Gln-Gly cleavage: a dominant dissociation site in the fragmentation of protonated peptides

An understanding of the gas-phase dissociation of protonated peptides within the mass spectrometer is essential for automated high-throughput protein identification. In this communication we describe a facile cleavage of the Gln-Gly peptide bond under low-collisional energy conditions. A variety of synthetic peptides have been analysed where key amino acids have been substituted within the sequence PQGPPQQGGR, which is a consensus repeat present in the tryptic peptides of acidic proline-rich protein 1 (PRP-1). The collision-induced dissociation spectra obtained from the PRP-1 tryptic peptides and the synthetic peptides indicate that facile Gln-Gly cleavage occurs when an X-Gln-Gly-Y sequence is present in a peptide, where X is any amino acid and Y any amino acid other than Gly.     

Characterisation of variant forms of prophenin: mechanistic aspects of the fragmentation of proline-rich peptides

Prophenin 1 (PF-1) is a 79-residue polypeptide originally isolated from porcine leukocytes. Its amino acid sequence has been determined by a combination of mass spectrometry and Edman degradation (Harwig SSL. et al. FEBS Lett. 1995; 362: 65). Prophenin (PF) and variants thereof are also found in organic extracts of porcine pulmonary tissue (Wang Y. et al. FEBS Lett. 1999; 460: 257). In the present study we have characterised the variant forms of PF found in these extracts using nano-electrospray (nano-ES) high resolution and tandem mass spectrometry. The major forms of PF found in these extracts by nano-ES mass spectrometry are the 80-residue polypeptides prophenin-2-Pyr (PF-2-Pyr) and prophenin-2-Gln (PF-2-Gln). Prophenin-2-Pyr is refractory to Edman degradation due to the presence of an N-terminal pyroglutamic residue. In PF-2-Gln the N-terminal residue is glutamine and the C-terminus is amidated. In porcine pulmonary extracts PF-1 is present to only a minor extent. Other shorter polypeptides are also found in these extracts including 18- and 17-residue C-terminal fragments of PF. The primary structure of PF is highly unusual in that it shows four almost perfect decamer repeats of FPPPN(V/F)PGPR and, out of the 79/80 residues, 42 are proline and 14 are phenylalanine. Tryptic digestion of PF gives peptides containing the decamer repeat and collision-induced dissociation of these peptides provides an insight into the fragmentation mechanisms of proline-rich peptides. Facile cleavage within the Pro-Pro-Pro sequence of these peptides suggests the involvement of a cyclic peptide in the fragmentation mechanism. Fragmentation mechanisms that account for the formation of fragment ions at other cleavage sites are also discussed.     

Peptidomic analysis of human acquired enamel pellicle

Human acquired enamel pellicle is the result of a selective interaction of salivary proteins and peptides with the tooth surface. In the present work, the characterization of the peptides as well as the type of interactions established with the enamel surface was performed. Peptides from in vivo bovine enamel implants in the human oral cavity were sequentially extracted using guanidine and trifluoroacetic acid solutions and the fractions obtained were analysed by LC-MS and LC-MS/MS. Based on the LC-MS data, six phosphorylated peptides were identified in an intact form, strongly adsorbed to the enamel surface. Data from the LC-MS/MS analyses allowed us to identified 30 fragment peptides non-covalently bonded to enamel [basic proline-rich proteins, histatins (1 and 3) and acidic proline-rich protein classes]. The tandem mass spectrometry experiments showed the existence of a pattern of amide bond cleavage for the different identified peptide classes suggesting a selective proteolytic activity. For histatins, a predominance of cleavage at Arg, Lys and His residues was observed, while for basic proline-rich proteins, cleavage at Arg and Pro residues prevailed. In the case of acidic proline-rich proteins, a clearly predominance of cleavage of the Gln-Gly amide bond was evident.     

Cyclic and Lasso Peptides: Sequence Determination,Topology Analysis,and Rotaxane Formation

A broadly applicable chemical cleavage methodology to facilitate MS/MS sequencing was developed for macrocyclic and lasso peptides, which hold promise as exciting new therapeutics. Existing methods such as Edman degradation, CNBr cleavage, and enzymatic digestion are either limited in scope or completely fail in cleavage of constrained nonribosomal peptides. Importantly, the new method was utilized for synthesizing a unique peptide‐based rotaxane (both cyclic and threaded) from the lasso peptide, benenodin‐1 ΔC5.     

Cyclic and Lasso Peptides: Sequence Determination,Topology Analysis,and Rotaxane Formation

A broadly applicable chemical cleavage methodology to facilitate MS/MS sequencing was developed for macrocyclic and lasso peptides, which hold promise as exciting new therapeutics. Existing methods such as Edman degradation, CNBr cleavage, and enzymatic digestion are either limited in scope or completely fail in cleavage of constrained nonribosomal peptides. Importantly, the new method was utilized for synthesizing a unique peptide‐based rotaxane (both cyclic and threaded) from the lasso peptide, benenodin‐1 ΔC5.     

Synthetic metal-binding protein surface domains for metal ion-dependent interaction chromatography. II. Immobilization of synthetic metal-binding peptides from metal ion transport proteins as model bioactive protein surface domains.

This preliminary investigation tests the premise that biologically relevant (1) peptide-metal ion interactions, and (2) metal ion-dependent macromolecular recognition events (e.g., peptide-peptide interactions) may be modeled by biomimetic affinity chromatography. Divinylsulfone-activated agarose (6%) was used to immobilize three different synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein histidine-rich glycoprotein (HRG). The synthetic peptides represented 1-3 multiple repeat units of the 5-residue sequence (Gly-His-His-Pro-His) found in the C-terminal of HRG. By frontal analyses, immobilized HRG peptides of the type (GHHPH)nG, where n = 1-3, were each found to have a similar binding capacity for both Cu(II) ions and Zn(II) ions (31-38 mumol/ml gel). The metal ion-dependent interaction of a variety of model peptides with each of the immobilized HRG peptide affinity columns demonstrated differences in selectivity despite the similar internal sequence homology and metal ion binding capacity. The immobilized 11-residue HRG peptide was loaded with Cu(II) ions and used to demonstrate selective adsorption and isolation of proteins from human plasma. These results suggest that immobilized metal-binding peptides selected from known solvent-exposed protein surface metal-binding domains may be useful model systems to evaluate the specificity of biologically relevant metal ion-dependent interaction and transfer events in vitro.     

Aromatic capping surprisingly stabilizes furan moieties in peptides against acidic degradation

We herein describe the synthesis of furan containing peptides for further post-synthetic derivatisation in solution through our recently developed furan-oxidation-labeling technology. Previously, it was reported by others that during acidic cleavage of furan-modified peptides, furan moieties can suffer from degradation. We demonstrate here that this degradation is position dependent and can be fully suppressed through introduction of proximate aromatic residues. Versatile introduction of 2-furylalanine at internal, C-terminal as well as the sensitive N-terminal positions has now been proven possible.     

Synthetic metal-binding protein surface domains for metal ion-dependent interaction chromatography. I. Analysis of bound metal ions by matrix-assisted UV laser desorption time-of-flight mass spectrometry.

To extend the analytical capabilities of immobilized metal ion affinity chromatography (IMAC) for evaluation of biologically relevant peptide-metal ion interactions, we have prepared synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein known as histidine-rich glycoprotein (HRG). Three synthetic peptides, representing multiples of a 5-residue repeat sequence (Gly-His-His-Pro-His) from within the histidine- and proline-rich region of the C-terminal domain were prepared. Prior to immobilization, the synthetic peptides were evaluated for identity and sample homogeneity by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), a method developed recently for the mass determination of high-molecular-mass biopolymers. 2,5-Dihydroxybenzoic acid was evaluated as a matrix to facilitate the laser desorption and ionization of intact peptides and was found to be ideally suited for determinations of mass within the low-mass region of interest (641.7 to 1772.8 dalton). We observed minimal chemical noise from photochemically generated peptide-matrix adduct signals, clustering, and multiply-charged peptide species. Peptides with bound sodium and potassium ions were observed; however, these signal intensities were reduced by immersion of the sample probe tip in water. Mixtures of the three different synthetic peptides were also evaluated by LDTOF-MS after their elution through a special immobilized peptide-metal ion column designed to investigate metal ion transfer. We found LDTOF-MS to be a useful new method to verify the presence of peptide-bound metal ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of Edman degradation sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry in designing substrates for matrix metalloproteinases

The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. We have been designing single-stranded peptides (SSPs) and triple-helical peptides (THPs) as potential discriminatory MMP substrates. Edman degradation sequence and matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analyses of proteolytic activity have been utilized to aid in further substrate design. THP models of the alpha1(I)772-786 sequence from type I collagen were synthesized to examine the triple-helical substrate specificity of MMP family members. Sequence and MALDI-MS analyses were used in conjunction with a fluorometric assay to determine the exact point of cleavage by each MMP. MMP-1 (interstitial collagenase) cleaved the substrates at a single Gly-Ile bond, analogous to the cleavage site in type I collagen. MMP-2 (Mr 72 000 type IV collagenase; gelatinase A) was found to cleave the substrates at two sites, a Gly-Ile bond and a Gly-Gln bond. MMP-3 (stromelysin 1) was found to cleave only one of the substrates after reaction for 48 h. Ultimately, sequence and MALDI-MS analyses allowed us to detect an additional cleavage site for MMP-2 in comparison to MMP-1, while MMP-3 was found to cleave a substrate after an extended time period. The second cleavage site would cause the kinetic parameters for MMP-2 to be overestimated by the fluorometric assay. Further design variations for these substrates need to consider the presence of more stable triple-helical conformation (to eliminate MMP-3 binding) and the removal of Gly-Gln bonds that may be susceptible to MMP-2.     

Antimicrobial activity and protease stability of peptides containing fluorinated amino acids

Selective fluorination of peptides results in increased chemical and thermal stability with simultaneously enhanced hydrophobicity. We demonstrate here that fluorinated derivatives of two host defense antimicrobial peptides, buforin and magainin, display moderately better protease stability while retaining, or exhibiting significantly increased bacteriostatic activity. Four fluorinated analogues in the buforin and two in the magainin series were prepared and analyzed for (1) their ability to resist hydrolytic cleavage by trypsin; (2) their antimicrobial activity against both gram-positive and gram-negative bacterial strains; and (3) their hemolytic activity. All but one fluorinated peptide (M2F5) showed retention, or significant enhancement, of antimicrobial activity. The peptides also showed modest increases in protease resistance, relative to the parent peptides. Only one of the six fluorinated peptides (BII1F2) was degraded by trypsin at a slightly faster rate than the parent peptide. Hemolytic activity of peptides in the buforin series was essentially null, while fluorinated magainin analogues displayed an increase in hemolysis compared to the parent peptides. These results suggest that fluorination may be an effective strategy to increase the stability of biologically active peptides where proteolytic degradation limits therapeutic value.     

Gas-phase cleavage of PTC-derivatized electrosprayed tryptic peptides in an FT-ICR trapped-ion cell: Mass-based protein identification without liquid chromatographic separation

Condensed phase protein sequencing typically relies on N-terminal labeling with phenylisothiocyanate ("Edman" reagent), followed by cleavage of the N-terminal amino acid. Similar Edman degradation has been observed in the gas phase by collision-activated dissociation of the N-terminal phenyl thiocarbamoyl protonated peptide [1] to yield complementary b1 and y(n-1) fragments, identifying the N-terminal amino acid. By use of infrared multiphoton (rather than collisional) activation, and Fourier transform ion cyclotron resonance (rather than quadrupole) mass analysis, we extend the method to direct analysis of a mixture of tryptic peptides. We validate the approach with bradykinin as a test peptide, and go on to analyze a mixture of 25 peptides produced by tryptic digestion of apomyoglobin. A b1+ ion is observed for three of the Edman-derivatized peptides, thereby identifying their N-terminal amino-acids. Search of the SWISS-PROT database gave a single hit (myoglobin, from the correct biological species), based on accurate-mass FT-ICR MS for as few as one Edman-derivatized tryptic peptide. The method is robust-it succeeds even with partial tryptic digestion, partial Edman derivatization, and partial MS/MS IRMPD cleavage. Improved efficiency and automation should be straightforward.     

Primary structural determination of N-terminally blocked peptides from the bark of Eucommia ulmoides Oliv by mass spectrometric analysis

Sequencing of N-terminally blocked proteins/peptides is a challenge since these molecules inhibit processing by Edman degradation. By using high accuracy Fourier transform ion cyclotron resonance (FTICR) tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), the primary structures of two novel N-terminally blocked antifungal peptides (EAFP1 and EAFP2), purified from the bark of Eucommia ulmoides Oliv, have been determined. The results show that the high mass accuracy provided by FTICR mass spectrometry is effective to determine the N-terminally blocking group, and can simplify the task of spectral interpretation and improve the precision of sequence determination. The combination of MALDI-TOFMS with carboxyl peptidase Y digestion was used to determine the C-terminal 36- and 27-residue sequences of EAFP1 and EAFP2, respectively, to provide the sequence linkage information for tryptic fragments. Compared with traditional peptide chemistry the advantage of mass spectrometric techniques is their simplicity, speed and sensitivity.     

Collision Induced Dissociation Products of Disulfide-Bonded Peptides: Ions Result from the Cleavage of More Than One Bond

Disulfide bonds are a post-translational modification (PTM) that can be scrambled or shuffled to non-native bonds during recombinant expression, sample handling, or sample purification. Currently, mapping of disulfide bonds is not easy because of various sample requirements and data analysis difficulties. One step towards facilitating this difficult work is developing a better understanding of how disulfide-bonded peptides fragment during collision induced dissociation (CID). Most automated analysis algorithms function based on the assumption that the preponderance of product ions observed during the dissociation of disulfide-bonded peptides result from the cleavage of just one peptide bond, and in this report we tested that assumption by extensively analyzing the product ions generated when several disulfide-bonded peptides are subjected to CID on a quadrupole time of flight (QTOF) instrument. We found that one of the most common types of product ions generated resulted from two peptide bond cleavages, or a double cleavage. We found that for several of the disulfide-bonded peptides analyzed, the number of double cleavage product ions outnumbered those of single cleavages. The influence of charge state and precursor ion size was investigated, to determine if those parameters dictated the amount of double cleavage product ions formed. It was found in this sample set that no strong correlation existed between the charge state or peptide size and the portion of product ions assigned as double cleavages. These data show that these ions could account for many of the product ions detected in CID data of disulfide bonded peptides. We also showed the utility of double cleavage product ions on a peptide with multiple cysteines present. Double cleavage products were able to fully characterize the bonding pattern of each cysteine where typical single b/y cleavage products could not.     

Solid-state NMR spectroscopy method for determination of the backbone torsion angle psi in peptides with isolated uniformly labeled residues

We demonstrate a solid-state nuclear magnetic resonance technique, with the acronym ROCSA-LG, for the determination of backbone torsion angles psi in peptides with multiple, but isolated, uniformly labeled residues. The method correlates the 13C' chemical shift anisotropy and the 13Calpha-1Halpha heteronuclear dipolar tensors within a single uniformly labeled residue in a two-dimensional (2D) experiment. The technique requires the measurement of only five 2D spectra and is compatible with high-speed magic-angle spinning. Experimental results are presented for the 17-residue alpha-helical peptide MB(i+4)EK and for amyloid fibrils formed by the 15-residue peptide Abeta11-25.     

Peptide sequence prediction supported by correlation-associated networks in human cerebrospinal fluid

During the course of biosynthesis, processing and degradation of a peptide, many structurally related intermediate peptide products are generated. Human body fluids and tissues contain several thousand peptides that can be profiled by reversed-phase chromatography and subsequent MALDI-ToF-mass spectrometry. Correlation-Associated Peptide Networks (CAN) efficiently detect structural and biological relations of peptides, based on statistical analysis of peptide concentrations. We combined CAN with recognition of probable cleavage sites for peptidases and proteases in cerebrospinal fluid, resulting in a model able to predict the sequence of unknown peptides with high accuracy. On the basis of this approach, identification of peptide coordinates can be prioritized, and a rapid overview of the peptide content of a novel sample source can be obtained.     

An unusual cleavage reaction of a peptide observed during dithiotreitol and tris(2-carboxyethyl)phosphine reduction: application to sequencing of HpTx2 spider toxin using nanospray tandem mass spectrometry

A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem mass spectrometry (MS/MS). This 30-residue toxin possesses a highly knotted structure with cystines arranged in close proximity. The low-energy collision-induced dissociation MS/MS spectrum of the [M+4H](4+) ion permitted characterization of the C-terminal sequence of HpTx2 up to Cys(26) that is involved in a disulfide bridge. Chemical pre-treatment with DTT or TCEP was then investigated, and it was found that an unexpected cleavage reaction of HpTx2 gave two smaller peptides which were completely sequenced by MS/MS experiments using a Qq-TOF mass spectrometer. This unusual hydrolysis reaction facilitated the determination of the complete sequence of the HpTx2 toxin.     

Synthesis and characterization by fast atom bombardment mass spectrometry of peptides related to the B-domain of staphylococcal protein A

Solid-phase synthesis was used to prepare several peptides related to Staphylococcal protein A, including a 58-residue sequence corresponding to the entire B-domain of the protein. These materials were characterized by a combination of fast atom bombardment mass spectrometry and microchemical methods, including Edman degradation and tryptic digestion. The errors identified included incomplete removal of blocking groups, premature chain termination and the dehydration of an aspartic add residue in the sequence. All of these problems would have been extremely difficult to elucidate by conventional techniques of protein chemistry, and demonstrate that mass spectrometry is the method of choice for verifying the structural integrity of the products of solid-phase synthesis.     

Evolutionary screening of collagen-like peptides that nucleate hydroxyapatite crystals

The biogenesis of inorganic/organic composite materials such as bone typically involves the process of templated mineralization. Biomimetic synthesis of bone-like materials therefore requires the development of organic scaffolds that mediate mineralization of hydroxyapatite (HAP), the major inorganic component of bone. Using phage display, we identified a 12-residue peptide that bound to single-crystal HAP and templated the nucleation and growth of crystalline HAP mineral in a sequence- and composition-dependent manner. The sequence responsible for the mineralizing activity resembled the tripeptide repeat (Gly-Pro-Hyp) of type I collagen, a major component of bone extracellular matrix. Using a panel of synthetic peptides, we defined the structural features required for mineralizing activity. The results support a model for the cooperative noncovalent interaction of the peptide with HAP and suggest that native collagen may have a mineral-templating function in vivo. We expect this short HAP-binding peptide to be useful in the synthesis of three-dimensional bone-like materials.     

Thermodynamic stability of polyacrylamide and poly(N,N‐dimethyl acrylamide)

Based on theoretical thermodynamic principles, the possibility of environmental degradation of polyacrylamide to its starting monomer was investigated. Theoretical electronic structure studies on the geometry and fragmentation energy of acrylamide and N,N‐dimethyl acrylamide tetramer models were carried out using a first principles gradient corrected density functional approach. Thermal degradation to form a radical would require the cleavage of carbon–carbon bonds in the polymer chain; the energy needed for this cleavage was found to depend on the structure of the repeat unit which ranged from low of 72.5 kcal for a rare head‐to‐head construct to 86.2 kcal for a normal head‐to‐tail polymer construct (therefore, for the cleavage of a normal head‐to‐tail repeat unit, temperatures of approximately 450°C would be required). The thermodynamics of the unzipping, disproportionation, and back‐biting reactions in the resulting radical fragments were also investigated; the back‐biting process was found to require the least energy and provided the most stable radical fragment with a low probability for disproportionation or releasing of monomer to occur. The effect of solvation on the hydrogen‐bonding network in the acrylamide tetramer was studied by adding explicit molecules of water to the tetramer models. The addition of water had a significant effect on the stability of the model polymer slightly stabilizing the head‐to‐head polymer, and slightly destabilizing the head to tail polymer. Copyright © 2007 John Wiley & Sons, Ltd.