Alternative First Dimension Approaches

Adkins, J.N., Vamum, S.M., Auberry, K.J., Moore, R.J., Angell, N.H., Smith, R.D., Springer, D.L., Pounds, J.G. (2002). Toward a human blood serum proteome analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics 1,47-955. 

Ballif, B.A., Villen, J., Beausoleil, S.A., Schwartz, D., Gygi, S.P. (2004). Phosphoproteomic analysis of the developing mouse brain. Mol. Cell. Proteomics 3, 1093-1101. 

Blonder, J., Rodriguez-Galan, M.C., Chan, K.C., Lucas, D.A., Yu, L.R., Conrads, T.P, Issaq, H. J., Young, H.A., Veenstra, T.D. (2004). Analysis of murine natural killer cell microsomal proteins using two-dimensional liquid chromatography coupled to tandem electrospray ionization mass spectrometry. J. Proteome Res. 3, 862-870. 

Hattrup, E., Keeler, M., Letarte, J., Johnson, R., Haynes, PA. (2005). Comprehensive proteomics in yeast using chromatographic fractionation, gas phase fractionation, protein gel electrophoresis, and isoelectric focusing. Proteomics 5, 2018-2028. 

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For the analysis of nonvolatile compounds, on-line coupled microcolumn SEC-PyGC has been described [979]. Alternatively, on-line p,SEC coupled to a conventional-size LC system can be used for separation and quantitative determination of compounds, in which volatility may not allow analysis via capillary GC [976]. An automated SEC-gradient HPLC flow system for polymer analysis has been developed [980]. The high sample loading capacity available in SEC makes it an attractive technique for intermediate sample cleanup [981] prior to a more sensitive RPLC technique. Hence, this intermediate step is especially interesting for experimental purposes whenever polymer matrix interference cannot be separated from the peak of interest. Coupling of SEC to RPLC is expected to benefit from the miniaturised approach in the first dimension (no broadening). Development of the first separation step in SEC-HPLC is usually quite short, unless problems are encountered with sample/column compatibility. 

The bottom-up approach very much resembles classical protein identification strategies. The proteins in the proteome are first separated by 2D-GE (Ch. 17.3), or in some cases by SCX, size-exclusion (SEC), or affinity (AfC) chromatography. Specific proteins are excised from the gel, blotted, or electroeluted. The protein is digested, and the digest is analysed by LC-MS. The EC separation involves either RPLC with microcapillary or nano-LC columns (Ch. 17.5.2), or 2D-LC with typically SEC or SCX in the first dimension and RPLC in the second (Ch. 17.5.4). Alternatively, the sample may be introduced via either direct-infusion nano-ESl (Ch. 17.2), CE-MS (Ch. 17.5.6), or a microfluidic device coupled to MS (Ch. 17.5.5). 

Another approach to overcoming the limitations inherent in the lEF dimension of 2-DE is to use alternative types of 2-D separations. 2-D blue native (BN) electrophoresis (Schagger and von Jagow, 1991) can be used to separate membrane and other functional protein complexes as intact, enzymatically active complexes in the first dimension. This is followed with a second-dimension separation by Tricine-SDS-PAGE to separate the complexes into their component subunits. This method, combined with protein identification by MALDI PMF, has been applied to several studies of the mitochondrial proteome (Brookes et al., 2002 Kraft et al., 2001). In a study of human heart mitochondria using BN/SDS-PAGE, the individual subunits of all five complexes of the oxidative phosphorylation system were represented and a novel variant of cytochrome c oxidase subunit Vic was reported (Devreese et al., 2002). 

Alternatively, when, across-the-board screening of an entire sample is required, MDGC is too time-consuming and complicated, and a comprehensive, i.e., a GC x GC, approach has to be used. " In this case, the entire first-column (first-dimension) eluate, cut into small adjacent fractions to maintain the first-dimension resolution, is subjected to further analysis on the second (second-dimension) column. GC X GC provide superior analyses... 

Computer simulations of bulk liquids are usually performed by employing periodic boundary conditions in all three directions of space, in order to eliminate artificial surface effects due to the small number of molecules. Most simulations of interfaces employ parallel planar interfaces. In such simulations, periodic boundary conditions in three dimensions can still be used. The two phases of interest occupy different parts of the simulation cell and two equivalent interfaces are formed. The simulation cell consists of an infinite stack of alternating phases. Care needs to be taken that the two phases are thick enough to allow the neglect of interaction between an interface and its images. An alternative is to use periodic boundary conditions in two dimensions only. The first approach allows the use of readily available programs for three-dimensional lattice sums if, for typical systems, the distance between equivalent interfaces is at least equal to three to five times the width of the cell parallel to the interfaces. The second approach prevents possible interactions between interfaces and their periodic images. 

The lower, chloroform-rich phase is separated carefully from the protein-containing interface, and then it is washed twice with methanol-water (10 9, v/v) and the washes are discarded. The chloroform layer contains the phosphatidic acid (as a sodium salt) and can be isolated by acetone precipitation. The yields can be of the order of 90-95%. One alternative route to identification of the chloroform-soluble material is to analyze it for total phosphorus and total fatty acid ester (see procedures described earlier). In the case of diacylphosphatidylcholine as the substrate, the fatty acid ester/P molar ratio should be 2.0. Another approach is to subject the chloroform-soluble fraction to preparative thin-layer chromatography on silica gel H (calcium ion free) in a two-dimensional system with a solvent system of chloroform-methanol-28% ammonium hydroxide (65 35 6, v/v) in the first direction and a solvent system of chloroform-acetone-methanol-glacial acetic acid-water (4.5 2 1 1.3 0.5, v/v) in the second direction. The phosphatidic acid will not migrate far in the basic solvent Rf 0.10) and will show an Rf value one-half of that of any remaining starting substrate (fyO.40) in the second solvent. Of course with a simple substrate system, one can use the basic solvent in one dimension only... 

For Maxwell models, where Tj = 0, we need to distinguish the sub- and the supercritical ceises. In the subcritical case (i.e., U < yT]j pX)) and in two space dimensions, one can prescribe the diagonal components o-p and 7P, whereas in three space dimensions a correct choice of boundary conditions for tP is not simple. A possible choice of four boundary conditions is a nonlocal one (in terms of the Fourier components of rP—see [29]). An alternative approach leading to first order differential boundary conditions at the inflow boundary is described in [30]. 

First, one can experimentally measure as many fluxes (or more) as the dimension of the null-space, so as to uniquely calculate the remaining fluxesJ " This approach is called metabolic flux analysis. Alternatively, an objective of the metabolic network can be chosen to computationally explore the best use of the metabolic network by a given metabolic genotype. Herein, we pursue the second option. The solution to Eq. (7) subject to the linear inequality constraints can be formulated as a linear programming (LP) problem, in which one finds fhe flux distribution that minimizes a particular objective. Mathematically, the LP problem is stated as ... 

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