Reconstitution of Fragmentary Form of Thermostable Alanine Racemase

The fragmentary form of alanine racemase from Bacillus stearothermophilus is composed of two sets of two different polypeptides corresponding to the two domains of the subunit of wild-type enzyme. It was denatured with 6 M guanidium hydrochloride to be separated into pieces, and renatured by dilution with about 50% recovery of the activity. The two kinds of polypeptides (i.e., large and a small fragments) were isolated by gel filtration in the presence of 4 M guanidium hydrochloride. The CD spectra obtained by summation of the spectra of the refolded fragments were closely similar to that of the native fragmentary enzyme. The lysine residue to which PLP is bound in the wild-type enzyme occurs in the large peptide of the fragmentary enzyme containing the amino terminus of the wild-type enzyme. The visible spectrum of the large peptide refolded, however, indicated that PLP was not bound to it. The large peptide alone had no significant activity, but it was activated by incubation with the small peptide. Accordingly, co-existene of both peptide fragments is necessary for folding of a complex of the two kinds of peptide to form an active structure.     

Schistosoma mansoni dermaseptin-like peptide: structural and functional characterization

Analysis of the Schistosoma mansoni peptidome for immunomodulatory molecules by solvent extraction and reverse-phase HPLC revealed a 27-amino-acid residue peptide from an extract of cercariae. Using matrix-assisted, laser desorption-ionization, time-of-flight mass spectrometry, the peptide yielded a protonated molecular ion [M + H]+ of m/z 2789. The unequivocal sequence was deduced by automated Edman degradation as: DLWNSIKDMAAAAGRAALNAVTGMVNQ. The peptide exhibited an 80.76% identity with dermaseptin 3.1 from the leaf frog Agalychnis annae, and was therefore named Schistosoma mansoni dermaseptin-like peptide (SmDLP). Immunocytochemical staining using a primary antidermaseptin B2 antibody located SmDLP in acetabular glands of cercariae, in and around schistosomula, and in adult worms and their eggs. Dot-blotting confirmed its presence in extracts (cercariae and worms) and excretion/secretion (E/S) products (transforming cercariae and eggs). This was corroborated by use of a MALDI-ToF spectra database of E/S products from cercariae. Functional characterization of the peptide indicated that SmDLP had typical amphipathic antimicrobial peptide properties, i.e., the ability to lyse human erythrocytes causing a decrease in the levels of nitric oxide produced by monocytic cells. This last function strongly suggests that SmDLP plays a vital role in the parasite's immunoevasion strategy. The possibility that schistosomes acquired this gene from amphibians has been discussed by constructing a phylogenetic tree.     

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTip_C18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2′-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures.     

Outcome prediction in pneumonia induced ALI/ARDS by clinical features and peptide patterns of BALF determined by mass spectrometry

Background

Peptide patterns of bronchoalveolar lavage fluid (BALF) were assumed to reflect the complex pathology of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) better than clinical and inflammatory parameters and may be superior for outcome prediction.

Methodology/Principal Findings

A training group of patients suffering from ALI/ARDS was compiled from equal numbers of survivors and nonsurvivors. Clinical history, ventilation parameters, Murray''s lung injury severity score (Murray''s LISS) and interleukins in BALF were gathered. In addition, samples of bronchoalveolar lavage fluid were analyzed by means of hydrophobic chromatography and MALDI-ToF mass spectrometry (MALDI-ToF MS).Receiver operating characteristic (ROC) analysis for each clinical and cytokine parameter revealed interleukin-6>interleukin-8>diabetes mellitus>Murray''s LISS as the best outcome predictors. Outcome predicted on the basis of BALF levels of interleukin-6 resulted in 79.4% accuracy, 82.7% sensitivity and 76.1% specificity (area under the ROC curve, AUC, 0.853). Both clinical parameters and cytokines as well as peptide patterns determined by MALDI-ToF MS were analyzed by classification and regression tree (CART) analysis and support vector machine (SVM) algorithms. CART analysis including Murray''s LISS, interleukin-6 and interleukin-8 in combination was correct in 78.0%. MALDI-ToF MS of BALF peptides did not reveal a single identifiable biomarker for ARDS. However, classification of patients was successfully achieved based on the entire peptide pattern analyzed using SVM. This method resulted in 90% accuracy, 93.3% sensitivity and 86.7% specificity following a 10-fold cross validation (AUC = 0.953). Subsequent validation of the optimized SVM algorithm with a test group of patients with unknown prognosis yielded 87.5% accuracy, 83.3% sensitivity and 90.0% specificity.

Conclusions/Significance

MALDI-ToF MS peptide patterns of BALF, evaluated by appropriate mathematical methods can be of value in predicting outcome in pneumonia induced ALI/ARDS.     

A specific radioimmunoassay for the novel opioid peptide dynorphin

Dynorphin was recently isolated from porcine pituitary extracts and shown to be the most potent known opioid peptide. Antisera were prepared to synthetic dynorphin-(1–13), the biologically active NH₂-terminal fragment of the peptide. A high-titer, sensitive antiserum was characterized with fragments from dynorphin-(1–13). Leucine-enkephalin, which is contained in dynorphin, is not recognized at all by the antiserum. To study distribution in tissue, a procedure using hot acidified methanol extraction of rat pituitary neurointermediate lobe preparations was developed and validated. ¹²⁵I-labelled dynorphin-(1–13), when added to tissue, remained intact throughout this extraction procedure, and added dynorphin-(1–13) was almost completely recovered. There was no destruction of radiolabelled peptide during incubation in the radioimmunoassay. Serial dilutions of pituitary extracts yielded curves that were parallel to the dynorphin-(1–13) standard curve. The immunoreactivity from tissue was completely destroyed by papain treatment.     

Peptide end sequencing by orthogonal MALDI tandem mass spectrometry

Highly sensitive peptide fragmentation and identification in sequence databases is a cornerstone of proteomics. Previously, a two-layered strategy consisting of MALDI peptide mass fingerprinting followed by electrospray tandem mass spectrometry of the unidentified proteins has been successfully employed. Here, we describe a high-sensitivity/high-throughput system based on orthogonal MALDI tandem mass spectrometry (o-MALDI) and the automated recognition of fragments corresponding to the N- and C-terminal amino acid residues. Robotic deposition of samples onto hydrophobic anchor substrates is employed, and peptide spectra are acquired automatically. The pulsing feature of the QSTAR o-MALDI mass spectrometer enhances the low mass region of the spectra by approximately 1 order of magnitude. Software has been developed to automatically recognize characteristic features in the low mass region (such as the y1 ion of tryptic peptides), maintaining high mass accuracy even with very low count events. Typically, the sum of the N-terminal two ions (b2 ion), the third N-terminal ion (b3 ion), and the two C-terminal fragments of the peptide (y1 and y2) can be determined. Given mass accuracy in the low ppm range, peptide end sequencing on one or two tryptic peptides is sufficient to uniquely identify a protein from gel samples in the low silver-stained range.     

Influence of hemin on the conformation of cyanogen bromide-cleaved peptides of apomyoglobin

The complexes of the three BrCN-cleaved fragments of sperm whale apomyoglobin with hemin were studied by circular dichroism (CD). In native myoglobin, the heme is located in the middle fragment; the isolated peptide (residues 56–131), however, produces little extrinsic Cotton effects by the addition of hemin, although about four molecules of hemin are bound to this peptide. In marked contrast, the COOH-terminal peptide (residues 132–153), which binds three hemin molecules, shows strong Cotton effects in the Soret bands and drastically changes its conformation from unordered to highly helical. The Arg-modified or Lys-deaminated peptide no longer undergoes conformational changes by the addition of hemin, suggesting that the two propionic acid groups of one hemin molecule interact with the Arg residue and one of the Lys residues, which stabilizes the induced helical conformation. The NH₂-terminal peptide (residues 1–55) binds one hemin molecules, and the helicity of this fragment is slightly enhanced by the addition of hemin. Both the CD and difference absorption spectra indicate that the mode of interaction between the peptides and hemin are different for the three apomyoglobin fragments.     

Conformation of disulfide bridges in peptides with pepsin partial sequences.

On the basis of Raman spectra investigation of two model heterodetic cyclic peptides, containing partial sequences of pepsin fragments 45--50 and 206--210 of the chain, it was concluded that the disulfide bridge conformation in pepsin is determined not only by the size and conformation of the peptide loops created by disulfide bridges, but also by the peptide fragments located outside these loops.     

Inactivation of yeast ornithine decarboxylase by polyamines in vivo does not result from the incorporation of polyamines into enzyme protein

The peptide mixture obtained from controlled proteolytic digestion of ligandin with proteinase K or subtilisin retained 40% of glutathione-S-transferase and steroid isomerase activities, immunological reactivity and lower affinity bilirubin binding but binding at the primary site was abolished. When these limited proteolytic digests, which had no intact ligandin as determined by SDS gel electrophoresis, were subjected to Sephadex G-75 column chromatography, 40–50% of the peptide fragments were recovered in fractions where intact ligandin eluted. The results suggest that intact ligandin is not required for enzymatic activities, binding of bilirubin at the secondary site, or immunological reactivity; steroid isomerase and glutathione-S-transferase activities are modulated in a parallel manner and may be mediated by the same region of the protein, and primary and secondary binding sites for bilirubin are distinct and independent, despite nicks introduced by proteolysis in ligandin's subunits, some of the fragments remain associated under non-denaturing conditions and the susceptibility of the two subunits to the proteases is different.     

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K channels

The membrane location of two fragments in two different K⁺-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T₁ and ¹³C-¹H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. ²H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.     

Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectral Analysis of Marine Lipopeptides with Potential Therapeutic Implications

Marine Bacillus circulans DMS-2 (MTCC 8281) was found to produce lipopeptides, which reduced the surface tension of the medium from 69 to 28 mNm⁻¹. The methanol extracts of the lipopeptides were resolved in RP-HPLC, which resulted in the presence of two major surface active fractions corresponding to the peaks E and F were eluted at the retention times of 16.8 and 18 min, respectively. Fourier transform infrared spectroscopy and matrix assisted laser desorption ionization time of flight (MALDI-ToF) mass spectral analysis showed that the produced lipopeptides belonged to surfactin family. In the MALDI-ToF spectrum, the major molecular mass of the HPLC purified isoforms were identified at m/z 1,044 and 1,058 Da, respectively. In addition, this marine strain also produced new variants of surfactins with molecular weights ranging from m/z 1,066 to 1,098 Da, which have not been reported earlier. Both surface-active fractions were found to have potent antimicrobial activity against the Gram-positive and Gram-negative bacterial strains.     

Analysis of MALDI-TOF mass spectrometry data for discovery of peptide and glycan biomarkers of hepatocellular carcinoma

This paper presents computational methods to analyze MALDI-TOF mass spectrometry data for quantitative comparison of peptides and glycans in serum. The methods are applied to identify candidate biomarkers in serum samples of 203 participants from Egypt; 73 hepatocellular carcinoma (HCC) cases, 52 patients with chronic liver disease (CLD) consisting of cirrhosis and fibrosis cases, and 78 population controls. Two complementary sample preparation methods were applied prior to generating mass spectra: (1) low molecular weight (LMW) enrichment of each serum sample was carried out for MALDI-TOF quantification of peptides, and (2) glycans were enzymatically released from proteins in each serum sample and permethylated for MALDI-TOF quantification of glycans. A peak selection algorithm was applied to identify the most useful peptide and glycan peaks for accurate detection of HCC cases from high-risk population of patients with CLD. In addition to global peaks selected by the whole population based approach, where identically labeled patients are treated as a single group, subgroup-specific peaks were identified by searching for peaks that are differentially abundant in a subgroup of patients only. The peak selection process was preceded by peak screening, where we eliminated peaks that have significant association with covariates such as age, gender, and viral infection based on the peptide and glycan spectra from population controls. The performance of the selected peptide and glycan peaks was evaluated in terms of their ability in detecting HCC cases from patients with CLD in a blinded validation set and through the cross-validation method. Finally, we investigated the possibility of using both peptides and glycans in a panel to enhance the diagnostic capability of these candidate markers. Further evaluation is needed to examine the potential clinical utility of the candidate peptide and glycan markers identified in this study.     

N-Terminal Protein Characterization by Mass Spectrometry after Cyanogen Bromide Cleavage using Combined Microscale Liquid- and Solid-Phase Derivatization

A sample preparation method for protein N-terminal peptide isolation from cyanogen bromide (CNBr) protein digests has been developed. In this strategy, the CNBr cleavage was preceded by protein α- and ε-amine acetylation and carboxyamidomethylation in a one-pot reaction scheme. The peptide mixture was adsorbed on ZipTip_C18 pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-2-(biotinamido) ethyl-1, 3-dithiopropionate. In the subsequent steps, the peptides were exposed in situ to hydroxylamine for reversal of potential hydroxyl group acylation, followed by reductive release of the disulfide-linked biotinamido moiety from the derivatives. The selectively thiol group-functionalized internal and C-terminal peptides were reversibly captured by covalent chromatography on activated thiol-sepharose, leaving the N-terminal fragment in the flow-through fraction. The use of the reversed-phase support as a venue for postcleavage serial modification proved instrumental to ensure throughput and completeness of derivatization. By this sequence of solid-phase reactions, the N-terminal peptide could be recognized uniquely in the MALDI-mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-picomole amounts of model protein. The N-terminal CNBr fragments were retrieved selectively from the affinity support. The sample preparation method provides for robustness and simplicity of operation using standard equipment available in most biological laboratories and is anticipated to be readily expanded to gel-separated proteins.     

Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism

A TOF MS/MS method to directly determine the amino acid sequence in a cyclic lipopeptide without its hydrolysis is described. The fragments of the peptide and the hydrocarbon chains were identified through comparing the MS of two analogues of the lipopeptide; the connecting relationship of amino acid residues in the lipopeptide was determined based on the difference of mass to charge ratio between peaks in the MS spectra and the amino acid analysis; and finally, according to the mechanism of double hydrogen transfer(DHT) the C-terminal of peptide and hydroxy aliphatic acid in the lipopeptide was directly determined without the hydrolysis. The determined sequence of amino acid residues in the cyclic lipopeptide is also supported by the rest peaks in the MS spectra grounded on simple fragmenting mechanism. This method can be used to determine the amino acid sequence in any aliphatic acid loop-inlaying cyclic lipopeptides.     

Conformational analysis of peptide fragments derived from the peripheral subunit-binding domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: evidence for nonrandom structure in the unfolded state

There is currently a great deal of interest in the early events in protein folding. Two issues that have generated particular interest are the nature of the unfolded state under native conditions and the role of local interactions in folding. Here, we report the results of a study of a set of peptides derived from a small two-helix protein, the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex. Five peptides of overlapping sequence were prepared, including sequences corresponding to each of the helices and to the region connecting them. The peptides were characterized by CD and, where possible, nmr. A peptide corresponding to the second helix is between 12 and 17% helical at neutral pH. CD also indicates a lower percentage of helical structure in the peptide corresponding to the first alpha-helix, although the values of the alpha-proton chemical shifts suggest some preference for nonrandom structure. Peptides corresponding to the interhelical loop, which in the full domain contains two overlapping beta-turns and a 5-residue 3(10)-helix, are less structured. There is no significant change in the helicity of any of these peptides with pH. To test for fragment complementation, CD spectra of the two peptides derived from each helix and the long connecting peptide were compared to the spectra of each possible pair, as well as to a mixture containing all three. No increase in structure was observed. We complement our peptide studies by characterizing a point mutant, D34V, which disrupts a critical hydrogen bonding network. This mutant is unable to fold and provides a useful model of the denatured state. The mutant is between 9 and 16% helical as judged by CD. The modest amount of helical structure formed in some of the peptide fragments and in the point mutant suggests that the denatured state of the peripheral subunit binding domain is not completely unstructured. This may contribute to the very rapid folding observed for the intact protein.     

Combination of a SAW-biosensor with MALDI mass spectrometric analysis

A S-sens K5 surface acoustic wave biosensor was coupled with mass spectrometry (SAW-MS) for the analysis of a protein complex consisting of human blood clotting cascade factor alpha-thrombin and human antithrombin III, a specific blood plasma inhibitor of thrombin. Specific binding of antithrombin III to thrombin was recorded as a function of time with a S-sens K5 biosensor. Two out of five elements of the sensor chip were used as references. To the remaining three elements coated with RNA anti-thrombin aptamers, thrombin and antithrombin III were bound consecutively. The biosensor measures mass changes on the chip surface showing that 20% of about 400fmol/cm2 thrombin formed a complex with the 1.7-times larger antithrombin III. Mass spectrometry (MS) was applied to identify the bound proteins. Sensor chips with aptamer-captured (1) thrombin and (2) thrombin-antithrombin III complex (TAT-complex) were digested with proteases on the sensor element and subsequently identified by peptide mass fingerprint (PMF) with matrix assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry. A significant identification of thrombin was achieved by measuring the entire digest with MALDI-ToF MS directly from the sensor chip surface. For the significant identification of both proteins in the TAT-complex, the proteolytic peptides had to be separated by nano-capillary-HPLC prior to MALDI-ToF MS. SAW-MS is applicable to protein interaction analysis as in functional proteomics and to miniaturized diagnostics.     

Generation of peptides by human erythrocytes: facts and artifacts

Previously reported data on peptide composition of human erythrocyte lysate were obtained under conditions that did not exclude proteolytic degradation of hemoglobin in the process of peptide isolation. Comparative chromatographic analysis of the diluted erythrocyte lysate incubated in acidic conditions with or without proteolytic enzyme inhibitors showed that several peptides earlier identified as intraerythrocyte ones in fact result from hemoglobin degradation by erythrocyte acidic protease(s) during incubation of the lysate. A rational scheme excluding postlysis proteolysis was developed for isolation of peptide fraction. Further analysis resulted in determination of structure and content of about 50 endogenous intraerythrocyte hemoglobin fragments. A primary endopeptidase splitting of alpha- and beta-globin chains followed by consecutive exopeptidase trimming of primary fragments is suggested as a degradation mechanism. The intraerythrocyte peptides were shown to differ from peptides excreted by the erythrocytes to the extracellular medium in the primary culture. It was also found that intraerythrocyte peptides cannot play the role of precursors of hemoglobin fragments present in tissue extracts.     

CNBr/Formic Acid Reactions of Methionine- and Trifluoromethionine-Containing Lambda Lysozyme: Probing Chemical and Positional Reactivity and Formylation Side Reactions by Mass Spectrometry

The cyanogen bromide (CNBr)/formic acid cleavage reactions of wild-type and trifluoromethionine (TFM)-containing recombinant lambda lysozyme were studied utilizing ESI and MALDI mass spectrometry. Detailed analysis of the mass spectra of reverse-phase HPLC-purified cleavage fragments produced from treatment of the wild-type and labeled proteins with CNBr indicated cleavage solely of methionyl peptide bonds with no observation of cleavage at TFM. N-Acetyl-TFM was also found to be resistant to reaction with CNBr, in contrast to N-acetyl-methionine. The analysis also indicated differential reactivity among the three methionine positions in the wild-type enzyme. Additionally, formylation of intact enzyme as well as peptide fragments were observed and characterized and indicated that serine, threonine, as well as C-terminal homoserine side chains are partially formylated under standard cleavage protocols.     

Limited trypsinolysis of beta-haemocyanin of Helix pomatia. Characterization of the fragments and heterogeneity of the copper groups by circular dichroism.

A limited trypsinolysis of the tenths of beta-haemocyanin of Helix pomatia was performed at pH 8.2. The absorbance at 346 nm remained constant, indicating a preservation of the oxygen-binding sites. The five tryptic fragments were separated by chromatography on Sephadex G-100 and on DEAE-cellulose. They contained 2 Cu per 50000 daltons and showed different mobilities in agar electrophoresis. The molecular weights indicated that one fragment was constituted of three functional domains of about 50000 daltons, that two fragments were constituted of two domains, and two others of one domain. Twentieths of beta-haemocyanin seemed thus to be made up of 9 domains. The circular dichroic spectra of the fragments indicated the presence of two classes of copper groups according to their positive maximum at 455 or at 500 nm. The circular dichroic spectra also showed that no fragment could have originated from a larger one, confirming the presence of nine domains in the twentieths.