Peptide multidimensional chromatography

Other reviews of multidimensional separations have been published. These include a book on polymer characterization by hyphenated and multidimensional techniques (Provder et al., 1995), a review on polymer analysis by 2DLC (van der Horst and Schoenmakers, 2003), and two reviews on two-dimensional techniques in peptide and protein separations (Issaq et al., 2005 Stroink et al., 2005). Reviews on multidimensional separations in biomedical and pharmaceutical analysis (Dixon et al. 2006) and multidimensional column selectivity (Jandera, 2006) were recently published. Suggested nomenclature and conventions for comprehensive multidimensional chromatography were published in 2003 (Schoenmakers et al., 2003), and a book chapter in the Advances in Chromatography series on MDLC was published in 2006 (Shalliker and Gray 2006). 

COUPLED MULTIDIMENSIONAL CHROMATOGRAPHY AND TANDEM MASS SPECTROMETRY SYSTEMS FOR COMPLEX PEPTIDE MIXTURE ANALYSIS... 

An inherent drawback of this methodology is that quantitative information is only obtained from those peptides which are subjected to MS/MS analysis. This fact stresses the necessity for efficient peptide separation via multidimensional chromatography (e.g., SCX/reversed-phase HPLC) prior to tandem MS analysis. Moreover, ion trap instruments cannot be employed in quantitative iTRAQ experiments since their peptide fragmentation spectra do not contain information... 

Protein quantitation by MS makes it possible to use in-line liquid-phase separation methods such as multidimensional chromatography and capillary electrophoresis. Quantitation can be carried out by comparing peptide ion currents but this is inherently inaccurate and is biased by instrument design. Instead, quantitation is often based on the use of stable isotopes. The general approach is to label alternative samples with equivalent reagents, one of which contains a heavy isotope and one of which contains a light isotope. The samples are mixed, separated into fractions, and analyzed by MS. The ratio of the two isotopic variants can be determined from the heights... 

Other groups have also used EC and CE to perform non-comprehensive multidimensional separations (15, 16). A three-dimensional separation was performed by Stromqvist in 1994, where size exclusion chromatography (SEC), reverse-phase HPLC, and CZE were used in an off-line manner to separate peptides (17). The most useful information gained from all of these non-comprehensive studies was knowledge of the orthogonality and compatibility of EC and CE. 

Figure 15.6 Chromatogram of a plasma standard of human leukocyte elastase inhibitors obtained by using LC-LC. Adapted from Journal of Liquid Chromatography and Related Technologies, 19, R. A. Earley and L. R Tini, Versatile multidimensional chromatographic system for di ug discovery as exemplified by the analysis of a non-peptidic inhibitor of human leukocyte elastase , pp. 2527-2540, 1996, by courtesy of Marcel DekkeiTnc.

One attempt to overcome these disadvantages has been to use multidimensional liquid chromatography (LC) followed directly by tandem mass spectrometry to separate, fragment and identify proteins (Link et al., 1999). In this process, a denatured and reduced protein mixture is digested with a protease to create a collection of peptides (Fig. 2.6). The peptide mixture is applied to a cation exchange column and a fraction of these peptides are eluted based on charge onto a reverse-phase column. The... 

Valentine, S.J., Kulchania, M., Srebalus Barnes, C.A., Clemmer, D.E. (2001). Multidimensional separations of complex peptide mixtures a combined high-performance hquid chromatography/ion mobility/time-of-flight mass spectrometry approach. Int. J. Mass Spectrom. 212, 97-109. 

Wienkoop, S., Glinski, M., Tanaka, N., Tolstikov, V.V., Fiehn, O., Weckwerth, W. (2004). Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins. Rapid Commun. Mass Spectrom. 18, 643-650. 

Elution with salt pulses A multiple step elution is performed by the introduction of, for example, 5%, 10%, 25%, 50%, and 100% of 1.5 M sodium chloride in 19 mM phosphate buffer (pH 2.5) containing 5% methanol. Each step is for 10 min and run at 0.5 mL/min. This elution method compromises analytical system dimensionality, as the peak capacity of the ion-exchange chromatography (IEX) step is equal at most to the number of salt steps. However, in the second dimension only one or two columns are needed and there is no particular limitation in the second dimension separation time as peptides are eluted in portions in a controlled manner. However, the number of salt steps is limited by the total analysis time. In this case the multidimensional system is relatively simple. 

FIGURE 15.3 Outline of experimental protocol used for ICAT differential protein expression profiling. Protein mixtures from two cell populations are labeled with light or heavy isotopic versions of a cleavable ICAT reagent. Labeled proteins are combined, subject to multidimensional separation by SCX, RP, and avidin affinity chromatography, then analyzed by tandem MS for peptide and protein identification. Based on the relative ratio of the two isotopically labeled peptides, a relative abundance of protein expression can be determined. 

Reversed-phase HPLC is widely utilized to generate a peptide map from digested protein, and the MS online method provides rapid identification of the molecular mass of peptides. The HPLC-MS-FAB online system is a sensitive and precise method for low-MW peptides (<3000 Da) even picomol quantities can be detected. However, as the MW of the analytes increases, the ionization of peptides becomes more difficult and decreases the sensibility of the FAB-MS (112). Electrospray ionization (ESI-MS) was found to be an efficient method for the determination of molecular masses up to 200,000 Da of labile biomolecules, with a precision of better than 0.1%. Molecular weights of peptide standards and an extensive hydrolysate of whey protein were determined by the HPLC-MS-FAB online system and supported by MALDI-TOF (112). Furthermore, HPLC-MS-FAB results were compared with those of Fast Performance Liquid Chro-motography (FPLC) analysis. Mass spectrometry coupled with multidimensional automated chromatography for peptide mapping has also been developed (9f,l 12a). 

Figure 4.8. MuDPIT platform. The MuDPIT or multidimensional gel protein identification system is an multidimensional liquid chromatography (LC)-based system of separation prior to tandem mass spectrometry (MS/MS). It is not necessary to purify proteins prior MuDPIT, although a reduction in protein complexity by some prior purification is helpful in obtaining interpretable spectra. Protein lysates are digested into peptides that are loaded onto a strong cation exchange (SCX) support. Peptides are sequentially eluted onto a reverse-phase column for a second separation. Eluted peptides from the reverse-phase column are electrosprayed into a tandem mass spectrometer for amino acid sequencing and identification of proteins in the sample.

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