Created: 1st June 2000, last updated: 30th August 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.
Soga T, Heiger DN. Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry. Anal Chem 2000;72:1236-1241.
A method is described for determining underivatized amino acids by capillary electrophoresis coupled to electrospray (ESI) mass spectrometry. Electrophoresis is performed in 1 M formic acid, and sheath liquid containing 5 mM ammonium acetate in 50% methanol-water is added in the source to promote ionization. Nineteen of the amino acids commonly found in proteins and several physiologic amino acids are discriminated in 17 minutes. The detection sensitivity (signal-to-noise ratio of 3) is 0.3 to 11.0 µmol/L using vacuum injections of 3 seconds (3 nL), a performance equal to or better than HPLC methods with UV detection but 10- to 100-fold less sensitive than fluorescence detection with o-phthalaldehyde reagent.
Samyn B, Hardeman K, Van der Eycken J, Van Beeumen J. Applicability of the alkylation chemistry for chemical C-terminal protein sequence analysis. Anal Chem 2000; 72:1389-1399.
Automated methods for the cyclical removal of C-terminal amino acids from a polypeptide have been described. The C-terminal amino acid is first activated. This is performed by conversion to an oxazolinone by reaction with acetic anhydride in the presence of lutidine and then with treatment with the thiocyanate anion to form an isothiocyanate, which cyclizes to yield a thiohydantoin. Next the protein thiohydantoin is S-alkylated with 2-bromomethylnaphthalene. The alkylated thiohydantoin is finally cleaved with tetrabutylammonium thiocyanate. This study identifies a side product of the alkylation reaction that is resistant to cleavage and therefore reduces repetitive yield, indicating that development of a procedure that avoids the alkylation step would be beneficial. Despite this limitation of the chemistry, C-terminal sequencing of proteins immobilized on PVDF membranes in low-picomole quantities is demonstrated. N-terminal sequence analysis of proteins by automated Edman degradation, followed by C-terminal sequence analysis of the same, immobilized protein sample is therefore practical and is demonstrated in the study.
Goodlett DR, Bruce JE, Anderson GA, Rist B, Pasa-Tolic L, Fiehn O, Smith RD, Aebersold R. Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching. Anal Chem 2000; 72:1112-1118.
Protein identification is accomplished by measuring the mass of a single tryptic peptide. This is accomplished firstly by measuring the tryptic peptide mass to an accuracy of 1 ppm using a Fourier transform ion cyclotron resonance mass spectrometer. The stringency of database searching is further enhanced by identifying the presence of cysteine within the peptide sequence by alkylating with 2,4-dichlorobezyliodoacetamide. This introduces chlorine into cysteine-containing peptides. Chlorine is an atom normally absent from proteins, which confers a distinctive isotopic pattern permitting automatic screening of mass spectra. Secondary constraints on database searching include the specificity of the protease used for protein cleavage and the molecular weight of the protein estimated from its mobility during SDS gel electrophoresis. This strategy has the advantage, compared with data-dependent acquisition of information in MS/MS analyses, of reliably permitting the identification of low abundance components of protein mixtures.
Zhou W, Merrick SA, Khaledi MG, Tomer KB. Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry. J Am Soc Mass Spectrom 2000;11:273-282.
In experiments to identify phosphorylation sites on proteins by mass spectrometric analysis of tryptic peptides, the phosphopeptides are sometimes difficult to detect in complex peptide mixtures derived from large proteins. The presence of numerous peptides in the mixture may cause suppression of phosphopeptide signals of interest, and the presence of the phosphate groups may diminish the formation of positively charged phosphopeptide ions. Metal ion affinity chromatography has been used to purify phosphopeptides from tryptic peptide mixtures to facilitate identification of phosphorylation sites, but the bound phosphopeptides may be difficult to elute from the chromatographic stationary phase. This study demonstrates that phosphopeptides bound to agarose beads loaded with Fe2+ or Ga3+ can be detected directly by matrix-assisted laser desorption/ionization (MALDI) without first eluting the peptides. The bound phosphopeptides can be treated with alkaline phosphatase to count the number of phosphorylation sites by determining the incremental loss of 80-dalton units. The peptides can also be digested with carboxypeptidase to acquire C-terminal sequence information as an aid to their identification.
Cohen SL, Padovan JC, Chait BT. Mass spectrometric analysis of mercury incorporation into proteins for X-ray diffraction phase determination. Anal Chem 2000; 72:574-579.
A comparison is presented of MALDI and ESI for determining the extent of incorporation of a heavy metal, mercury, introduced by derivatizing cysteine side chains for the purpose of solving the phase problem during x-ray diffraction studies. ESI provides a more accurate quantitative measurement of the extent of mercury incorporation, but demands that the protein be stringently desalted before mass spectrometry. MALDI does not require removal of excess derivatizing reagents, salts, and buffers and can therefore be used for high-throughput studies and for the analysis of single protein crystals. However, it may give misleading quantitative results because of sensitivity to the choice of matrix, matrix acidity, and the identity of the mercury substituent. It is useful for determining only the lower limit of derivatization.
Rappsilber J, Siniossoglou S, Hurt EC, Mann M. A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry. Anal Chem 2000;72:267-275.
The spatial organization of multiprotein complexes may be investigated by nearest-neighbor analysis, in which pairs of proteins lying close together are chemically cross-linked and the cross-linked species identified after isolation in an appropriate separation system, such as gel electrophoresis. This study demonstrates the utility of peptide mass fingerprinting by MALDI-TOF mass spectrometry in this context. The technique permits the identification of proteins in simple mixtures and can be used to identify pairs of cross-linked proteins, revealing nearest-neighbor relationships. The approach is illustrated by analysis of the six-member Nup85p subcomplex of the nuclear pore.
Van Berkel WJH, Van den Heuvel RHH, Versluis C, Heck AJR. Detection of intact megaDalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry. Protein Sci 2000;9:435-439.
ESI mass spectra of very large protein complexes of the Penicillium flavoprotein, vanillyl-alcohol oxidase (VAO), are described. The VAO monomer is a 63,591-dalton polypeptide and exists as an 8-mer in aqueous solution, described as a tetramer of dimers. VAO is sprayed from aqueous 40 mM ammonium acetate solution, pH 6.8, at a VAO monomer concentration of 28 µM. The source region of the Q-TOF mass spectrometer employed in the study is operated at pressures substantially increased from normal values by leaking in dry nitrogen to optimally transmit large protein assemblies. The 8-mers are the most abundant species observed. However, 2-mers, 16-mers, and 24-mers are also detected. These species show narrow, nonoverlapping envelopes of multiple charge states, with molecular masses extending above 1.5 million daltons. The data demonstrate the feasibility of studying weak interactions between polypeptides by ESI mass spectrometry.
Charlwood J, Birrell H, Bouvier ESP, Langridge J, Camilleri P. Analysis of oligosaccharides by microbore high-performance liquid chromatography. Anal Chem 2000;72: 1469-1474.
An improvement is described for the normal methods of glycan analysis in which glycan mixtures released from proteins are fractionated by HPLC and the individual fractions are analyzed by mass spectrometry. In this study, a microbore hydrophilic interaction column is used to separate fluorophore-labeled glycans, and on-line mass spectrometry is employed to perform molecular weight and fragmentation analyses to assist in sequence determination.
Wilkinson TA, Yin J, Pidgeon C, Post CB. Alkylation of cysteine-containing peptides to mimic palmitoylation. J Peptide Res 2000;55:140-147.
Palmitoylation of cysteine residues by means of thioester linkage is an important modification that affects the function of a variety of proteins. Established methods for synthesizing palmitoylated peptides for use in functional studies are not specific for cysteines or rely on careful protection-deprotection strategies during solid-phase synthesis. In this study, a method for alkylating peptides with saturated C16 groups at cysteine residues is used to mimic palmitoylation. The method employs disulfide chemistry to modify cysteines specifically. C16 groups are linked by disulfide bridges using a new thioalkylating reagent, hexyldexyldithiopyridine. Protection of other peptide functional groups is unnecessary. The resulting modification mimics palmitoylation in conferring a membrane anchoring capacity on modified peptides.
Thorn KS, Naber N, Matuska M, Vale RD, Cooke R. A novel method of affinity-purifying proteins using a bis-arsenical fluorescein. Protein Sci 2000;9:213-217.
Genetically encoded affinity tags for purifying proteins are in widespread use. Most are proteins that bind small molecules, such as maltose binding protein or glutathione-S-transferase. The size of the added protein moiety, however, may affect the properties of the molecule being purified. Short peptides are also used, including the 6-histidine tag and the FLAG tag (a 6-residue antibody-binding epitope). The 6-His method requires rather harsh conditions for elution from immobilized metal ions and may lack specificity. The FLAG tag requires expensive antibody affinity matrix. In this study, an affinity matrix is described that is based on the bis-arsenical fluorescein dye, FlAsH (Fluorescein Arsenical Helix binder), which recognizes short alpha-helical peptides containing the sequence CCXXCC. Kinesin tagged with this motif binds to FlAsH resin and can be eluted in fully active form using small dithiols. The protein is purer than that obtained with nickel ion affinity chromatography of 6-His tagged kinesin and is eluted under gentler conditions.
Görg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000;21:1037-1053.
Methods for two-dimensional (2D) electrophoresis using immobilized pH gradient strips are reviewed and updated. Different methods for sample solubilization, sample application, and gel staining are compared, and guidelines for running conditions are provided. This review will be of interest to those performing 2D separations in the course of proteomic studies.
Santoni V, Molloy M, Rabilloud T. Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000;21:1054-1070.
The origin of difficulties in analyzing membrane proteins by two-dimensional electrophoresis are reviewed, including their relatively low abundance, high isoelectric point, and poor solubility of these proteins. Remedies are critically discussed.