Multidimensional protein identification MUDPIT

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... 

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). 

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. 

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. 

PS—alkyne probe, followed by a CC reaction to introduce a biotin tag with a tobacco etch virus (TEV) protease cleavage site. Tagged proteins were then enriched with streptavidin beads and subsequendy digested by trypsin. The supernatant contained peptide fragments of the enriched proteins and was, in contrast to the previous procedure, saved for subsequent analysis by multidimensional protein identification technology (MudPIT), a novel two-dimensional LC—MS/MS analysis method that will be discussed in more detail... 

Washburn MP (2004) Utilisation of proteomics datasets generated via multidimensional protein identification technology (MudPIT). Brief Funct Genomic Proteomic 3(3) 280-286... 

At times, the effect of separation by charge and by mass is combined in the same column with the use of a biphasic matrix the distal half of the column is filled with HPLC resin for separation based on the mass of proteins, and the proximal half of the column is packed with ion exchange matrix separating the proteins based on the charge. This biphasic column is used when the elution buffer for the two systems is the same or compatible. Such separation by a biphasic column is suitable for the multidimensional protein identification technology (mudPIT) by mass spectroscopy. 

T. Kislinger, A. O. Gramolini, D. H. MacLennan, and A. Emili, Multidimensional protein identification technology (MUDPIT) technical overview of a profiling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue, J. Am. Soc. Mass Spectrom. 16, 1207-1220 (2005). 

An improvement over 2D PAGE involves two-dimensional microcapillary HPLC coupled with mass spectrometry (see Fig. 24.24). In this technique, called MudPIT (multidimensional protein identification technology, developed by John Yates and co-workers at The Scripps Research Institute), a microcapillary HPLC column is used that has been packed first with a strong cation-exchange resin and then a reversed-phase (hydrophobic) material. The two packing materials used in sequence and with different resolving... 

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