically incorporates a strong cationic exchange (SCX) column in tandem with an RP column to achieve maximal resolution and exquisite sensitivity. MudPIT is effective for studying complex proteomes such as mammalian cellular samples. It has been applied to large-scale protein characterization with identification of up to 1484 proteins from yeast in a single experiment.12... 

In the case of peptide separation by HPLC, separation modes are combined in series. This approach is called tandem LC. For instance, ion exchange associated with RP is used for peptide separation. Multidimensional protein identification technique (MudPIT) involving use of microcapillary columns (SCX cationic column and RP column) linked in series and eluted into MS is preferred for separation of complex peptide mixtures (Figure 5.4). 

Bioinformatics tools involving computer-based statistical analyses are essential for data management and analysis. When a complex biological sample containing thousands of different proteins is analyzed by multifaceted approaches, such as multidimensional protein identification technology, the identification of the proteins in the mixture is extremely complicated. Even multiple peptide identification methods, such as using both MS and... 

MS, either after electrophoretic separation and proteolysis or independently of 2DE [e.g., as in Multidimensional Protein Identification Technology (MudPIT), Ref. 21, also see Sec. 3.1.3), as a stand-alone system for protein identification. 

Yates III, J. R. (2001). An automated multidimensional protein identification technology for shotgun proteomics. 

Another subsynaptic fraction derived directly from presynaptic specializations, the presynaptic particle fraction (PPF) can be separated from PSD by adjusting the pH of Triton X-100 extraction of isolated trans-synaptic scaffolds. As one might expect, it has been shown that the major proteins of the PPF, clathrin and dynamin, are concentrated in the presynaptic compartment (Phillips et al. 2005). These investigators used multidimensional protein identification technology (MudPIT) to compare the PPF and the PSD fraction. Of 341 proteins identified, 50 localized in the PPF, 231 in the PSD fraction, and 60 were found to be common to both fractions. The PPF was also characterized by a low proportion of actin and actin-associated proteins along with a high proportion of vesicle proteins. The authors concluded that the PPF consists of presynaptic proteins not cormected to the actin-based synaptic framework and that clathrin may be an anchorage scaffold for many presynaptic proteins. 

Washburn MP. Utilisation of proteomics datasets generated via multidimensional protein identification technology (MudPlT). Brief. Funct. Genom. Proteom. 2004 3 280-286. 

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