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1.
Quinton L Gilles N Smargiasso N Kiehne A De Pauw E 《Journal of the American Society for Mass Spectrometry》2011,22(11):1891-1897
This study describes the structural characterization of a totally new family of peptides from the venom of the snake green
mamba (Dendroaspis angusticeps). Interestingly, these peptides differ in several points from other already known mamba toxins. First of all, they exhibit
very small molecular masses, ranging from 1.3 to 2.4 kDa. The molecular mass of classical mamba toxins is in the range of
7 to 25 kDa. Second, the new peptides do not contain disulfide bonds, a post-translational modification commonly encountered
in animal toxins. The third difference is the very high proportion of proline residues in the sequence accounting for about
one-third of the sequence. Finally, these new peptides reveal a carbohydrate moiety, indicating a glycosylation in the sequence.
The last two features have made the structural characterization of the new peptides by mass spectrometry a real analytical
challenge. Peptides were characterized by a combined use of MALDI- TOF/TOF and nanoESI-IT-ETD experiments to determine not
only the peptide sequence but also the composition and the position of the carbohydrate moiety. Anyway, such small glycosylated
and proline-rich toxins are totally different from any other known snake peptide and form, as a consequence, a new family
of peptides. 相似文献
2.
R. E. Hamilton G. V. Patil K. Jayasimhulu R. A. Day 《Journal of mass spectrometry : JMS》1974,9(2):211-220
We have been able to extend the use of Schiff base derivatives in peptide sequencing to N-terminal prolyl peptides. Earlier studies from this laboratory revealed that certain aromatic Schiff bases of peptide esters gave electron-impact mass spectra with relatively intense molecular, sequence and internal fragment ions. We observed that the reaction of N-terminal prolyl peptide esters with 4-dimethylaminonaphthaldehyde, p-dimethylaminobenzaldehyde and 2-pyridinecarboxaldehyde gave cyclization products which were found to be 2-substituted-1-keto-3-aryl-5H-imidazo-[1,5-a]-pyrrole derivatives. The molecular ion and many of the expected cleavages were prominent in the mass spectra. Deuterium labeling at the α-carbon, amide nitrogen, or other exchangeable positions has been used in assigning the structure. It was also confirmed by the fragmentation pattern of the products derived by permethylation of the peptide derivative with tetramethylammonium hydroxide. Comparable cleavage patterns were seen among the N-terminal prolyl peptides examined. Proline amide gave the corresponding cyclized product. With the inclusion of N-terminal prolyl peptides in the list of peptides that we have examined, we may now prepare volatile derivatives of peptides containing any of the protein amino acids in two steps: esterification and treatment with the appropriate aromatic aldehyde. 相似文献
3.
Recently various methods for the N-terminal sulfonation of peptides have been developed for the mass spectrometric analyses of proteomic samples to facilitate de novo sequencing of the peptides produced. This paper describes the isotope-coded N-terminal sulfonation (ICenS) of peptides; this procedure allows both de novo peptide sequencing and quantitative proteomics to be studied simultaneously. As N-terminal sulfonation reagents, 13C-labeled 4-sulfophenyl[13C6]isothiocyanate (13C-SPITC) and unlabeled 4-sulfophenyl isothiocyanate (12C-SPITC) were synthesized. The experimental and reference peptide mixtures were derivatized independently using 13C-SPITC and 12C-SPITC and then combined to generate an isotopically labeled peptide mixture in which each isotopic pair differs in mass by 6 Da. Capillary reverse-phase liquid chromatography/tandem mass spectrometry experiments on the resulting peptide mixtures revealed several immediate advantages of ICenS in addition to the de novo sequencing capability of N-terminal sulfonation, namely, differentiation between N-terminal sulfonated peptides and unmodified peptides in mass spectra, differentiation between N- and C-terminal fragments in tandem mass spectra of multiply protonated peptides by comparing fragmentations of the isotopic pairs, and relative peptide quantification between proteome samples. We demonstrate that the combination of N-terminal sulfonation and isotope coding in the mass spectrometric analysis of proteomic samples is a viable method that overcomes many problems associated with current N-terminal sulfonation methods. 相似文献
4.
Thierry Le Bihan Ramon Grima Sarah Martin Thorsten Forster Yann Le Bihan 《Rapid communications in mass spectrometry : RCM》2010,24(7):1093-1104
We present the application of a targeted liquid chromatography/mass spectrometry (LC/MS) approach developed on a linear ion trap for the evaluation of the abundance of cytoplasmic proteins from a HeLa cell extract. Using a standard data‐dependent approach, we identified some specific peptides from this extract which were also commercially available in their AQUA form (use for absolute quantitation). For some of the peptides, we observed a non‐linear response between the intensity and the added quantity which was then fitted using a quadratic fit. All AQUA peptides spiked into a mix of 3 µg of the HeLa cell digest extract were detected down to 16 fmol. We placed an emphasis on peptide detection which, in this study, is performed using a combination of properties such as three specific Q3‐like ion signatures (for a given Q1‐like selection) and co‐elution with the AQUA peptide counterparts. Detecting a peptide without necessarily identifying it using a search engine imposes less constraint in terms of tandem mass (MS/MS) spectra purity. An example is shown where a peptide is detected using those criteria but could not be identified by Mascot due to its lower abundance. To complement this observation, we used a cross‐correlation analysis approach in order to separate two populations of MS/MS fragments based on differences in their elution patterns. Such an approach opens the door to new strategies to analyse lower intensity peptide fragments. An in silico analysis of the human trypsinosome allows the evaluation of how unique are the sets of features that we are using for peptide detection. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
5.
Thompson AH Bjourson AJ Shaw C McClean S 《Rapid communications in mass spectrometry : RCM》2006,20(24):3780-3788
Amphibian skin secretions contain a plethora of bioactive compounds, many of which are understood to act to deter ingestion by predators. Bradykinins in particular are constitutively expressed in many amphibian skin secretions, mediating a variety of effects including hyperalgesia and contraction of gastric smooth muscle. Using a variety of proteomic techniques (high-performance liquid chromatography (HPLC) separation, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), and quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS)) the current study identified 13 bradykinin-like peptides in the skin secretions of Phyllomedusa hypochondrialis azurea, including several new C-terminally extended isoforms (VPPGFTPFRLT, VHypPGFTPFRQT) and a novel phyllokinin-like peptide (RPPGFTPFRVY). Identification of the cDNA sequences encoding these peptides led to the deduction that the peptides were derived from differential post-translational processing and modification of five different precursors. Such an event emphasises the metabolic efficiency of peptide production in amphibian venom, with multiple products perhaps selective to different receptors in a variety of predators generated from a single precursor. An unusual modification was also recognised in the present study, with several bradykinin-like peptides featuring hydroxyprolination of the first proline residue rather than the commonly targeted second. This alteration may be mediated by the structural organisation of N-terminal amino acids prior to precursor processing. 相似文献
6.
Hideaki Tsunematsu Shizuko Nakashima Magobei Yamamoto 《Journal of mass spectrometry : JMS》1989,24(10):943-945
Fragmentations of N-benzyloxycarbonyl-protected tripeptide ethyl esters containing proline were compared with those of the corresponding peptide derivatives not containing proline in negative-ion fast atom bombardment mass spectrometry. The fragment ion [M – 109]? due to loss of the benzyloxy group followed by dehydrogenation from the peptide molecule was the base peak in the negative-ion mass spectra for the peptides not containing proline, whilst it was a very weak fragment ion or not observed at all in those for the peptides containing proline. These results suggest that the fragmentations of the peptide derivatives in negative-ion fast atom bombardment mass spectrometry depend on the conformational difference of the peptide derivatives owing to the existence of proline in the derivative. 相似文献
7.
Ruibing Chen Mingming Ma Limei Hui Jiang Zhang Lingjun Li 《Journal of the American Society for Mass Spectrometry》2009,20(4):708-718
Neuropeptides are often released into circulatory fluid (hemolymph) to act as circulating hormones and regulate many physiological
processes. However, the detection of these low-level peptide hormones in circulation is often complicated by high salt interference
and rapid degradation of proteins and peptides in crude hemolymph extracts. In this study, we systematically evaluated three
different neuropeptide extraction protocols and developed a simple and effective hemolymph preparation method suitable for
MALDI MS profiling of neuropeptides by combining acid-induced abundant protein precipitation/depletion, ultrafiltration, and
C18 micro-column desalting. In hemolymph samples collected from the crab Cancer borealis, several secreted neuropeptides have been detected, including members from at least five neuropeptide families, such as RFamide,
allatostatin, orcokinin, tachykinin-related peptide (TRP), and crustacean cardioactive peptide (CCAP). Furthermore, two TRPs
were detected in the hemolymph collected from food-deprived animals, suggesting the potential role of these neuropeptides
in feeding regulation. In addition, a novel peptide with a Lys-Phe-amide C-terminus was identified and de novo sequenced directly
from the Cancer borealis hemolymph sample. To better characterize the hemolymph peptidome, we also identified several abundant peptide signals in
C. borealis hemolymph that were assigned to protein degradation products. Collectively, our study describes a simple and effective sample
preparation method for neuropeptide analysis directly from crude crustacean hemolymph. Numerous endogenous neuropeptides were
detected, including both known ones and new peptides whose functions remain to be characterized. 相似文献
8.
Dr. Silja Mordhorst Assist. Prof. Dr. Brandon I. Morinaka Dr. Anna L. Vagstad Prof. Dr. Jörn Piel 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2020,132(48):21626-21631
Ornithine is a component of many bioactive nonribosomal peptides but is challenging to incorporate into ribosomal products. We recently identified OspR, a cyanobacterial arginase-like enzyme that installs ornithines in the antiviral ribosomally synthesised and posttranslationally modified peptide (RiPP) landornamide A. Here we report that OspR belongs to a larger family of peptide arginases from diverse organisms and RiPP types. In E. coli, seven selected enzymes converted arginine into ornithine with little preference for the leader type. A broad range of peptide sequences was modified, including polyarginine repeats. We also generated analogues of ornithine-containing nonribosomal peptides using RiPP technology. Five pseudo-nonribosomal products with ornithines at the correct positions were obtained, including a brevicidine analogue containing ornithine and a d -amino acid installed by the peptide epimerase OspD. These results suggest new opportunities for peptide bioengineering. 相似文献
9.
Manfred Raida Peter Schulz-Knappe Gabriele Heine Wolf-Georg Forssmann 《Journal of the American Society for Mass Spectrometry》1999,10(1):45-54
We present a multidimensional approach to map the composition of complex peptide mixtures obtained as crude extract from biological liquids by (1) cation exchange chromatography and (2) subsequent microbore reversed-phase liquid chromatography and electrospray mass spectrometry coupling (LC-MS). Human hemofiltrate is an equivalent to blood and is used to obtain peptide material in large quantities from patients with chronic renal failure. The upper exclusion limit of the filtration membranes used results in a protein-free filtrate containing peptides in a range up to 20 ku. Using this unique peptide source, several thousand peptides were detected and an LC-MS data base of circulating human peptides was created. The search for known peptides by their molecular mass is a reliable method to guide peptide purification. 相似文献
10.
Amadeu H. Iglesias Luiz F. A. Santos Fabio C. Gozzo 《Journal of the American Society for Mass Spectrometry》2009,20(4):557-566
The use of chemical crosslinking is an attractive tool that presents many advantages in the application of mass spectrometry
to structural biology. The correct assignment of crosslinked peptides, however, is still a challenge because of the lack of
detailed fragmentation studies on resultant species. In this work, the fragmentation patterns of intramolecular crosslinked
peptides with disuccinimidyl suberate (DSS) has been devised by using a set of versatile, model peptides that resemble species
found in crosslinking experiments with proteins. These peptides contain an acetylated N-terminus followed by a random sequence
of residues containing two lysine residues separated by an arginine. After the crosslinking reaction, controlled trypsin digestion
yields both intra- and intermolecular crosslinked peptides. In the present study we analyzed the fragmentation of matrix-assisted
laser desorption/ionization-generated peptides crosslinked with DSS in which both lysines are found in the same peptide. Fragmentation
starts in the linear moiety of the peptide, yielding regular b and y ions. Once it reaches the cyclic portion of the molecule, fragmentation was observed to occur either at the following peptide
bond or at the peptide crosslinker amide bond. If the peptide crosslinker bond is cleaved, it fragments as a regular modified
peptide, in which the DSS backbone remains attached to the first lysine. This fragmentation pattern resembles the fragmentation
of modified peptides and may be identified by common automated search engines using DSS as a modification. If, on the other
hand, fragmentation happens at the peptide bond itself, rearrangement of the last crosslinked lysine is observed and a product
ion containing the crosslinker backbone and lysine (m/z 222) is formed. The detailed identification of fragment ions can help the development of softwares devoted to the MS/MS data
analysis of crosslinked peptides. 相似文献
11.
Adding energy minimization strategy to peptide‐design algorithm enables better search for RNA‐binding peptides: Redesigned λ N peptide binds boxB RNA
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Xingqing Xiao Michelle E. Hung Joshua N. Leonard Carol K. Hall 《Journal of computational chemistry》2016,37(27):2423-2435
Our previously developed peptide‐design algorithm was improved by adding an energy minimization strategy which allows the amino acid sidechains to move in a broad configuration space during sequence evolution. In this work, the new algorithm was used to generate a library of 21‐mer peptides which could substitute for λ N peptide in binding to boxB RNA. Six potential peptides were obtained from the algorithm, all of which exhibited good binding capability with boxB RNA. Atomistic molecular dynamics simulations were then conducted to examine the ability of the λ N peptide and three best evolved peptides, viz. Pept01, Pept26, and Pept28, to bind to boxB RNA. Simulation results demonstrated that our evolved peptides are better at binding to boxB RNA than the λ N peptide. Sequence searches using the old (without energy minimization strategy) and new (with energy minimization strategy) algorithms confirm that the new algorithm is more effective at finding good RNA‐binding peptides than the old algorithm. © 2016 Wiley Periodicals, Inc. 相似文献
12.
Hongmei Yang Ning Liu Xiaoyan Qiu Shuying Liu 《Journal of the American Society for Mass Spectrometry》2009,20(12):2284-2293
A simple and high-throughput method for the identification of disulfide-containing peptides utilizing peptide-matrix adducts
is described. Some commonly used matrices in MALDI mass spectrometry were found to specifically react with sulfhydryl groups
within peptide, thus allowing the observation of the peptide-matrix adduct ion [M+n+n′ matrix+H]+ or [M+n+n′ matrix+Na]+ (n = the number of cysteine residues, n′=1, 2,…, n) in MALDI mass spectra after chemical reduction of disulfide-linked peptides. Among several matrices tested, α-cyano-4-hydroxycinnamic
acid (CHCA, molecular mass 189 Da) and α-cyano-3-hydroxycinnamic acid (3-HCCA) were found to be more effective for MALDI analysis
of disulfide-containing peptides/proteins. Two reduced cysteines involved in a disulfide bridge resulted in a mass shift of
189 Da per cysteine, so the number of disulfide bonds could then be determined, while for the other matrices (sinapinic acid,
ferulic acid, and caffeic acid), a similar addition reaction could not occur unless the reaction was carried out under alkaline
conditions. The underlying mechanism of the reaction of the matrix addition at sulfhydryl groups is proposed, and several
factors that might affect the formation of the peptide-matrix adducts were investigated. In general, this method is fast,
effective, and robust to identify disulfide bonds in proteins/peptides. 相似文献
13.
Neuropeptides are heavily posttranslationally modified (PTM) gene products that are often characterized by a variety of mass spectrometric approaches. Recently, the occurrence of amino acids in the D-form has been documented in several neuropeptides. As this modification has no associated mass shift, this particular PTM is difficult to evaluate using mass spectrometry (MS) alone. Here we demonstrate several approaches using capillary electrophoresis (CE) with absorbance and laser-induced fluorescence (LIF) for the separation of native and derivatized molluscan peptides containing D-amino acids. The combination of peptide derivatization followed by CE/LIF is well suited for single cell measurements because of its ability to characterize the peptides in such small samples. In order to verify this approach, the D-Trp-containing peptide NdWFa (NH2-Asn-D-Trp-Phe-CONH2), present in individual neurons from the marine mollusk Aplysia californica, has been characterized. The mass spectra show that NdWFa and/or NWFa are present in specific neurons; CE/LIF analysis of these cells demonstrates that NdWFa is the dominant form of the peptide. 相似文献
14.
Rima Erviana Yutthakan Saengkun Prapenpuksiri Rungsa Nisachon Jangpromma Patcharaporn Tippayawat Sompong Klaynongsruang Jureerut Daduang Sakda Daduang 《Molecules (Basel, Switzerland)》2021,26(19)
The increasing antimicrobial-resistant prevalence has become a severe health problem. It has led to the invention of a new antimicrobial agent such as antimicrobial peptides. Heteroscorpine-1 is an antimicrobial peptide that has the ability to kill many bacterial strains. It consists of 76 amino acid residues with a cecropin-like region in N-terminal and a defensin-like region in the C-terminal. The cecropin-like region from heteroscorpine-1 (CeHS-1) is similar to cecropin B, but it lost its glycine-proline hinge region. The bioinformatics prediction was used to help the designing of mutant peptides. The addition of glycine-proline hinge and positively charged amino acids, the deletion of negatively charged amino acids, and the optimization of the hydrophobicity of the peptide resulted in two mutant peptides, namely, CeHS-1 GP and CeHS-1 GPK. The new mutant peptide showed higher antimicrobial activity than the native peptide without increasing toxicity. The interaction of the peptides with the membrane showed that the peptides were capable of disrupting both the inner and outer bacterial cell membrane. Furthermore, the SEM analysis showed that the peptides created the pore in the bacterial cell membrane resulted in cell membrane disruption. In conclusion, the mutants of CeHS-1 had the potential to develop as novel antimicrobial peptides. 相似文献
15.
Mendes MA Monson de Souza B Delazari dos Santos L Palma MS 《Rapid communications in mass spectrometry : RCM》2004,18(6):636-642
High-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) techniques were applied for the detection, purification, monitoring, and sequencing of two novel and biologically active peptides occurring at very low levels in the venom of the wasp Agelaia pallipes pallipes. These peptides were sequenced under LC/ESI-MS/MS conditions and designated as Agelaia-CP (I/L-L-G-T-I-L-G-L-L-K-G-I/L-NH2, MW 1207.8 Da) and Agelaia-MP (I/L-N-W-L-K-L-G-K-A-I-I-D-A-I/L-NH2, MW 1565.0 Da). The peptide Agelaia-CP showed no hemolytic activity, but it behaved as a mast cell degranulator and induced a potent chemotaxis in polymorphonucleated leukocyte (PMNL) cells, typical of a wasp chemotactic peptide. The peptide Agelaia-MP showed both powerful mast cell degranulation and hemolysis of washed rat red blood cells, and is thus assigned as a new member of the mastoparan family of peptides. Both peptides seem to be directly involved in the strong inflammatory reactions associated with wasp stings. 相似文献
16.
Characterization of the dehydration products due to thermal decomposition of peptides by liquid chromatography‐tandem mass spectrometry
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Chenglin Liu Elena Topchiy Teresa Lehmann Franco Basile 《Journal of mass spectrometry : JMS》2015,50(3):625-632
Thermal decomposition (TD) of proteins is being investigated as a rapid digestion step for bottom‐up proteomics. Mass spectrometry (MS) analyses of the TD products of simple peptides and intact proteins have revealed several nonvolatile products at masses lower than the precursor biomolecule (M). In addition to products stemming from site‐specific cleavages, many signals are also observed at a corresponding M‐18, most likely because of dehydration (M‐H2O) during the heating process. Understanding the structural nature of the water loss product is important in establishing the utility of their tandem mass spectra (collision‐induced dissociation) in determining the precursor ion amino acid sequence in a bottom‐up proteomic workflow. Dehydration of a peptide can take place from a variety of sources including side chain groups, C‐terminus, and/or intramolecular cyclization (C to N‐terminus cyclization). In this work, liquid chromatography‐tandem MS (LC‐MS/MS) and a series of standard peptides (angiotensin II, DRVYIHPF and its cyclic analog) are implemented to decipher the structure of the TD dehydration product. In addition, a derivatization strategy incorporating N‐terminus acetylation was developed that allowed the direct comparison of tandem mass spectra of standard cyclic peptides with those resulting from the TD process, thus eliminating any ambiguity from the direct comparison of their mass spectra (due to gas‐phase cyclization of b‐ions, which can result in sequence scrambling of the precursor ion). Results from these investigations indicated that peptide dehydrated TD products were mostly linear in nature, and water loss was favored from the C‐terminus carboxyl group or, when present, the aspartic acid side chain. Given the predictable nature of the formation of TD dehydration products, their MS/MS analysis can be of utility in providing complementary and confirmatory sequence information of the precursor peptide. Copyright © 2015 John Wiley & Sons, Ltd. 相似文献
17.
Saikat Pahan Dr. Sanjit Dey Dr. Gijo George Souvik Panda Mahapatra Dr. DRGKoppalu R. Puneeth Kumar Prof. Hosahudya. N. Gopi 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2024,136(2):e202316309
Chirality is ubiquitous in nature, and homochirality is manifested in many biomolecules. Although β-double helices are rare in peptides and proteins, they consist of alternating L- and D-amino acids. No peptide double helices with homochiral amino acids have been observed. Here, we report chiral β-double helices constructed from γ-peptides consisting of alternating achiral (E)-α,β-unsaturated 4,4-dimethyl γ-amino acids and chiral (E)-α,β-unsaturated γ-amino acids in both single crystals and in solution. The two independent strands of the same peptide intertwine to form a β-double helix structure, and it is stabilized by inter-strand hydrogen bonds. The peptides with chiral (E)-α,β-unsaturated γ-amino acids derived from α-L-amino acids adopt a (P)-β-double helix, whereas peptides consisting of (E)-α,β-unsaturated γ-amino acids derived from α-D-amino acids adopt an (M)-β-double helix conformation. The circular dichroism (CD) signature of the (P) and (M)-β-double helices and the stability of these peptides at higher temperatures were examined. Furthermore, ion transport studies suggested that these peptides transport ions across membranes. Even though the structural analogy suggests that these new β-double helices are structurally different from those of the α-peptide β-double helices, they retain ion transport activity. The results reported here may open new avenues in the design of functional foldamers. 相似文献
18.
T. Yu. Samgina K. A. Artemenko V. A. Gorshkov S. V. Ogourtsov R. A. Zubarev A. T. Lebedev 《Rapid communications in mass spectrometry : RCM》2009,23(9):1241-1248
A high‐performance liquid chromatography nano‐electrospray ionization Fourier transform mass spectrometry (HPLC/nanoESI‐FTMS) approach involving recording of collision‐activated dissociation (CAD) and electron‐capture dissociation (ECD) spectra of an intact sample and two its modifications after performic oxidation and reduction followed by carboxamidomethylation helps to establish peptide profiles in the crude secretion of frog species at mid‐throughput level, including de novo sequencing. The proposed derivatization procedures allow increasing of the general sequence coverage in the backbone, providing complementary information and, what is more important, reveal the amino acid sequence in the cystine ring (‘rana box’). Thus purely mass spectrometric efficient sequencing becomes possible for longer than usual proteolytic peptides. Seventeen peptides belonging to four known families were identified in the secretion of the European brown frog Rana arvalis inhabiting the Moscow region in Russia. Ranatuerins, considered previously a unique feature of the North American species, as well as a new melittin‐related peptide, are worth special mention. The developed approach was previously successfully used for the identification of peptides in the skin secretion of the Caucasian green frog Rana ridibunda. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
19.
Rumachik NG McAlister GC Russell JD Bailey DJ Wenger CD Coon JJ 《Journal of the American Society for Mass Spectrometry》2012,23(4):718-727
We implemented negative electron-transfer dissociation (NETD) on a hybrid ion trap/Orbitrap mass spectrometer to conduct ion/ion
reactions using peptide anions and radical reagent cations. In addition to sequence-informative ladders of a•- and x-type fragment ions, NETD generated intense neutral loss peaks corresponding to the entire or partial side-chain cleavage
from amino acids constituting a given peptide. Thus, a critical step towards the characterization of this recently introduced
fragmentation technique is a systematic study of synthetic peptides to identify common neutral losses and preferential fragmentation
pathways. Examining 46 synthetic peptides with high mass accuracy and high resolution analysis permitted facile determination
of the chemical composition of each neutral loss. We identified 19 unique neutral losses from 14 amino acids and three modified
amino acids, and assessed the specificity and sensitivity of each neutral loss using a database of 1542 confidently identified
peptides generated from NETD shotgun experiments employing high-pH separations and negative electrospray ionization. As residue-specific
neutral losses indicate the presence of certain amino acids, we determined that many neutral losses have potential diagnostic
utility. We envision this catalogue of neutral losses being incorporated into database search algorithms to improve peptide
identification specificity and to further advance characterization of the acidic proteome. 相似文献
20.
Danielle Vellucci Athit Kao Robyn M. Kaake Scott D. Rychnovsky Lan Huang 《Journal of the American Society for Mass Spectrometry》2010,21(8):1432-1445
Protein-protein interaction is one of the key regulatory mechanisms for controlling protein function in various cellular processes.
Chemical cross-linking coupled with mass spectrometry has proven to be a powerful method not only for mapping protein-protein
interactions of all natures, including weak and transient ones, but also for determining their interaction interfaces. One
critical challenge remaining in this approach is how to effectively isolate and identify cross-linked products from a complex
peptide mixture. In this work, we have developed a novel strategy using conjugation chemistry for selective enrichment of
cross-linked products. An azide-tagged cross-linker along with two biotinylated conjugation reagents were designed and synthesized.
Cross-linking of model peptides and cytochrome c as well as enrichment of the resulting cross-linked peptides has been assessed. Selective conjugation of azide-tagged cross-linked
peptides has been demonstrated using two strategies: copper catalyzed cycloaddition and Staudinger ligation. While both methods
are effective, Staudinger ligation is better suited for enriching the cross-linked peptides since there are fewer issues with
sample handling. LC MSn analysis coupled with database searching using the Protein Prospector software package allowed identification of 58 cytochrome
c cross-linked peptides after enrichment and affinity purification. The new enrichment strategy developed in this work provides
useful tools for facilitating identification of cross-linked peptides in a peptide mixture by MS, thus presenting a step forward
in future studies of protein-protein interactions of protein complexes by cross-linking and mass spectrometry. 相似文献