Structural Analysis Of Peptides Research Articles

Integration of mass spectrometry in analytical biotechnology

Mass spectrometry (MS) has become an indispensable tool for peptide and protein structure analysis because of three unique capabilities that enable it to be used to solve structural problems not easily handled by conventional techniques. First, MS is able to provide accurate molecular weight information on low-picomole amounts of peptides and proteins independent of covalent modifications that may be present. Second, this information is obtainable for peptides present in complex mixtures such as those that result from a proteolytic digest of a protein. Third, by using tandem MS, partial to complete sequence information may be obtained for peptides containing up to 25 amino acid residues, even if the peptides are present in mixtures. Sensitivity and speed of the MS-based approaches now equal (and in some cases exceed) that of Edman-based sequence analysis. In this perspective we discuss how MS, tandem high-performance MS, and on-line liquid chromatography/MS using fast atom bombardment or electrospray ionization have been integrated with more conventional techniques in order to increase the accuracy and speed of peptide and protein structure characterization. The expanding role of matrix-assisted laser desorption MS in protein analysis is also described. The unique niche that MS occupies for locating and structurally characterizing posttranslational modifications of proteins is emphasized. Examples chosen from the authors' laboratory illustrate how MS is used to sequence blocked proteins, define N- and C-terminal sequence heterogeneity, locate and correct errors in DNA- and cDNA-deduced protein sequences, identify sites of deamidation, isoaspartyl formation, phosphorylation, oxidation, disulfide bond formation, and glycosylation, and define the structural class of carbohydrate at specific attachment sites in glycoproteins.

Application of photodiode array detection and fast atom bombardment mass spectrometry for the identification of the arginine residue in neuropeptides

Chemical derivatization by phenylglyoxal (PGX) was applied to the identification of arginine in the neuropeptides dynorphin A (1-6) and substance P. The obtained products were separated on a short reversed phase C18 column and analysed on-line with the photodiode array UV technique. The selective attachment of a chromogenic molecule into the arginine residue resulted in significant change in the absorbance spectra around 250 nm, depending on the number of PGX molecules attracted. Further analysis employed fast atom bombardment mass spectrometry (FAB MS) and C-terminal sequencing for detailed verification of the derivatives formed during modification with PGX. The results clearly demonstrated that the photodiode array technique, when combined with chemical modification of certain amino acids, provides new possibilities for the analysis of peptide structures.

Structural analysis of proteins by laser desorption and electrospray mass spectrometry.

The development of electrospray and matrix-assisted laser desorption mass spectrometry has provided protein chemists with tools for peptide and protein structure analysis with unprecedented sensitivity and molecular weight range. The two technologies can be viewed as competitive with respect to their molecular mass determinations, but complementary with respect to their differences in instrumentation, sample preparation methods, and nature of spectra produced.

Biochemical determinants of clearance of tissue-type plasminogen activator from the circulation.

Biochemical modification of tissue-type plasminogen activator (t-PA) designed to alter pharmacokinetics and pharmacodynamics offers promise for development of pharmaceuticals particularly suitable for treatment of specific disorders and for induction of coronary thrombolysis by intramuscular as well as intravenous administration. Accordingly, to identify biochemical determinants of clearance of t-PA from the circulation, we injected rabbits intravenously with three different preparations of t-PA synthesized from the same human gene and expressed in Chinese hamster ovary cells cultured under disparate conditions. Influences of glycosylation on clearance were defined by experiments with enzymatically treated t-PA in which clearance was assessed with concomitant administration of selected neoglycoproteins that compete with t-PA for specific glycoprotein receptors. The role of an intact active catalytic site, as reflected by differences in clearance with and without prior treatment of t-PA with the protease inhibitor PPACK, was defined also. Results indicate that clearance is altered by inhibition of the active site and that the nature and extent of glycosylation--not evident simply by analysis of peptide structure--influence clearance as well. These findings suggest that mannose/N-acetylglucosamine-specific glycoprotein receptors expressed on hepatic reticuloendothelial cells participate in clearance of t-PA from the circulation but that galactose-specific glycoprotein receptors probably do not. The observations may explain differences in clearance seen with different preparations of t-PA that have been seen in clinical pilot studies and may identify biochemical determinants of clearance amenable to modification for development of agents with potentially desirable, specific biological properties.

Observations on the reaction of 2-hydroxy-5-nitrobenzyl bromide with a peptide-bound tryptophanyl residue

Previous studies on the isolation of peptides containing tryptophanyl residues modified with 2-hydroxy-5-nitrobenzyl bromide demonstrated multiple products of reaction at the same residue as well as technical difficulties in the primary structure analysis of peptides containing the modified tryptophanyl residue. The present study was undertaken to explore the reaction of 2-hydroxy-5-nitrobenzyl bromide with the single tryptophanyl residue in a synthetic peptide, experimental allergenic encephalitogenic peptide. The modification of this peptide was accomplished in sodium acetate, pH 4.75, and reagent removed by gel filtration. Amino acid analysis of the modified peptide suggested that only the tryptophanyl residue had been modified under these experimental conditions. The modified peptide could be separated into multiple derivatives by high-performance liquid chromatography. Although it is clear that some of the observed heterogeneity reflects a difference in the degree of substitution at the single tryptophanyl residue, several of the derivatives appear to have the same extent of substitution. It is suggested that the heterogeneity observed is a reflection of the establishment of a new diastereoisomeric center in the peptide. These results are consistent with previous observations from other laboratories and provide a basis for the explanation of apparent heterogeneity of peptides obtained from modified proteins.

ChemInform Abstract: ALKALI CATION COMPLEXATION AND TRANSPORT PROPERTIES OF SYNTHETIC BICYCLIC DECAPEPTIDES: STRUCTURAL, THERMODYNAMIC, AND KINETIC ANALYSIS

AbstractMit Hilfe spektroskopischer Methoden (NMR und UV) sowie durch Mikrocalorimetrie wurde die Komplexierung von Alkalimetallkationen durch die bicyclischen Decapeptide S,S′‐ Bis‐[cyclo‐glycyl‐L‐hemicystyll‐glycyl‐glycyl‐L‐prolyl], S,S′‐Bis‐[cyclo‐glycyl‐L‐ hemicystyl‐glycyl‐glycyl‐D‐prc)lyl], S,S′‐Bis‐[cyclo‐glycyl‐L‐hemicystyl‐sarcosyl‐sarcosyl‐ ‐L‐prolyl‐] und S,S′‐Bis‐[cyclo‐glycyl‐L‐hemicystyl‐sarcosyl‐sarcosyl‐D‐prolyl] unter strukturellen, thermodynamischen und kinetischen Gesichtspunkten sowie die Eigenschaften der Peptide in bezug auf den Ionentransport in einem künstlichen Lipoid‐ Zweischichten‐System untersucht.

15N NMR spectroscopy, 22. Influence of shift reagents and paramagnetic ions on the spectra of peptides and polyamides

AbstractThe influence of Eu(dpm)3 and Dy(fod)3 on the 15N NMR spectra of Z‐Gly‐Leu‐Leu‐OMe or Z‐Ala‐Leu‐Leu‐OMe was investigated; however, solvent effects were found to be more useful than shift reagents to improve the spectroscopic structure analysis of peptides. Furthermore, the influence of manganese and copper ions on the Nuclear Overhauser Effect and on the line width of benzyloxycarbonylglycine, polysarcosine, polyglycine and poly(β‐alanine) was investigated in various solvents. The nature of the solvent was found to have a strong influence on the relaxation efficiency of the paramagnetic ions. Manganese ions can be used to shorten the pulse interval without risk of line broadening, when the measurements are carried out under the conditions of “inverse‐gated‐decoupling”.