Created: 1st September 1999, last updated: 12th November 1999, © 1999 ABRF
This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, HHMI/University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9050; Tel: (214) 648-5051; Fax: (214) 648-9477; email: slaugh01@utsw.swmed.edu; or to any member of the editorial board. Article summaries reflect the reviewers' opinions and not necessarily those of the Association.
Bauer MD, Sun Y, Wang F. Sequencing of gel-isolated proteins using microblotter capillary chromatography-electrospray mass spectrometry. J Protein Chem 1999;18: 337-347.
An automated method for obtaining mass spectral information on peptides that are being collected for Edman degradation is described. Peptides derived from gel-isolated proteins are subjected to capillary HPLC. A stream-splitting tee is introduced between the column and UV detector, sending part of the stream to an electrospray source for mass spectrometric analysis and part to a microblotter for automated collection on PVDF membranes for later Edman degradation. The mass spectral information is useful for identifying fractions that are likely to contain peptides pure enough to give useful amino acid sequence information, provides data useful for confirming sequence assignments, and facilitates the verification of single amino acid replacements introduced into proteins by site-directed mutagenesis.
Reuter G, Gabius H-J. Eukaryotic glycosylation: whim of nature or multipurpose tool? Cell Mol Life Sci 1999; 55:368-422.
This article is a comprehensive review of glycoscience. A short introduction to glycan attachment, including rare attachment modes, is followed by descriptions of glycan analysis methods, glycan conformation, biosynthesis, glycosamines and proteoglycans, glycosphingolipids, glycans and disease, and glycan recognition. The text is richly illustrated and includes 671 references with full titles. This review may be particularly useful to nonspecialists.
Jeannot MA, Zheng J, Li L. Observation of sodium gel-induced protein modifications in dodecylsulfate polyacrylamide gel electrophoresis and its implications for accurate molecular weight determination of gel-separated proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. J Am Soc Mass Spectrom 1999;10:512-520.
The affects of commonly occurring contaminants of proteins separated by SDS-PAGE are investigated with regard to sensitivity, resolution, and mass accuracy of MALDI-TOF mass spectrometry. Glycerol and Tris have little effect, but SDS degrades performance even at low concentrations. Proteins containing cysteines become modified by formation of adducts with unpolymerized acrylamide. A rapid method is described for extracting proteins from gels following copper staining. Gel bands are cut into small pieces and incubated for only 30 seconds in a small volume of a saturated solution of sinapinic acid in 70% acetonitrile; 1 M HCl is also included to lower the pH and keep the matrix in its acidic form. The gel is then crushed in the eluting solvent and the supernatant recovered for mass spectrometry following centrifugation. The procedure removes most of the SDS from the gel.
Keough T, Youngquist RS, Lacey MP. A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry. Proc Natl Acad Sci USA 1999;96:7131-7136.
Fragmentation of peptides to yield product ions informative for sequence depends largely on the presence of ionizing protons at the peptide bonds. In tryptic peptides, however, a single positive charge resides preferentially on the side chain of the C-terminal Lys or Arg residue, where it does not contribute to the formation of informative fragments. The multiply charged ions produced by electrospray contain additional protons that are distributed along the peptide backbone where they can promote the formation of sequence fragments, but MALDI gives poor yields of such multiply charged ions. This makes peptide sequencing by postsource decay on MALDI-TOF instruments difficult. In this study, a negative charge is added to tryptic peptides by addition of a sulfate group on the free N-terminal amine by treatment with either sulfobenzoic acid cyclic anhydride or chlorosulfonylacetyl chloride. The resulting negative charge exactly counterbalances the positive charge on the side chain of the C-terminal residue. Thus, only those ions that have an additional proton--one residing on backbone amide bonds--are observed by MALDI. Peptides derivatized in this way therefore display greatly enhanced fragmentation. Moreover, fragments of the y series are strongly favored because the introduced negative charge suppresses ions that include the N-terminal residue. The resulting peptides are therefore readily fragmented to yield spectra that are easily interpretable.
Yoshida T, Okada T. Peptide separation in normal-phase liquid chromatography: study of selectivity and mobile phase effects on various columns. J Chromatogr 1999; 840:1-9.
In previous studies, the authors of this article developed a normal phase separation method for peptides. This method is capable of retaining and separating hydrophilic peptides that are not retained by octadecyl silica columns under reverse-phase conditions. Acetonitrile is used as the mobile phase, and peptides are eluted from hydrophilic stationary phases with a gradient of increasing concentration of water. Selectivities obtained in this way differ significantly from those obtained by reverse-phase methods. The extent of hydrophilic interactions between peptides and the stationary phase can be modified using salts and organic acids in the same way ion exchange effects can be controlled during reverse-phase HPLC. In this study, trifluoroacetic acid, triethylamine, and mixtures of the two were tested with various hydrophilic stationary phases, including TSK gel Amide-80, TSK gel OH-120 (containing glyceryl groups bonded to silica), and TSK gel Silica-60. Recoveries of peptides is satisfactory with all three columns. Diol is best, followed by amide and then silica. Optimal modifier combinations are described for each column. This method will be of interest to readers seeking to augment the repertoire of procedures available for characterizing synthetic peptides.
Oda Y, Huang K, Cross FR, Cowburn D, Chait BT. Accurate quantitation of protein expression and site-specific phosphorylation. Proc Natl Acad Sci USA 1999;96: 6591-6596.
Stable isotope labeling is used in conjunction with mass spectrometry to quantitate differences in the abundance of proteins between cell lines. Protein extracted from one cell population is mixed with protein from another population that has been grown in 15N-enriched medium. After mixing, individual proteins in the combined pool are separated by one- or two-dimensional gel electrophoresis, without, of course, resolving the isotopically distinct species. Bands or spots are digested with trypsin and subjected to peptide mass fingerprinting by MALDI-TOF. Signals for peptides derived from the 15N-enriched and unenriched media are distinct, and their relative intensities provide measures of the relative abundance of the proteins in the corresponding cell pools. Protein identification is achieved by selecting peptide mass values from the unenriched protein components for database searching. A similar approach is used to measure the relative abundance of proteins that become differentially phosphorylated when grown under alternative conditions. In this case, the strength of signals from phosphorylated peptides are compared with their unphosphorylated counterparts. Differences are normalized to the relative strengths of signals from peptides that remain unmodified.
Rogniaux H, Van Dorsselaer A, Barth P, et al. Binding of aldose reductase inhibitors: correlation of crystallographic and mass spectrometric studies. J Am Soc Mass Spectrom 1999;10:635-647.
The interaction between an enzyme and a representative series of inhibitors is studied by a combination of mass spectrometric and x-ray crystallographic techniques. Mass spectrometry is used to assess the relative stability of enzyme-inhibitor complexes. The extent of dissociation of these noncovalent complexes is controlled by adjusting the accelerating voltage of ions in the source-analyzer interface region of the mass spectrometer, and the value for the voltage required to dissociate 50% of the complexes is used as a measure of gas-phase stability. This stability value correlates with the energy of the electrostatic and hydrogen bond interactions between enzyme and inhibitor computed from x-ray crystallographic data. Mass spectrometry is therefore proposed as a rapid assay for the effects of chemical modifications introduced during the design of new inhibitors, whereas detailed x-ray crystallographic analysis is employed to determine the details of the ligand contacts and to rationalize design. In this way, mass spectrometry can be used to select candidates that are worthy of further crystallographic study.
Kiselar JG, Downard KM. Direct identification of protein epitopes by mass spectrometry without immobilization of antibody and isolation of antibody-peptide complexes. Anal Chem 1999;71:1792-1801.
A rapid method for identifying linear sequence epitopes on defined protein antigens is described. The antigenic protein is first digested with a sequence-specific protease such as trypsin. Antibody is then added to an aliquot of the peptide mixture. MALDI spectra of the aliquots with and without antibody are then compared. Peptides containing antigenic epitopes remain bound to antibody under the conditions of sample preparation and subsequent ionization, and signals from these peptides are thus diminished or absent from the spectrum of the antibody-containing sample. They are identified by their mass values. A critical aspect of this method is the need to obtain uniform deposition of sample on the MALDI target to ensure that spectra are highly reproducible. This is achieved by heat-assisted or electrospray deposition of sample-matrix mixtures.