C-terminal sequencing by mass spectrometry: application to gelatine-derived proline-rich peptides

Protonated peptides derived from proline‐rich proteins (PRP) are often difficult to sequence by standard collision‐induced dissociation (CID) mass spectrometry (MS) due to preferential amide bond cleavage N‐terminal to proline. In connection with bovine spongiform encephalopathy regulations, proteolytic products derived from the PRP collagen have been suggested as markers for contamination of animal feedstuffs with processed animal protein (Fernandez Ocaña, M. et al., Analyst 2004, 129, 111–115). Herein, we report the identification of these marker peptides using the strategy of C‐terminal sequencing by CID MS from their sodium and lithium adducts. Upon fragmentation a new cationized peptide was produced that is one C‐terminal amino acid shorter in length. This dissociation pathway allowed for the facile identification of the C‐terminal residue by matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry. Each newly formed cationized peptide was further fragmented by up to seven stages of electrospray ionization ion trap MS. Proline‐rich C‐terminal sequence tags were established which permitted successful database identification of collagen alpha type I proteins.     

PTM MarkerFinder,a software tool to detect and validate spectra from peptides carrying post‐translational modifications

Mass spectrometry (MS) analysis of peptides carrying post‐translational modifications is challenging due to the instability of some modifications during MS analysis. However, glycopeptides as well as acetylated, methylated and other modified peptides release specific fragment ions during CID (collision‐induced dissociation) and HCD (higher energy collisional dissociation) fragmentation. These fragment ions can be used to validate the presence of the PTM on the peptide. Here, we present PTM MarkerFinder, a software tool that takes advantage of such marker ions. PTM MarkerFinder screens the MS/MS spectra in the output of a database search (i.e., Mascot) for marker ions specific for selected PTMs. Moreover, it reports and annotates the HCD and the corresponding electron transfer dissociation (ETD) spectrum (when present), and summarizes information on the type, number, and ratios of marker ions found in the data set. In the present work, a sample containing enriched N‐acetylhexosamine (HexNAc) glycopeptides from yeast has been analyzed by liquid chromatography‐mass spectrometry on an LTQ Orbitrap Velos using both HCD and ETD fragmentation techniques. The identification result (Mascot .dat file) was submitted as input to PTM MarkerFinder and screened for HexNAc oxonium ions. The software output has been used for high‐throughput validation of the identification results.     

Evaluation of Chemical Derivatisation Methods for Protein Identification using MALDI MS/MS

In proteomic studies, assigning protein identity from organisms whose genomes are yet to be completely sequenced remains a challenging task. For these organisms, protein identification is typically based on cross species matching of amino acid sequence obtained from collision induced dissociation (CID) of peptides using mass spectrometry. The most direct approach of de novo sequencing is slow and often difficult, due to the complexity of the resultant CID spectra. For MALDI-MS, this problem has been addressed by using chemical derivatisation to direct peptide fragmentation, thereby simplifying CID spectra and facilitating de novo interpretation. In this study, milk whey proteins from the tammar wallaby (Macropus eugenii) were used to evaluate three chemical derivatisation methods compatible with MALDI MS/MS. These methods included (i) guanidination and sulfonation using chemically-assisted fragmentation (CAF), (ii) guanidination and sulfonation using 4-sulfophenyl isothiocyanate (SPITC) and (iii) derivatising the epsilon-amino group of lysine residues with Lys Tag 4H. Derivatisation with CAF and SPITC resulted in more protein identification than Lys Tag 4H. Sulfonation using SPITC was the preferred method due to the low cost per experiment, the reactivity with both lysine and arginine terminated peptides and the resultant simplified MS/MS spectra.*Australian Peptide Conference Issue.**This project was funded by an ARC Linkage grant to Deane supported by TGR Biosciences and facilitated by access to the Australian Proteome Analysis Facility established under the Australian Government’s Major National Research Facilities program.     

Studies on the Selective Trifluoroacetolytic Scission of Native Peptaibols and Model Peptides Using HPLC and ESI‐CID‐MS

Representative members of a group of linear, N‐acylated polypeptide antibiotics (peptaibols) containing α‐aminoisobutyric acid (Aib) and, in part, isovaline (Iva), as well as proteinogenic amino acids and a C‐terminal‐bonded 2‐amino alcohol, were treated with anhydrous trifluoroacetic acid (TFA) at 37° for 0.5–26 h. The resulting fragments were separated by HPLC and characterized by electrospray ionization collision‐induced dissociation mass spectrometry (ESI‐CID‐MS). The following 16–20‐residue peptaibols were investigated: natural, microheterogeneous mixtures of antiamoebins and alamethicin F50, uniform paracelsin A, and synthetic trichotoxin A50/E. In the natural peptides, bonds formed between Aib (Iva) and Pro (Hyp) were rapidly and selectively cleaved within 0.5 h. Furthermore, TFA esters of the C‐terminal amino alcohols were formed. Depending on time, release of C‐terminal tri‐ and tetrapeptides as well as amino acids from the major fragments was observed. Synthetic homooligopeptides, namely Z‐ and Ac‐(Aib)₁₀‐O^tBu and Z‐(Aib)₇‐O^tBu, were analyzed for comparison. On treatment with TFA, a regular series of Z‐(Aib)_10–5‐OH from Z‐(Aib)₁₀‐O^tBu were detected within 0.5 h, and, after 3 h, release of a regular series of Z‐(Aib)_7–3‐OH from Z‐(Aib)₇‐O^tBu were observed. Moreover, concomitant release of the series of H‐(Aib)_10–3‐OH from the decapeptide occurred. From these data, a repetitive cleavage mechanism via intermediate formation of C‐terminal oxazolones on trifluoroacetolysis is proposed. Furthermore, their formation and stability in native peptaibols are correlated with subtle structural differences in protein amino acids linked to Aib. From the conspicuous concordance of the formation and abundance of regular series of trifluoroacetolytic fragments and of positive ions of the b‐series in CID‐MS, the generation of intermediate oxazolonium ions in both gas and liquid phase is concluded.     

Drug‐binding energetics of human α‐1‐acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations

This study utilizes sensitive, modern isothermal titration calorimetric methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α‐1‐acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug–AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine, all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug‐binding thermodynamics was characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed that an enthalpy–entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (?Hº) and favorable (positive) entropic (?Sº) contributions to the Gibbs free energy (?Gº). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side‐chain sub‐domains within the multi‐lobed AGP ligand binding cavity.Copyright © 2012 John Wiley & Sons, Ltd.     

PAS‐cal: A repetitive peptide sequence calibration standard for MALDI mass spectrometry

Mass spectrometers equipped with matrix‐assisted laser desorption/ionization (MALDI‐MS) require frequent multipoint calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS‐cal tailored for MALDI‐MS based bottom‐up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C‐terminal arginine residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multipoint calibration of MS spectra using PAS‐cal peptides compares well to current commercial reagents for protein identification by PMF. Calibration of tandem mass spectra from LC‐MALDI experiments using the longest peptide, PAS‐cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS‐cal standard generically useful for bottom‐up proteomics.     

Electron transfer dissociation of peptides generated by microwave D-cleavage digestion of proteins

The nonenzymatic digestion of proteins by microwave D-cleavage is an effective technique for site-specific cleavage at aspartic acid (D). This specific cleavage C-terminal to D residues leads to inherently large peptides (15-25 amino acids) that are usually relatively highly charged (above +3) when ionized by electrospray ionization (ESI) due to the presence of several basic amino acids within their sequences. It is well-documented that highly charged peptide ions generated by ESI are well-suited for electron transfer dissociation (ETD), which produces c- and z-type fragment ions via gas-phase ion/ion reactions. In this paper, we describe the sequence analysis by ETD tandem mass spectrometry (MS/MS) of multiply charged peptides generated by microwave D-cleavage of several standard proteins. Results from ETD measurements are directly compared to CID MS/MS of the same multiply charged precursor ions. Our results demonstrate that the nonenzymatic microwave D-cleavage technique is a rapid (<6 min) and specific alternative to enzymatic cleavage with Lys-C or Asp-N to produce highly charged peptides that are amenable to informative ETD.     

Effectiveness of CID, HCD, and ETD with FT MS/MS for degradomic-peptidomic analysis: comparison of peptide identification methods

We report on the effectiveness of CID, HCD, and ETD for LC-FT MS/MS analysis of peptides using a tandem linear ion trap-Orbitrap mass spectrometer. A range of software tools and analysis parameters were employed to explore the use of CID, HCD, and ETD to identify peptides (isolated from human blood plasma) without the use of specific "enzyme rules". In the evaluation of an FDR-controlled SEQUEST scoring method, the use of accurate masses for fragments increased the number of identified peptides (by ~50%) compared to the use of conventional low accuracy fragment mass information, and CID provided the largest contribution to the identified peptide data sets compared to HCD and ETD. The FDR-controlled Mascot scoring method provided significantly fewer peptide identifications than SEQUEST (by 1.3-2.3 fold) and CID, HCD, and ETD provided similar contributions to identified peptides. Evaluation of de novo sequencing and the UStags method for more intense fragment ions revealed that HCD afforded more contiguous residues (e.g., ≥ 7 amino acids) than either CID or ETD. Both the FDR-controlled SEQUEST and Mascot scoring methods provided peptide data sets that were affected by the decoy database used and mass tolerances applied (e.g., identical peptides between data sets could be limited to ~70%), while the UStags method provided the most consistent peptide data sets (>90% overlap). The m/z ranges in which CID, HCD, and ETD contributed the largest number of peptide identifications were substantially overlapping. This work suggests that the three peptide ion fragmentation methods are complementary and that maximizing the number of peptide identifications benefits significantly from a careful match with the informatics tools and methods applied. These results also suggest that the decoy strategy may inaccurately estimate identification FDRs.     

Octanol–water partition of nonzwitterionic peptides: Predictive power of a molecular size-based model

A remarkably simple, molecular size-based model developed to predict octanol–water partition coefficients for organic compounds is tested on a set of 188 neutral peptides with available experimental partition data. Despite using only two parameters, it gives a promising correlation (r² = 0.914; σ = 0.455, F = 1978.0), and predictions are in a realistic range even for larger peptides (cyclosporin, melanotan, sandostatin) where common, overparametrized fragment methods become quite unreliable. Ion-pair partitioning and the extraction constant formalism is briefly reviewed to describe the sigmoidal lipophilicity profile of ionizable, nonzwitterionic peptides. It seems possible to extend the present model to estimate apparent partition coefficients measured around neutral pH and physiological conditions for monoionic peptides; however, as no standard conditions are yet defined and only relatively small number of experimental data are available, the situation here is more complex. Proteins 30:86–99, 1998. © 1998 Wiley-Liss, Inc.     

Application of electrospray ionization MS/MS and matrix-assisted laser desorption/ionization-time of flight mass spectrometry to structural analysis of the glycosyl-phosphatidylinositol-anchored protein.

We applied the improved sensitivity and soft ionization characteristics of electrospray Ionization (ESI)-MS/MS and matrix-assisted laser desorption/ionization(MALDI)-time of flight (TOF) mass spectrometry (MS) to analysis of the GPI-anchored C-terminal peptide derived from 5'-nucleotidase. ESI-MS/MS analysis was applied to the core structure (MW, 2,743). In the collision-induced dissociation (CID) spectrum, single-charged ions such as m/z 162 (glucosamine), 286 (mannose-phosphate-ethanolamine), and 447 ([mannose-phosphate-ethanolamine]-glucosamine) were clearly detected as characteristic fragment ions of the GPI-anchored peptide. On MALDI-TOF-MS analysis, heterogeneous peaks of GPI-anchored peptides were detected as single-charged ions in the positive mode. Product ions were obtained by post-source decay (PSD) of m/z 2,905 using curved field reflectron of TOF-MS. Most of the expected product ions derived from the GPI-anchored peptide, containing the core structure and an additional mannose side chain, were successively obtained. Thus, ESI-MS/MS and MALDI-TOF-PSD-MS proved to be effective and sensitive methods for analyzing the GPI-anchored peptide structure with less than 10 pmol of sample. These characteristic fragments or fragmentation patterns seem to be very useful for identification of GPI-anchored C-terminal peptides derived from any kind of GPI-anchored protein.     

Using the knowns to discover the unknowns: MS‐based dereplication uncovers structural diversity in 17‐hydroxygeranyllinalool diterpene glycoside production in the Solanaceae

Exploring the diversity of plant secondary metabolism requires efficient methods to obtain sufficient structural insights to discriminate previously known from unknown metabolites. De novo structure elucidation and confirmation of known metabolites (dereplication) remain a major bottleneck for mass spectrometry‐based metabolomic workflows, and few systematic dereplication strategies have been developed for the analysis of entire compound classes across plant families, partly due to the complexity of plant metabolic profiles that complicates cross‐species comparisons. 17‐hydroxygeranyllinalool diterpene glycosides (HGL‐DTGs) are abundant defensive secondary metabolites whose malonyl and glycosyl decorations are induced by jasmonate signaling in the ecological model plant Nicotiana attenuata. The multiple labile glycosidic bonds of HGL‐DTGs result in extensive in‐source fragmentation (IS‐CID) during ionization. To reconstruct these IS‐CID clusters from profiling data and identify precursor ions, we applied a deconvolution algorithm and created an MS/MS library from positive‐ion spectra of purified HGL‐DTGs. From this library, 251 non‐redundant fragments were annotated, and a workflow to characterize leaf, flower and fruit extracts of 35 solanaceous species was established. These analyses predicted 105 novel HGL‐DTGs that were restricted to Nicotiana, Capsicum and Lycium species. Interestingly, malonylation is a highly conserved step in HGL‐DTG metabolism, but is differentially affected by jasmonate signaling among Nicotiana species. This MS‐based workflow is readily applicable for cross‐species re‐identification/annotation of other compound classes with sufficient fragmentation knowledge, and therefore has the potential to support hypotheses regarding secondary metabolism diversification.     

Lipopeptides Produced by a Soil <Emphasis Type="Italic">Bacillus Megaterium</Emphasis> Strain

A soil microorganism identified as Bacillum megaterium was found to produce several antibiotics substances after growth for 20 h at 37°C in a mineral culture medium. Analysis both by electron spray ionization (ESI) and matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) mass spectrometry (MS) identified these substances as lipopeptides. Predominant peaks at m/z 1,041 and m/z 1,065 revealed ions which are compatible with surfactins and lichenysins, respectively. Two other ions m/z 1,057 and m/z 1,464 were further studied by collision-induced dissociation (CID) unveiling an iturin A at the first and fengycins A and B at the second m/z peaks. The CID spectrum of the m/z 1,464 ion also suggests the existence of fengycins A and B variants in which Ile was changed to Val in the position 10 of the peptide moiety. Raw mixtures of all these compounds were also assayed for antibiotic features. The data enlighten the unusual diversity of the lipopeptide mixture produced by a sole Bacillus species.     

Protein lysine‐Nζ alkylation and O‐phosphorylation mediated by DTT‐generated reactive oxygen species

Reactive oxygen species (ROS) play crucial roles in physiology and pathology. In this report, we use NMR spectroscopy and mass spectrometry (MS) to demonstrate that proteins (galectin-1, ubiquitin, RNase, cytochrome c, myoglobin, and lysozyme) under reducing conditions with dithiothreitol (DTT) become alkylated at lysine-N_ζ groups and O-phosphorylated at serine and threonine residues. These adduction reactions only occur in the presence of monophosphate, potassium, trace metals Fe/Cu, and oxygen, and are promoted by reactive oxygen species (ROS) generated via DTT oxidation. Superoxide mediates the chemistry, because superoxide dismutase inhibits the reaction, and hydroxyl and phosphoryl radicals are also likely involved. While lysine alkylation accounts for most of the adduction, low levels of phosphorylation are also observed at some serine and threonine residues, as determined by western blotting and MS fingerprinting. The adducted alkyl group is found to be a fragment of DTT that forms a Schiff base at lysine N_ζ groups. Although its exact chemical structure remains unknown, the DTT fragment includes a SH group and a –CHOH–CH₂– group. Chemical adduction appears to be promoted in the context of a well-folded protein, because some adducted sites in the proteins studied are considerably more reactive than others and the reaction occurs to a lesser extent with shorter, unfolded peptides and not at all with small organic molecules. A structural signature involving clusters of positively charged and other polar groups appears to facilitate the reaction. Overall, our findings demonstrate a novel reaction for DTT-mediated ROS chemistry with proteins.     

Effective novel dissociation methods for intact protein: heat-assisted nozzle-skimmer collisionally induced dissociation and infrared multiphoton dissociation using a Fourier transform ion cyclotron resonance mass spectrometer equipped with a micrometal electrospray ionization emitter

Heating of a nano-electrospray ionization (nanoESI) source can improve the dissociation efficiency of collisionally induced dissociation (CID) methods, such as nozzle-skimmer CID (NS-CID) and infrared multiphoton dissociation (IRMPD), for large biomolecule fragmentation. A metal nanoESI emitter was used due to its resistance to heating above 250 degrees C. This novel method for the dissociation of large biomolecular ions is termed "heat-assisted NS-CID" (HANS-CID) or "heat-assisted IRMPD" (HA-IRMPD). Multiple charged nonreduced protein ions (8.6 Da ubiquitin, 14 kDa lysozyme, and 67 kDa bovine serum albumin) were directly dissociated by HANS-CID and HA-IRMPD to effectively yield fragment ions that could be assigned. The fragment ions of ubiquitin by HANS-CID can be analyzed by tandem mass spectrometry (MS/MS) using sustained off-resonance irradiation CID (SORI-CID) and IRMPD. In addition, a native large protein, immunoglobulin G (IgG, 150 kDa), was efficiently dissociated by HA-IRMPD. The product ions that were obtained reflected the domain structure of IgG. However, these product ions of IgG and lysozyme were not dissociated by MS/MS using the same heating energetic methods such as IRMPD and SORI-CID.     

ITMSQ: A software tool for N‐ and C‐terminal fragment ion pairs based isobaric tandem mass spectrometry quantification

Tandem MS (MS2) quantification using the series of N‐ and C‐terminal fragment ion pairs generated from isobaric‐labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solutionfor N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantitative methods.     

Purification and structural characterization of fengycin homologues produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> LSFM-05 grown on raw glycerol

Raw glycerol is a byproduct of biodiesel production that currently has low to negative value for biodiesel producers. One option for increasing the value of raw glycerol is to use it as a feedstock for microbial production. Bacillus subtilis LSFM 05 was used for the production of fengycin in a mineral medium containing raw glycerol as the sole carbon source. Fengycin was isolated by acid precipitation at pH 2 and purified by silica gel column chromatography and characterized using electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) with collision-induced dissociation (CID). The mass spectrum revealed the presence of the ions of m/z 1,435.7, 1,449.9, 1,463.8, 1,477.8, 1,491.8 and 1,505.8, which were further fragmented by ESI-MS/MS. The CID profile showed the presence of a series of ions (m/z 1,080 and 966) and (m/z 1,108 and 994) that represented the different fengycin homologues A and B, respectively. Fengycin homologues A and B are variants that differ at position 6 of the peptide moiety, having either Ala or Val residues, respectively. Mass spectrometry analyses identified four fengycin A and three fengycin B variants with fatty acid components containing 14–17 carbons. These results demonstrate that raw glycerol can be used as feedstock to produce fengycin, and additional work should focus on the optimization of process conditions to increase productivity.     

Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks (<Emphasis Type="Italic">Lassi</Emphasis>) Fermented by <Emphasis Type="Italic">Lactobacillus acidophillus</Emphasis> with Consideration of Incubation Period and Simmering Treatment

The lassi, fermented milks product containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus acidophilus NCDC-15 and the incubation period and simmering effect was also optimized for production of ACE-inhibitory peptides. The time–temperature combination for the heat treatment was optimized using RSM. The biological activity was measured in the supernatant of the fermented milk after centrifugation. The lowest IC₅₀ values for the inhibition of angiotensin-converting enzyme (ACE) was found 28.9 ± 0.95 μg protein/ml in the supernatant of milk fermented by L. acidophilus and heated at 78 °C for 10 h. The fractions which showed the highest ACE-inhibitory indexes were further purified by different techniques including solid phase extraction, RP-HPLC and FPLC and the related peptides were identified by LC–MS/MS using the Ultimate 3000 nano HPLC system (Dionex) coupled to a 4000 Q TRAP electro-spray ionization mass spectrometry. The high ACE-inhibitory activity containing fractions of the milk fermented by L. acidophilus contained the sequences of b-casein (b-CN) fragment. The fraction-III showed minimum IC₅₀ value i.e. 14.57 ± 0.72 μg/ml compared with fraction-I and fraction-II. Among these peptides 14 peptides have been identified from the fraction-I of the lassi prepared from L. acidophilus i.e. β-CN f47–56, β-CN f47–57, β-CN f199–209, β-CN f176–182, β-CN f176–183, β-CN f176–184, β-CN f1–7, β-CN f57–68, β-CN f166–175, β-CN f195–206, β-CN f195–207, β-CN f195–209, β-CN f94–106 and β-CN f169–176 showed partially or completely homology to that the milk protein bioactive peptides having ACE inhibitory. The two peptides KVLPVPQK (β-CN f169–176) and YQEPVLGPVRGPFPIIV (β-CN f193–209) have the same sequence as ACE inhibitory peptides (Maeno et al. in J Dairy Sci 79(8):1316–1321, 1996; Yamamoto et al. in J Dairy Sci 77:917–922, 1994b).     

Sequencing of the thirteen structurally isomeric quartets of N‐terminal dipeptide motifs in peptides by collision‐induced dissociation

N‐terminal sequencing of protonated peptides is challenging, since each b₂ ion represents two sequence isomers, e.g., NE and EN. Additionally the occurrence of compositional isomers, such as NE and QD, further increases the number of isomers to four (NE, EN, QD, DQ). This leads to a subset of 13 b₂ ion masses where each value represents four individual species. The b₂ ions within such a quartet are characterized by the same elemental composition. To test the utility of CID for differentiation of isomeric b₂ ions, the CID spectra of 52 small synthetic peptides were recorded, representing the 13 isomeric b₂ ion quartets, which may be formed from unmodified amino acid residues. The CID spectra of protonated peptides containing these quartets were carefully inspected for N‐terminal sequence information. Below the m/z value of the b₂ ion, individual differences were found in the b₂ fragment ion signatures (neutral loss of CO, H₂O, NH₃, and other less common units). Recognition of N and Q in second position from the N‐terminus is based on c₁ ion formation. Relative intensities of immonium ions were also used for differentiation between sequence isomers. In the complementary high‐mass regions above the m/z value of the y_max‐2 ion, individual differences were observed in the formation of y_max‐1, x_max‐1 and z_max‐1 ions, which could be correlated to the complementary low‐mass ions. In summary, de novo sequencing of the N‐terminal dipeptide motif is feasible by considering all available sequence information present in CID spectra of protonated peptides.     

Mass spectrometric analysis of peptides in brain neurosecretory cells and neurohemal organs in the adult blowfly,Protophormia terraenovae

Neuropeptides in neurosecretory cells of the pars intercerebralis (PI) and pars lateralis (PL) in the brain, and those in the corpus cardiacum–hypocerebral ganglion complex (CC-HG) and corpus allatum (CA) were examined by mass spectrometry and immunocytochemistry in adult females of the blowfly, Protophormia terraenovae. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometry (ESI-Q-Tof MS) and MS/MS, 4 peptides (including myosuppressin and SIFamide) were detected in the PI, 12 peptides (including [Arg⁷]-corazonin and [Arg⁷]-corazonin^3–11) in the PL, 13 peptides (including myosuppressin, [Arg⁷]-corazonin and [Arg⁷]-corazonin^3–11) in the CC-HG, and 6 peptides in the CA. MALDI-TOF MS analysis of each tissue or organ was made in about 20 flies under diapause-inducing (LD 12:12 at 20 °C) and diapause-averting conditions (LD 18:6 at 25 °C). These molecular ion peaks did not distinctively differ between diapause-inducing and diapause-averting conditions. A peptide with an m/z value at 1395.1 was purified from 240 brains and the 2nd–10th amino acids were sequenced as –YRKPPFNGS–, corresponding to a partial sequence of SIFamide. Only two pairs of somata in the PI were immunoreactive to antisera against SIFamide, which were local neurons widely extending fibers throughout the brain neuropils.     

Synthesis and conformational studies of peptides containing TOAC,a spin-labelled Cα,α-disubstituted glycine

A variety of host L -alanine homo-peptides (to the pentamer) containing one or two spin-labelled TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) residues were synthesized by solution methods and fully characterized. The conformational features of the terminally blocked, doubly spin-labelled–TOAC–(Ala)₂–TOAC–Ala– pentapeptide were examined in the crystal state by X-ray diffraction and in solution using a combination of techniques (Fourier transform infrared, circular dichroism, cyclic voltammetry and electron spin resonance) in comparison with singly labelled shorter peptides. The 3₁₀-helical structure of the pentapeptide, promoted by the two C^α,α-disubstituted glycines under favourable experimental conditions, allows an interaction to take place between the two nitroxide TOAC side chains spaced by one turn of the helix. Taken together, these results suggest that TOAC is an excellent probe for exploring bends and helices in doubly labelled peptides.