Liquid-chromatography analysis proteins

C.J. Ward, K.A. Beattie, E.Y.C. Lee and G.A. Codd, Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria comparisons with high-performance liquid chromatography analysis for microcystins, FEMS Microbiol. Lett., 153 (1997) 465-473. 

Zavitsanos, P., and Goetz, H. (1991). The practical application of diode array UV-visible detectors to high-performance liquid chromatography analysis of peptides and proteins. In High-Performance Liquid Chromatography of Peptides and Proteins Separation, Analysis,... 

Pisano, J. J., Analysis of amino acid phenylthiohydantoins by gas chromatography and high-performance liquid chromatography, in Protein Sequence Determination, Needleman, S. B., Ed. Springer-Verlag, New York, also listed as Mol. Biol. Biochem. Biophys., 8, 280-296, 1975. 

For examples of peptide analysis using these techniques, see (a) Hean, M.T.W. Regnies, F.E. Wehr, C.T (Eds.), High Performance Liquid Chromatography of Proteins and Peptides, Academic Press, New York, 1983 (b) Hean, M.T.W. 

Multidimensional Liquid Chromatography of Proteins Separation of Amino Acids—Amino Acid Analysis References... 

Fig. 7 Dependence of the analysis time and pressure drop of the column on particle size at a given plate number for nonporous packings. Dm = 5 x 10 cm s t] = 1 cP, e = 0.4, i/r = 945, k = 3 and the required N is shown in the figure. " Reprinted from T. Issaeva, A. Kourganov, and K. Unger Super-high-speed liquid chromatography of proteins and peptides on non-porous Micra NPS-RPpackings, J. Chromatogr. A, 846 (1999) 13-23, Copyright 1999 with permission from Elsevier Science...

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

H. Yamamoto, T. Manabe and T. Okuyama, Apparatus for coupled high-performance liquid chromatography and capillary electrophoresis in the analysis of complex protein mixtures , 7. Chromatogr. 515 659-666 (1990). 

A. W. Moore-Jr, J. P. Lamiann-Jr, A. V. Lemmo and J. W. Jorgenson, Two-dimensional liquid chromatography-capillary electrophoresis teclmiques for analysis of proteins and peptides . Methods Enzymol. 270 401-419 (1996). 

Figure 5.6 Positive-ion electrospray spectrum obtained from the major component in the LC-MS analysis of a purified recombinant 62 kDa protein using a Cig microbore 50 X 1 mm column and a flow rate of 50 p.lmin . The starting buffer (buffer A ) was 0.1% TEA in water, while the gradient buffer (buffer B ) consisted of 0.1% TEA in acetonitrile-water (9 1 vol/vol). The running conditions consisted of 0% B for 5 min, followed by a linear gradient of 100% B for 55 min. Reprinted from J. Chromatogr., B, 685, McAtee, C. P., Zhang, Y., Yarbough, P. O., Fuerst, T. R., Stone, K. L., Samander, S. and Williams, K. R., Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry , 91-104, Copyright (1996), with permission from Elsevier Science.

Biinger, H., Kaufner, L., and Pison, U., Quantitative analysis of hydrophobic pulmonary surfactant proteins by high-performance liquid chromatography with light-scattering detection, /. Chromatogr. A, 870, 363, 2000. 

The introduction and eventual commercialization of matrix-assisted laser desorption/ionization (MALDI) and electrospray (ESI) allowed biomarker status to be extended to proteins in 1996.15"17 With a few exceptions, ESI has been used in conjunction with extractions and high-pressure liquid chromatography (UPLC) interfaced with mass spectrometry. MALDI, on the other hand, has been widely adapted for rapid analysis of intact organisms, supported by bioinformatics.1819... 

Murphy, R.E. (2001). Comprehensive two-dimensional liquid chromatography for comparative protein analysis. Presented at the International Symposium on the Separation of Proteins, Peptides and Polynucleotides. (ISPPP 2001) Orlando, FL. 

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. 

Opiteck, G.J., Jorgenson, J.W., MacNair, J.E., Moseley, M.A., 3rd (1997). Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides. Rapid separation and characterization of protein and peptide mixtures using 1.5 microns diameter non-porous silica in packed capillary liquid chromatography/mass spectrometry. Anal. Chem. 69, 2283-2291. 

Capillary Liquid Chromatography Coupled with Capillary Electrophoresis for Analysis of Unlabeled Peptides and Proteins... 

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