Created: 1st March 2000, last updated: 30th May 2000, © 2000 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.
Veenstra TD, Martinovic S, Anderson GA, Pasa-Tolic L, Smith RD. Proteome analysis using selective incorporation of isotopically labeled amino acids. J Am Soc Mass Spectrom 2000;11:78-82.
The potential for rapid and sensitive proteome analysis using a suitable protein fractionation method followed by protein identification through accurate mass measurement has remained unrealized because of issues such as unexpected posttranslational modifications. A method is described that supplements this approach by acquiring information about amino acid composition to facilitate unambiguous protein identification. An auxotrophic strain of Escherichia coli is grown on minimal medium with natural isotopic abundance and on medium containing isotopically labeled leucine (Leu-D10). Proteins extracted from the two cultures are mixed and fractionated by capillary isoelectric focusing, and protein masses measured by Fourier transform ion cyclotron resonance mass spectrometry. The difference in mass between natural isotopic abundance and isotopically labeled proteins provides a measure of the number of leucine residues present in the protein. This approach can be extended to the labeling of other amino acids and the analysis of proteolytic products.
Lennon JJ, Walsh KA. Locating and identifying posttranslational modifications by in-source decay during MALDI-TOF mass spectrometry. Protein Sci 1999;8: 2487-2493.
A method is described for identifying and locating the sites of posttranslational modification in peptides and proteins. The method is based on interpretation of signals from cn fragment ions generated by in-source decay during delayed extraction in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A semi-automated data analysis process assists the identification of mass shift between segments of sequence on either side of the modified site. The residue at the junction of the segments is identified as the modification site. Sites of phosphorylation in seven synthetic peptides are determined, and the location of the heme group and N,N,N-trimethyl lysine in yeast cytochrome c are identified.
Focus on H/D exchange of proteins in solution. J Am Soc Mass Spectrom 1999;10:671-731.
A collection of five papers focus on hydrogen/ deuterium exchange of proteins in solution as monitored by mass spectrometry. A general perspective on the present and future roles of mass spectrometry by Clare Woodward is also included. Deng et al. describe the use of quench-flow studies of protein folding in conjunction with electrospray ionization, and they provide new information about a large, multimeric protein, aldolase. Resing et al. use rapid proteolysis and peptide separation, together with electrospray ionization, to characterize the hydrogen exchange behavior of the signal transduction kinase, ERK2. Wang et al. demonstrate the detection of a small but functionally significant conformational change in troponin C on binding of calcium using electrospray ionization with Fourier transform ion cyclotron resonance mass spectrometry. Nemirovskiy et al., also using electrospray, document the response of calmodulin to calcium binding. Figueroa and Russell study conformational changes of bradykinin, alpha-melanocyte-stimulating hormone and melittin on changes in solvent polarity using matrix-assisted laser desorption and ionization (MALDI) time-of-flight mass spectrometry, and they discuss the relative merits of electrospray and MALDI for such studies.
Keller BO, Li L. Discerning matrix-cluster peaks in matrix-assisted laser desorption and ionization time-of-flight mass spectra of dilute peptide mixtures. J Am Soc Mass Spectrom 2000;11:88-93.
When peptide mixtures being subjected to matrix assisted laser desorption/ionization are very dilute or contain large amounts of salts, signals from matrix clusters may be prominent. A simple scheme for identifying matrix cluster ions is presented. Although the presence of matrix cluster signals can be predicted, the relative intensities of these signals depend on details of sample preparation. Discrimination of signals due to matrix clusters from those due to peptides facilitates protein identification.
Feng B, Smith RD. A simple nanoelectrospray arrangement with controllable flow rate for mass analysis of submicoliter protein samples. J Am Soc Mass Spectrom 2000;11:94-99.
A simple arrangement for nanoelectrospray ionization using a conventional syringe pump connected to a pulled capillary with a large orifice diameter (9 µm) and lacking metal coating is evaluated. The arrangement avoids several disadvantages associated with metal coated nanoelectrospray emitters, including poor flow-rate reproducibility, the need for a very small orifice that is subject to clogging, the occurrence of degradation of the metal coating during electrospay, the need for "tip touching" against a surface, and the formation of gas bubbles that terminate electrospray. Subattamole detection limits are demonstrated for nM protein solutions at flow rates of 5 to 10 nL/min using an LCQ mass spectrometer.
Zubin EM, Romanova EA, Volkov EM, Tashlitsky VN, Korshunova GA, Shabarova ZA, Oretskaya TS. Oligonucleotide-peptide conjugates as potential antisense agents. FEBS Lett 1999;456:59-62.
A method is described for linking peptides to oligonucleotides for use as antisense reagents, among other applications. A phosphoramidite derivative of a suitably protected nucleoside bearing a side arm on the 2' position in the carbohydrate ring is synthesized. This side arm terminates in a primary amine that is protected with a 9-fluoroenylmethyloxy carbonyl (Fmoc) group. The modified nucleoside is incorporated into an oligonucleotide under standard, solid-phase conditions of chain assembly. The Fmoc group is then removed by treatment with a base, and a presynthesized, Fmoc-protected peptide is linked by its carboxyl terminus to the resulting free amino function using HBTU. The conjugate is cleaved from its solid support and deprotected. The advantage of this method is that the peptide can be incorporated into the DNA at any position.
Southan C, Lavery P, Fantom GM. Disposable microbore high-pressure liquid chromatography columns for protein and peptide separations. Anal Chem 1999;271: 152-158.
Procedures are described for constructing microbore high-performance liquid chromatography (HPLC) columns with internal diameters of 0.25 to 1.8 mm using glass or plastic tubing. The tubing is secured with standard HPLC fittings, permitting the columns to be used in conventional HPLC systems. The simple construction and low cost of these columns allows them to be considered disposable. The columns are shown to be effective for peptide and protein separations by reverse phase and for protein separations by ion exchange and size exclusion.
Zambrano R, Briones E, Avila J, Ballesta JPG. Phosphorylation of P'1 serine inhibits peptide bond sensitivity to Staphylococcus aureus V8 protease. Arch Biochem Biophys 1999;368:207-209.
Suppression of cleavage by Staphylococcus aureus V8 protease is documented to occur at glutamic acid residues that are followed by a serine when the serine has been phosphorylated. The negatively charge on the phosphate moiety is suggested to render such sites refractory to cleavage.