Peptide mixture

Dynamic/continuous-flow FAB allows a continuous stream of liquid into the FAB source hence it constitutes an LC/MS interface for analyses of peptide mixtures. 

Gapreomycin, Viomycin, and Enviomycin. Capreomycin (Capastat, Lilly), a bacteriostatic, antimycobacterial peptide mixture isolated from Streptomjces capreolus was first reported in 1961 (106—108). This tuberactinomycin family member, shown in Table 4, was introduced into the U.S. market in 1971 where it has remained a usehil but nephrotoxic and ototoxic second-line alternative to first-line tuberculosis therapies. Because capreomycin is somewhat less toxic than viomycin (tuberoactinomycin B [32988-50-4]) C25H42N23O2Q (109,110), capreomycin has now displaced viomycin in the United States and most other markets. The stmcture of viomycin is shown in Figure 2. The related enviomycin (tuberactinomycin N [33103-22-9]), C23H43N23O2Q,... 

The separation of proteins and peptides mixtures is the objective of protein biochemisdy. Albumin (Mr 66 000) concentration in a biological fluid (seaim, urine or cerebrbrospinal fluid) is assayed as markers for a series disease, such as nephritic syndrome or chronic glomuleronephritis. In diabetic patients the progression of microalbuminuria is accompanied by an increase in urinary concentrations of human semm albumen. In normal the excretion of albumin is 20 (tg/ml, in pathology - 20-200 p.g/ml. 

A. J. Tomlinson and S. Naylor, Enhanced performance membrane preconcentration-capillary electiophoiesis-mass spectiometry (mPC-CE-MS) in conjunction with transient isotachophoresis for analysis of peptide mixtures, J. High Resolut. Chromatogr. 18 384-386(1995). 

In 1990, Bushey and Jorgenson developed the first automated system that eoupled HPLC with CZE (19). This orthogonal separation teehnique used differenees in hydrophobieity in the first dimension and moleeular eharge in the seeond dimension for the analysis of peptide mixtures. The LC separation employed a gradient at 20 p.L/min volumetrie flow rate, with a eolumn of 1.0 mm ID. The effluent from the ehromatographie eolumn filled a 10 p.L loop on a eomputer-eontrolled, six-port miero valve. At fixed intervals, the loop material was flushed over the anode end of the CZE eapillary, allowing eleetrokinetie injeetions to be made into the seeond dimension from the first. 

Figure 11.18 Schematic diagram of an in-line SPE unit for CE using (a) polyester wool frits to hold the sorbent, or (b) a paiticle-loaded membrane. Reprinted from Journal of Capillary Electrophoresis, 2, A. J. Tomlinson and S. Naylor, Enhanced performance membrane preconcenti ation-capillary electrophoresis-mass spectiometi y (mPC-CE-MS) in conjunction with ti ansient isotachophoresis for analysis of peptide mixtures, pp 225-233, 1995, with permission from ISC Teclmical Publications Inc.

K. Matsuoka, M. Taoka, T. Isobe, T. Okuyama and Y. Kato, Automated high-resolution two-dimensional liquid cliromatographic system for the rapid and sensitive separation of complex peptide mixtures , 7. Chromatogr. 515 313-320 (1990). 

Since the proline residue in peptides facilitates the cyclization, 3 sublibraries each containing 324 compounds were prepared with proline in each randomized position. Resolutions of 1.05 and 2.06 were observed for the CE separation of racemic DNP-glutamic acid using peptides with proline located on the first and second random position, while the peptide mixture with proline preceding the (i-alamine residue did not exhibit any enantioselectivity. Since the c(Arg-Lys-0-Pro-0-(i-Ala) library afforded the best separation, the next deconvolution was aimed at defining the best amino acid at position 3. A rigorous deconvolution process would have required the preparation of 18 libraries with each amino acid residue at this position. 

Peptide mass fingeiprinting (PMF) is a mass spectrometry based method for protein identification. The protein is cleaved by an enzyme with high specificity (trypsin, Lys-C, Asp-N, etc.) or chemical (CNBr). The peptide mixture generated is analyzed by matrix-assisted laser desorp-tion/ionization (MALDI) or electrospray ionization (ESI)... 

Matrix-associated laser desorption ionization with a time-of-flight mass analyser (MALDl-ToF) was used to examine the crude tryptic peptide mixture from a number of the proteins, without HPLC separation, to provide a mass map, i.e. a survey of the molecular weights of the peptides generated by the digestion process. 

What are the advantages of using MALDl-ToF to examine the crude tryptic peptide mixture ... 

Figure 5.31 LC-electrospray-MS-MS spectrum of the column eluate at around 22 min in the analysis of the peptide mixture from the tryptic digest of glycoprotein TIME-EA4 from silkworm diapause eggs. Reprinted from Bioorg. Med. Chem., 10, Kurahashi, T., Miyazaki, A., Murakami, Y., Suwan, S., Franz, T., Isobe, M., Tani, M. and Kai, H., Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS , 1703-1710, Copyright (2002), with permission from Elsevier Science.

Fig. 19 Lateral model for assembly of peptide mixtures producing laminated P-sheets. Residues in solid circles are on the upper face, residues in dashed circles are on the lower face of the P-sheeL Reproduced from Takahashi et al. [57] with permission. Copyright Wiley-VCH. Numbers refer to the peptide entries in Fig. 18. Arrows are directions of the (antiparallel) beta sheet strands beginning and end of arrow correspond to N- and C-peptide termini respectively...

Stadalius, M. A., Gold, H. S., and Snyder, L. R., Optimization model for the gradient elution separation of peptide mixtures by reversed-phase high-performance liquid chromatography. Verification of retention relationships, /. Chromatogr., 296, 31, 1984. 

Davis, M. T., Stahl, D. C., Hefta, S. A., and Lee, T. D., A microscale electrospray interface for on-line, capillary liquid chromatography/tandem mass spectrometry of complex peptide mixtures, Anal. Chem., 67, 4549, 1995. 

Figure 2.5. Tandem mass spectrometry. A. A peptide mixture is electrosprayed into the mass spectrometer. Individual peptides from the mixture are isolated (circled peptide) and fragmented. B. The fragments from the peptide are mass analyzed to obtain sequence information. The fragments obtained are derived from the N or C terminus of the peptide and are designated "b" or "y" ions, respectively. The spectrum shown indicates peptides that differ in size by the amino acids shown.

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. 

Gilar, M., Daly, A.E., Kele, M., Neue, U.D., Gebler, J.C. (2004). Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography. J. Chromatogr. A 1061, 183-192. 

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

In an off-line configuration, a complex peptide mixture from a proteomic sample is loaded onto a SCX column and fractions collected (Fig. 11.1). After the collection of fractions, they are then loaded into an autosampler and analyzed via the traditional RP/ MS/MS approach. Using this system, a variety of buffers and elution conditions may be used (Table 11.1). For example, one may use a volatile salt such as ammonium formate (Adkins et al., 2002 Blonder et al., 2004 Fujii et al., 2004 Yu et al., 2004 Qian et al., 2005a and b) or ammonium acetate (Cutillas et al., 2003 Coldham and Woodward, 2004), collect SCX fractions, lyophilize, resuspend in low acetonitrile and acid, and then directly analyze via RP/MS/MS. In most of the cases, when ammonium acetate or ammonium formate are used, a 20-minute wash period is used to remove the ammonium acetate or ammonium formate prior to the reversed-phase gradient (Table 11.1). However, because fractions are collected and can be buffer exchanged,... 

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