Characterization of combinatorial peptide libraries by electrospray ionization Fourier transform mass spectrometry

The advantages of Fourier transform mass spectrometry (FTMS) are precision high mass accuracy measurements and the capability of high resolution, multistage mass spectrometry together with a number of other advanced features. These powerful facilities can be used to rapidly screen complex mixtures without the necessity of chromatographic separations. The example shown here illustrates the use of the high resolving power and accurate mass capabilities of FTMS for the rapid, direct analysis of a complex mixture, which had been ionized by direct infusion electrospray ionization.     

Investigation of suramin-albumin binding by electrospray mass spectrometry

Suramin, an organic polyanion with six sulphonic groups, is under clinical trials as an agent against hormone-refractory prostate cancer. The drug binds strongly to serum albumin. The objectives were to use electrospray to measure the molecular masses of the intact complexes of albumin and suramin to determine the number of suramin molecules bound under different molar ratios, and to investigate the binding of suramin in human serum. With albumin in excess (2:1 to 25:1 ratio), only 1 and 2 bound suramins were found; with suramin excess (2:1 to 1000:1) up to 20 bound suramin molecules/albumin were found. Up to 5 bound suramins were found in human serum with 4:1 suramin:albumin ratio, which corresponds to recommended therapeutic doses (200-300 micrograms/mL). At 8:1 ratio, which would be toxic, complexes with up to ten bound suramin molecules were found, and unreacted albumin diminished.     

Investigation of beta-amyloid peptide by on-line high-performance liquid chromatography coupled to electrospray ionization mass spectrometry

beta(1-39) amyloid peptide is one of the components of the cerebral amyloid deposits that are characteristic of Alzheimer's disease. Solid-phase synthesis of this peptide resulted in a fairly complex crude product containing both the target peptide and a number of side products. High-performance liquid chromatography coupled to electrospray ionization mass spectrometry allowed rapid and reliable identification of both the desired peptide and most of the side products which were found to have relative molecular masses above and below that of the target peptide.     

Determination of diethylpyrocarbonate-modified amino acid residues in alpha 1-acid glycoprotein by high-performance liquid chromatography electrospray ionization-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry

Fertilization in invertebrates results in tyrosine (Tyr) phosphorylation of several egg proteins. However, the involvement of Tyr phosphorylation in mediating mammalian egg activation has not yet been investigated. Using an antibody specific for phosphotyrosine (P-Tyr), immunoblotting, and densitometric analysis, we found that maturation of the oocyte is accompanied by a generalized increase in the P-Tyr content of almost all egg proteins detected. After sperm penetration, 5 of the 17 protein bands detected demonstrated a small increase in their P-Tyr content, while at the pronuclear (PN) stage the signal was markedly reduced. Ionomycin emulated the changes observed at fertilization in most protein bands detected, demonstrating a small increase in their P-Tyr content within 15 min of exposure. Analysis of the involvement of the tyrosyl-phosphorylated, mitogen-activated protein (MAP) kinase during meiosis revealed comigration of the phosphotyrosyl bands with the protein and a good correlation with its enzyme activity. Maturation was accompanied by an increase in MAP kinase activity. The activity dropped partially after sperm penetration and furthermore later at the PN stage. A larger quantity accompanied by a more significant change in the P-Tyr content implies for extracellular regulated kinase (ERK) 2 being the dominant isoform present in the rat egg. Our results indicate that fertilization in mammals involves changes in activity of protein tyrosine kinases (PTKs) or in the balance between PTKs and protein tyrosine phosphatases. The single, ionomycin-induced Ca2+ rise is sufficient to imitate fertilization-induced changes in MAP kinase activity, as well as in tyrosine phosphorylation of other proteins within the egg.     

Rapid peptide mapping by reversed-phase liquid chromatography on nonporous silica with on-line electrospray time-of-flight mass spectrometry

Reversed-phase liquid chromatography (LC) using a nonporous silica support has been combined with electrospray (ES) time-of-flight (TOF) mass spectrometry (MS) for the fast separation and mass detection of peptides. Using this LC method, the resolution of a peptide mixture can be completed is less than 35 s. The resulting chromatographic peak widths are less than 1 s wide. Because of the unique nature of a TOF mass analyzer, complete mass spectra can be acquired at a rate which is sufficient to sample these narrow peaks. When compared with conventional LC, the same separation takes nearly 20 min to complete, and the signal-to-noise ratio observed in the total ion chromatogram is dramatically lower due to the influence of increased background noise in the mass spectra. The limit of detection for a low molecular weight peptide, Val-Pro-Leu, was found to be 6 pmol with the total ion chromatogram and 500 fmol with the reconstructed ion chromatogram. A peptide map of horse heart myoglobin, completed in 3.5 min, is shown as an example of the results which can be obtained from combining this fast LC method with fast ES/TOF/MS detection capability.     

Antigenic peptide binding by class I and class II histocompatibility proteins

Enhanced expression of the immediate early gene c-fos has been used as a marker of cellular activation in many different neuronal pathways. We wished to determine the neurochemical content and the connectivity of neurons, in which expression of c-fos is induced. For this purpose, a dual-immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chromogen for c-fos protein located in the nucleus, and benzidine dihydrochloride (BDHC) in the presence of sodium nitroprusside to reveal cytoplasmic antigens (neuropeptide or retrograde tracer) in the same section. The blue granular BDHC reaction product in the cytoplasm combined with the homogeneous brown nuclear DAB staining for c-fos protein provides excellent resolution of dual-labelled cells even in tissue sections of 40 microns in thickness. The high sensitivity of the avidin-biotin-peroxidase immunocytochemistry and the stability of the reaction products provide an excellent tool for quantitative analysis of stimulated cells within a neurochemically defined cell group. The BDHC/DAB protocol was developed to identify activated cells in three experimental situations. Firstly, to investigate the phenotype of light-activated cells in the suprachiasmatic nucleus of the hypothalamus, c-fos protein DAB staining was carried out together with BDHC staining for peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VIP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Capillary electrophoresis/electrospray ionization high mass accuracy time-of-flight mass spectrometry for protein identification using peptide mapping

Capillary electrophoresis/electrospray ionization (CE/ESI) high mass accuracy time-of-flight mass spectrometry was used for the first time to characterize small proteins using peptide mapping. To identify small proteins, the intact proteins were first analyzed to obtain their average molecular weights with errors less than 1 Da. On-line capillary electrophoresis mass spectrometry of the tryptic digests of these small proteins was then performed to obtain the accurate molecular weights of the peptides with accuracies of approximately 10 ppm. Next, this information was used for the identification of the proteins using a protein database. It was found that high mass accuracy is an effective tool in reducing the list of most-likely proteins generated by the database. In addition, on-line collision-induced dissociation of the completely or partially resolved capillary electrophoresis peaks of the protein digests was used to unambiguously identify the sequences of these peptides. Each CE/ESI-MS analysis used only 5 nL of sample containing approximately 120 fmol of each peptide in protein digests. The results indicate that the combination of capillary electrophoresis and high resolution, high mass accuracy time-of-flight mass spectrometry is a viable option for the identification of small proteins using peptide mapping.     

Analysis of peptides, proteins, protein digests, and whole human blood by capillary electrophoresis/electrospray ionization-mass spectrometry using an in-capillary electrode sheathless interface

An in-capillary electrode sheathless interface was applied to the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of mixtures of small peptides, proteins, and tryptic digests of proteins. The effects of different experimental parameters on the performance of this CE/ESI-MS interface were studied. The distance of the in-capillary electrode from the CE outlet and the length of the electrode inside the capillary had no significant effects on the CE separation and ESI behavior under the experimental conditions used. However, significant enhancement of the sensitivity resulted from the use of narrower CE capillaries. Using a quadrupole mass spectrometer, an aminopropylsilane-coated capillary, and a wide scan mass-to-charge ratio range of 500-1400, detection limits of approximately 4, 1, and 0.6 fmol for cytochrome c and myoglobin were achieved for 75-, 50-, and 30-micron inner diameter capillaries, respectively. Approximately one order of magnitude lower detection limits were achieved under the multiple-ion monitoring mode. The application of the in-capillary electrode sheathless interface to real-world samples was demonstrated by CE/ESI-MS analysis of a human blood sample.     

EPR mapping of interactions between spin-labeled variants of human carbonic anhydrase II and GroEL: evidence for increased flexibility of the hydrophobic core by the interaction

Human carbonic anhydrase II (HCA II) interacts weakly with GroEL at room temperature. To further investigate this interaction we used electron paramagnetic resonance (EPR) spectroscopy to study HCA II cysteine mutants spin-labeled at selected positions. From our results it is evident that protein-protein interactions can be specifically mapped by site-directed spin-labeling and EPR measurements. HCA II needs to be unfolded to about the same extent as a GuHCl-induced molten-globule intermediate of the enzyme to interact with GroEL. The interaction with GroEL includes interactions with outer parts of the HCA II molecule, such as peripheral beta-strands and the N-terminal domain, which have previously been shown to be rather unstable. As a result of the interaction, the rigid and compact hydrophobic core exhibits higher flexibility than in the molten globule, which is likely to facilitate rearrangements of misfolded structure during the folding process. The degree of binding to GroEL and accompanying inactivation of the enzyme depend on the stability of the HCA II variant, and nonspecific hydrophobic interactions appear to be most important in stabilizing the GroEL-substrate complex.     

Differentiation of lysine/glutamine in peptide sequence analysis by electrospray ionization sequential mass spectrometry coupled with a quadrupole ion trap

Electrospray ionization coupled to a quadrupole ion trap mass spectrometer is used to differentiate between the isobaric amino acids lysine and glutamine in sequence analysis of peptides. Collision-induced dissociation is used for fragmentation. Several isobaric peptides with one or more lysines or glutamines at different positions were investigated. The ambiguous amino acid either in the peptide chain or at the C- or N-terminus can be clearly identified based on specific side chain fragment ions resulting from MS3 or MS4 of B- and Y"-fragment ions.     

Purification and determination of intact molecular mass by electrospray ionization mass spectrometry of the photosystem II reaction center subunits

A reverse phase high pressure liquid chromatography purification system for the rapid separation of photosystem II reaction center proteins free of salts and detergents is described. This procedure results in the isolation of the three small subunits: alpha- and beta-subunits of cytochrome b559 and PsbI protein, with near base-line resolution between each peak, although the D1 and D2 proteins were partially deconvoluted. The molecular masses obtained by electrospray ionization mass spectrometry for the purified beta-subunit of cytochrome b559, alpha-subunit of cytochrome b559, and the PsbI protein, 4,394.8 +/- 0.4, 9,283.7 +/- 0.8, and 4,209.5 +/- 0.4 Da, respectively, are in excellent agreement with values obtained from previous characterization studies (Sharma, J., Panico, M., Barber, J., and Morris, H. R. (1997) J. Biol. Chem. 272, 3935-3943). Direct electrospray analysis of the D1 and D2 proteins suggests that these components exist in heterogeneous forms. The molecular mass ascribed to a predominant form of the D1 protein, 38, 040.9 +/- 6.5 Da, and the D2 protein, 39,456.1 +/- 7.7, are also in agreement with those expected for the mature nonphosphorylated states of these subunits.     

Ribosome binding to mitochondria is regulated by GTP and the transit peptide

The association between ribosomes and the pore proteins at the endoplasmic reticulum membrane is important to co-translational translocation. To determine if a similar association occurs between the ribosome and mitochondrial membrane protein(s) during protein import in higher eukaryotes, we examined ribosome-mitochondria binding. By using spectral measurements, analysis of mitochondrial associated RNA, and electron microscopy, we demonstrated that ribosomes stably bind to purified rat liver mitochondria in vitro. Binding of ribosomes to mitochondria was markedly reduced by GTP and nearly abolished by the non-hydrolyzable GTP analogue, guanosine-5'-[thio]-triphosphate (GTPgammaS), but was only modestly reduced by GDP or ATP and unaffected by CTP. The initial rate of GTP hydrolysis by mitochondria was increased by ribosomes, whereas the rate of ATP hydrolysis by mitochondria was not affected. Ribosomes programmed with mRNA for 92 amino acids of the N terminus of mitochondrial malate dehydrogenase bound to mitochondria, but unlike unprogrammed rat liver ribosomes, neither GTP nor GDP disrupted binding; however, GTPgammaS did. These data show that receptors specific for ribosomes are present on the mitochondrial membrane, and a GTP-dependent process mediates this binding. The presence of a nascent chain alters these binding characteristics. These findings support the hypothesis that a co-translational translocation pathway exists for import of proteins into mitochondria.     

Lowering the entropic barrier for binding conformationally flexible inhibitors to enzymes

The design of inhibitors with enhanced potency against proteolytic enzymes has many applications for the treatment of human diseases. In addition to the optimization of chemical interactions between the enzyme and inhibitor, the binding affinity can be increased by constraining the inhibitor to the conformation that is recognized by the enzyme, thus lowering the entropic barrier to complex formation. We have structurally characterized the complexes of a macrocyclic pentapeptide inhibitor and its acyclic analogue with penicillopepsin, an aspartic proteinase, to study the effect of conformational constraint on the binding affinity. The phosphonate-based macrocycle PPi4 (Ki = 0.10 nM) is covalently linked at the P2-Asn and P1'-Phe side chains [nomenclature of Schechter and Berger, Biochim. Biophys. Res. Commun. (1967) 27, 157-162] via an amide bond, relative to the acyclic compound PPi3 (Ki = 42 nM). Comparisons of the high-resolution crystal structures of PPi4-penicillopepsin (0.95 A) and PPi3-penicillopepsin (1.45 A) reveal that the conformations of the inhibitors and their interactions with the enzyme are similar. The 420-fold increase in the binding affinity of PPi4 is attributed to a reduction in its conformational flexibility, thus providing the first rigorous measure of the entropic contribution to the binding energy in a protein-ligand complex and stressing the advantages of the design strategy.     

Nanoflow solvent gradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry

Microfabrication technology offers the opportunity to construct microfluidic modules which are designed to perform specific, dedicated functions. Here we report the construction of a microfabricated device for the generation and delivery by electroosmotic pumping of solvent gradients at nanoliter per minute flow rates. The device consists of three solvent reservoirs and channels which were etched in glass. Solvent gradients and solvent flows were generated by computer controlled differential electroosmotic pumping of aqueous and organic phase, respectively, from the solvent reservoirs. The device was integrated into an analytical system consisting of the solvent gradient delivery module, a reverse phase microcolumn and an electrospray ionization ion trap mass spectrometer (MS). The system was used for the analysis at high sensitivity of peptides and peptide mixtures generated by proteolytic digestion of proteins. We have measured an absolute limit of detection as low as 1 fmol and a concentration limit of detection at the 100 amol/microL level. The system was also successfully used for the identification of proteins separated by 1D and 2D gel electrophoresis. This was achieved by gradient frontal analysis of the peptide mixture generated by proteolysis of the respective proteins, and the automated generation and interpretation of collision-induced dissociation spectra.     

Screening for the detection of angiotensin-converting enzyme inhibitors, their metabolites, and AT II receptor antagonists

A gas chromatography-mass spectrometry (GC-MS) screening procedure was developed for the detection of angiotensin-converting enzyme (ACE) inhibitors, their metabolites, and angiotensin (AT) II receptor antagonists in urine as part of a systematic toxicologic analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. Using mass chromatography with the ions m/z 157, 160, 172, 192, 204, 220, 234, 248, 249, and 262, the possible presence of ACE inhibitors, their metabolites, and AT II antagonists could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed detection of therapeutic concentrations of ACE inhibitors (benazepril, enalapril, perindopril, quinapril, ramipril, trandolapril, their metabolites, or both) and therapeutic concentrations of the AT II antagonist, valsartan, in human urine samples. Human urine samples were not available for testing cilazapril, moexipril, and losartan; they were detected only in rat urine. The overall recoveries of ACE inhibitors ranged between 80% and 88%, with a coefficient of variation (CV) of less than 10% and the limit of detection of at least 10 ng/ml (signal to noise ratio 3) in the full-scan mode. The overall recovery of the valsartan was 68%, with a CV of less than 10%; the limit of detection was at least 10 ng/ml (S/N 3) in the full scan mode.     

A convenient approach to the synthesis of trinucleotide phosphoramidites--synthons for the generation of oligonucleotide/peptide libraries

Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale. Precursors of those amidites--trinucleotide phosphotriesters--have been prepared using the phosphotriester approach without protection of the 3'-hydroxyl function. The structures of trinucleotide phosphotriesters and intermediates were confirmed by 1H- and 31P-NMR spectra, mass-spectra and by analysis of SPDE-hydrolysates of deprotected preparations. Purity of the target products has been confirmed by test reactions. The synthons have been used for automated synthesis of oligonucleotides and corresponding libraries by a phosphite-triester approach. A 54mer, containing 12 randomized internal bases, and a 72mer with 24 internal randomized bases have been synthesized.     

Estimation of binding free energies for HIV proteinase inhibitors by molecular dynamics simulations

Absolute binding free energies for three inhibitors of HIV-1 proteinase were estimated from molecular dynamics simulations by a recently reported linear approximation procedure. The results were in fairly good agreement with experimental binding data. Two of the inhibitors were very similar and, for comparison, their relative free energies of binding were also calculated by free energy perturbation methods, giving virtually the same result. Effects of cut-off radii and charge states of the protein model were examined. The effects of pH on binding of one of the inhibitors were predicted.