created: 11th December 1997, last updated: 6th February 1998,© 1998 ABRF
Amino Acid and Sequence Analysis
Yan, J.X., Packer, N.H., Tonella, L., Ou, K., Wilkins, M.R., Sanchez, J.-C., Gooley, A.A., Hochstrasser, D.F. and Williams, K.L. (1997) Journal of Chromatography A 764, 201-210. High sample throughput phosphoamino acid analysis of proteins separated by one-and two-dimensional gel electrophoresis.
This paper describes a rapid, semi-automated, non-radioactive method for analyzing proteins for phosphoserine, phosphothreonine, and phosphotyrosine. Proteins from biological extracts are separated by one- or two-dimensional gel electrophoresis and blotted to PVDF membrane. Individual protein spots are then subjected to a limited acid hydrolysis (4 hours at 110°C) to maximize recovery of acid-labile phosphoamino acids. After Fmoc derivatization, phosphoamino acids are identified based on their retention times during reversed-phase HPLC. Starting with 30 pmol of a phosphorylated protein, the authors were able to detect phosphoserine at a level of 4 pmol.
Gu, Q.M. and Prestwich, G.D. (1997) Journal of Peptide Research 49, 484-491. Efficient peptide ladder sequencing by MALDI-TOF mass spectrometry using allyl isothiocyanate.
In ladder sequencing, peptides are treated with reagents that generate nested truncation products; these accumulated products are then mass analyzed as a mixture in one step to deduce the peptide sequence. Unlike other recently described ladder sequencing reagents, the one described here--allyl isothiocyanate--is commercially available, is stable, and reacts well with lysine-containing peptides. In this paper the authors show this reagent can provide up to seven residues of sequence from low-pmol amounts of peptides.
Mo, B., Li, J. and Liang, S. (1997) Analytical Biochemistry 249, 207-211. A Method for Preparation of Amino Acid Thiohydantoins from Free Amino Acids Activated by Acetyl Chloride for Development of Protein C-Terminal Sequencing.
A uniform and efficient chemistry is presented for synthesizing amino acid standards for carboxyl-terminal sequencing. Amino acid carboxylate groups are activated with acetyl chloride and then derivatized with trimethylsilyl isothiocyanate. Twenty separate reactions are conducted, one for each standard amino acid, and the products are purified by reversed-phase HPLC and then mixed. This approach works well for all twenty amino acids, including Pro and Ser, which are difficult to derivative using other chemistries.
Capillary Electrophoresis
Kurosu, Y., Murayama, K., Shindo, N., Shisa, Y., Satou, Y. and Ishioka, N. (1997) Journal of Chromatography A 771, 311-317. Optical resolution of phenylthiohydantoin-amino acids by capillary electrophoresis for protein sequencing.
Biological peptide analogs with interesting properties can be made by substituting the L isomer of a specific residue with its D enantiomer counterpart. However, verifying the enantiomer structure after synthesis can be difficult. Here, chiral selectors are used to separate PTH-DL-amino acids during capillary electrophoresis, after Edman sequencing. A mixture of digitonin and o-trimethyl-[beta]-cyclodextrin resolved 8 D/L pairs, and a mixture of digitonin and [beta]-escin was used to separate 13 pairs. The procedure was verified by sequencing and assigning the correct enantiomers for 200 pmol of [D-Ala2]-methionine enkephalin.
Carbohydrates and Glycoproteins
Kuster, B., Wheeler, S.F., Hunter, A.P., Dwek, R.A. and Harvey, D.J. (1997) Analytical Biochemistry 250, 82-101. Sequencing of N-Linked Oligosaccharides Directly from Protein Gels: In-Gel Deglycosylation Followed by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Normal-Phase High-Performance Liquid Chromatography.
The authors describe a method for generating carbohydrate profiles for glycoproteins in SDS-PAGE gel slices. In-gel deglycosylation is performed with PNGase F, the digestion products are fluorescently tagged, and oligosaccharides are identified by normal-phase HPLC coupled off-line to MALDI-TOF MS. The method is demonstrated by analyzing the neutral glycans of bovine ribonuclease B, chicken ovalbumin, and human IgG and the sialic acid-containing sugars of bovine fetuin and bovine [alpha]-acid glycoprotein, beginning with an initial gel load of 1.5 to 15 µg glycoprotein.
Mass Spectrometry
Fountoulakis, M. and Langen, H. (1997) Analytical Biochemistry 250, 153-156. Identification of Proteins by Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Following In-Gel Digestion in Low-Salt, Nonvolatile Buffer and Simplified Peptide Recovery.
A distinguishing feature of MALDI-MS is its ability to tolerate salt contamination, however removing salts from samples usually provides higher quality spectra. Here the authors describe how to prepare peptides from proteins in gel slices in a manner that keeps salt concentrations low. Dried gel pieces are swollen by rehydration in 5 mM Tris containing Lys-C protease, and after digestion the peptides are extracted for MALDI-MS. In many instances signals were clearly evident for digests in 5 mM Tris but not for digests in the more commonly used 100 mM Tris.
Eckerskorn, C., Strupat, K., Schleuder, D., Hochstrasser, D., Sanchez, J.-C., Lottspecich, F. and Hillenkamp, F. (1997) Analytical Chemistry 69, 2888-2892. Analysis of Proteins by Direct-Scanning Infrared-MALDI Mass Spectrometry after 2D-Page Separation and Electroblotting.
A procedure is described for measuring the masses of proteins bound to PVDF membrane after two-dimensional electrophoresis. The unstained membrane is doped with aqueous 0.3M succinic acid and then subjected to MALDI-TOF MS, but using an infrared ER-YAG 2.94 µm laser instead of the more commonly used ultraviolet laser. The sensitivity of the method is comparable to silver-staining. The authors hope to extend the method so doped spots can be digested with proteases and then analyzed by post-source decay to obtain internal sequences.
Peptides-Synthesis and Purification
Yoshida, T. (1997) Analytical Chemistry 69, 3038-3043. Peptide Separation in Normal Phase Liquid Chromatography.
The predominant method for separating peptides is reversed-phase HPLC, even though the options for rechromatography are somewhat limited: changes in support, counterions, or solvent pH. In this paper, the authors compare the separation of peptide mixtures by normal-phase and reversed-phase HPLC. Normal-phase HPLC used a TSK amide-80 column with carbamoyl groups bonded to a silica gel matrix, developed with a linear gradient of 0.1% trifluoroacetic acid/97% acetonitrile (solvent A) and 0.1% trifluoroacetic acid/55% acetonitrile (solvent B). Useful separations were achieved, complementary to the reversed-phase separations and with potential for other biological compounds, such as sugars and nucleic acids.
Matsumoto, H., Kahn, E.S. and Komori, N. (1997) Analytical Biochemistry 251, 116-119. Separation of Phosphopeptides from Their Nonphosphorylated Forms by Reversed-Phase POROS Perfusion Chromatography at Alkaline pH.
Phosphopeptides are usually purified and analyzed under conditions that deliberately avoid high pH, which can remove phosphate from some phosphopeptides. This paper shows that phosphorylated (and non-phosphorylated) peptides can be purified at pH 11.5, using ultra-fast, reversed-phase HPLC. Separations were accomplished in minutes, minimizing exposure to high pH, and collected fractions were quickly neutralized to minimize degradation.
Medzihradszky, K.F., Phillips, N.J., Senderowicz, L., Wang, P. and Turck, C.W. (1997) Protein Science 6, 1405-1411. Synthesis and characterization of histidine-phosphorylated peptides.
A post-translational modification that is becoming more widely recognized is phosphorylation of proteins at histidine residues. In fact, some now estimate that 6% of all protein phosphorylations may occur at histidine residues. In this paper, the authors describe the synthesis and exhaustive characterization of two peptides containing phosphohistidine.
Protein Characterization
Huhmer, A.F.R., Aced, G.I., Perkins, M.D., Gursoy, R.N., Seetharama Jois, D.S., Larive, C., Siahaan, T.J. and Schoneich, C. (1997) Analytical Chemistry 69, 29R-57R. Separation and Analysis of Peptides and Proteins.
This journal's annual review of peptide and protein articles, with major sections for Liquid Chromatography, Preparative Chromatography, Mass Spectrometry, Fourier Transform Infrared Spectroscopy, Nuclear Magnetic Resonance, Circular Dichroism, and Non-enzymatic Posttranslational Modifications of Proteins. Well over 500 references dating primarily from 1995-1996.
Ziegler, J., Vogt, T. Miersch, O. and Strack, D. (1997) Analytical Biochemistry 250, 257-260. Concentration of Dilute Protein Solutions Prior to Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.
Molecular biologists use hydrophobic resins when purifying DNA because the resins tightly bind protein, facilitating its removal from DNA samples. Here the authors show these commercially available resins can be used as protein concentrating reagents. Resin is added to highly dilute protein samples and, after a brief incubation step, collected by centrifugation. The protein-containing resin is then resuspended in SDS-PAGE sample buffer and heated at 65°C to release the protein, which can then be applied to an SDS-PAGE gel as usual.
Dainese Hatt, P., Quadroni, M., Staudenmann, W. and James, P. (1997) European Journal of Biochemistry 246, 336-343. Concentration of, and SDS removal from proteins isolated from multiple two-dimensional electrophoresis gels.
This paper describes a method for concentrating up to 50 stained spots from two-dimensional gels and for exchanging detergents. Protein spots stained with Coomassie brilliant blue, silver, or SYPRO are incubated in sample buffer overnight, to solubilize the fixed proteins. The large-volume samples are then concentrated into a single lane using funnel-shaped stacking gels. These funnel gels contain 0.1% octyl glucoside in the stacking and separation gel, so SDS is replaced with a detergent that is more compatible with downstream analytical methods, such as protease digestion and mass analysis. For 14 different proteins, the recoveries were 85-95% for Coomassie-stained samples, 65-88% for silver-stained samples, and 80-97% for SYPRO-stained samples.
Opitecik, G.J., Jorgenson, J.W. and Anderegg, R.J. (1997) Analytical Chemistry 69, 2283-2291. Two-Dimensional SEC/RPLC Coupled to Mass Spectrometry for the Analysis of Peptides.
Another report from this laboratory on the feasibility of two-dimensional chromatographic separation methods. In this study, a mixture of tryptic peptides from ovalbumin (1 nmol) and BSA (750 nmol) was analyzed by size exclusion and reversed-phase chromatography, with ultraviolet absorbance and mass analysis detection. System reproducibility was good, and the peptides were readily identified by their masses.
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