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Alexander Leitner 《Analytica chimica acta》2005,528(2):165-173
The presence of arginine as the naturally occurring amino acid with the highest gas-phase basicity strongly influences the fragmentation behavior of peptides undergoing collision-induced dissociation. Using a derivatization procedure recently developed in our group, based on a reversible reaction of the guanidino group with 2,3-butanedione and an arylboronic acid, we examined how this label affects the fragmentation patterns of labeled versus unlabeled peptides in MS/MS experiments. As part of this fundamental study, two groups of model peptides (angiotensins and bradykinins) as well as tryptic peptides were labeled according to our protocol and subjected to collision-induced dissociation (CID) in both a triple quadrupole and a quadrupole ion trap instrument. It was found that for angiotensins containing an AspArg sequence, C-terminal cleavage at Asp that occurs for native peptides was completely inhibited in Arg-labeled peptides. For bradykinins and peptides obtained from tryptic digests of standard proteins, some sample peptides were little affected by the tagging of arginine residues. Others, in contrast, exhibited an almost total loss of nonspecific backbone cleavage and their fragment ion spectra were dominated by loss of the arginine tag. These and other experimental results are discussed in view of the nature of the arginine tag and the concept of proton mobility. 相似文献
3.
De novo sequencing of peptides using MALDI/TOF-TOF 总被引:6,自引:0,他引:6
Yergey AL Coorssen JR Backlund PS Blank PS Humphrey GA Zimmerberg J Campbell JM Vestal ML 《Journal of the American Society for Mass Spectrometry》2002,13(7):784-791
The recently developed MALDI TOF-TOF instrument yields relatively complex but interpretable fragmentation spectra. When coupled with a straightforward sequence extension algorithm, it is possible to develop complete peptide sequences de novo from the spectra. This approach has been applied to a set of peptides derived from typtic digestion of electrophoretically separated sea urchin egg membrane proteins. When directed to proteins that have been described previously, the results were in essential agreement with those obtained by conventional data base searching approaches, with certain important exceptions. The present method detected errors in published sequences and was able to develop sequences from peptides differing in mass by one dalton (Da). These results show both the power of the present approach and the need for using de novo methods more frequently than may be otherwise appreciated. 相似文献
4.
Site-specific characterisation of mucin-type O-linked glycosylation is an analytical challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high density of occupied glycosylation sites. Here, we report the use of electron transfer dissociation (ETD) for the site-specific characterisation of densely glycosylated mucin-type O-linked glycopeptides using ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem repeat region containing a range of O-linked, tumour-associated carbohydrate antigens, namely Tn, T and sialyl T, with different glycosylation site occupancies and an increasing number of tandem repeats were studied. In addition, a glycopeptide from the anti-freeze glycoprotein of Antarctic and Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was characterised. ETD MS/MS of infused or capillary LC-separated glycopeptides provided broad peptide sequence coverage (c/z·-type fragment ions) with intact glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites were unambiguously determined, while simultaneously obtaining information about the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides showed less efficient peptide fragmentation, but some sequence and glycosylation site information was still obtained. This study demonstrates the capabilities of ETD MS/MS for site-specific characterisation of mucin-type glycopeptides containing high-density O-linked glycan clusters, using accessible and relative low-resolution/low-mass accuracy IT MS instrumentation. 相似文献
5.
Unnithan AG Myer MJ Veale CJ Danell AS 《Journal of the American Society for Mass Spectrometry》2007,18(12):2198-2203
A unique collision-induced dissociation pattern was observed for protonated polyproline peptides of length n in which y(n-2) and/or y(n-4) ions were formed in much higher abundance than any other product ions. Cleavage occurs only at every other amide bond, such that product ions are formed only from the losses of even numbers of proline residues. Exclusive losses of even numbers of proline residues were not observed from sodiated peptides. Further study of the tandem mass spectrometry (MS/MS) patterns of protonated proline-rich peptides showed that the substitution of alanine in the second position of polyproline peptides did not prevent the dominant formation of y(n-2) and y(n-4) ions. The loss of ProAla to form the y(8) ion from (ProAlaPro(8)NH(2)+H)(+) was as abundant as the loss of ProPro from (Pro(10)NH(2)+H)(+). However, modification of the peptides that presumably affected the location of the proton on the peptide did alter the MS/MS spectra. Pro(10) and Pro(5) with blocked N-termini or with arginine substituted for the first proline residue did not form abundant y(n-2) or y(n-4) ions. MS(3) and double resonance experiments showed that dissociation of intermediate y(n) product ions can produce y(n-2) ions, but are not necessary dissociation pathway intermediates. This analysis suggests that the ionizing proton must be located at the N-terminus for the peptide ion to dissociate in this manner. 相似文献
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Improved procedures for N-terminal sulfonation of peptides for matrix-assisted laser desorption/ionization post-source decay peptide sequencing 总被引:3,自引:0,他引:3
Post source decay (PSD) analysis of precursor ions generated from matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a powerful tool for amino acid sequencing and primary structure analysis of proteins. N-Terminal sulfonation has become an effective derivatization strategy in facilitating de novo peptide sequencing by the formation of predominate y-type ion series in MALDI PSD spectra. Recently, an effective and inexpensive N-terminal derivatization method has been reported using 4-sulfophenyl isothiocyanate (SPITC) as the derivatization reagent (J. Mass. Spectrom. 2003; 38: 373-377). In this paper, we report an improvement in the derivatization procedure with this reagent that involves replacing an organic co-reagent with other chemicals and eliminating the use of organic solvent. The method is demonstrated on a model peptide and on tryptic digests of two proteins. The results indicate that the improved sulfonation reaction can be implemented with high efficiency under aqueous conditions and that the sensitivity of mass detection can be increased considerably. 相似文献
9.
Hilpert K Elliott MR Volkmer-Engert R Henklein P Donini O Zhou Q Winkler DF Hancock RE 《Chemistry & biology》2006,13(10):1101-1107
Short antimicrobial host-defense peptides represent a possible alternative as lead structures to fight antibiotic resistant bacterial infections. Bac2A is a 12-mer linear variant of the naturally occurring bovine host defense peptide, bactenecin, and demonstrates moderate, broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria as well as against the yeast Candida albicans. With the assistance of a method involving peptide synthesis on a cellulose support, the primary sequence requirements for antimicrobial activity against the human pathogen Pseudomonas aeruginosa of 277 Bac2A variants were investigated by using a luciferase-based assay. Sequence scrambling of Bac2A led to activities ranging from superior or equivalent to Bac2A to inactive, indicating that good activity was not solely dependent on the composition of amino acids or the overall charge or hydrophobicity, but rather required particular linear sequence patterns. A QSAR computational analysis was applied to analyze the data resulting in a model that supported this sequence pattern hypothesis. The activity of selected peptides was confirmed by conventional minimal inhibitory concentration (MIC) analyses with a panel of human pathogen bacteria and fungi. Circular-dichroism (CD) spectroscopy with selected peptides in liposomes and membrane depolarization assays were consistent with a relationship between structure and activity. An additional optimization process was performed involving systematic amino acid substitutions of one of the optimal scrambled peptide variants, resulting in superior active peptide variants. This process provides a cost and time effective enrichment of new candidates for drug development, increasing the chances of finding pharmacologically relevant peptides. 相似文献
10.
Andreas Bertsch Andreas Leinenbach Anton Pervukhin Markus Lubeck Ralf Hartmer Carsten Baessmann Yasser Abbas Elnakady Rolf Müller Sebastian Böcker Christian G. Huber Oliver Kohlbacher 《Electrophoresis》2009,30(21):3736-3747
De novo sequencing of peptides using tandem MS is difficult due to missing fragment ions in the spectra commonly obtained after CID of peptide precursor ions. Complementing CID spectra with spectra obtained in an ion‐trap mass spectrometer upon electron transfer dissociation (ETD) significantly increases the sequence coverage with diagnostic ions. In the de novo sequencing algorithm CompNovo presented here, a divide‐and‐conquer approach was combined with an efficient mass decomposition algorithm to exploit the complementary information contained in CID and ETD spectra. After optimizing the parameters for the algorithm on a well‐defined training data set obtained for peptides from nine known proteins, the CompNovo algorithm was applied to the de novo sequencing of peptides derived from a whole protein extract of Sorangium cellulosum bacteria. To 2406 pairs of CID and ETD spectra contained in this data set, 675 fully correct sequences were assigned, which represent a success rate of 28.1%. It is shown that the CompNovo algorithm yields significantly improved sequencing accuracy as compared with published approaches using only CID spectra or combined CID and ETD spectra. 相似文献
11.
LiJunLi ZeperABLIZ MitsuoTAKAYAMA YunXIANG 《中国化学快报》2003,14(5):499-502
Positive ion ESI-MS has been used to examine the fragmentation pathways of the complex ions of deoxydinucleotides with H^ ,Na^ ,K^ by LCQ instrument.It had been found that the dissociation Varied markedly due to the differences of the base sequence.The alkali-metal ion binding site and the charcaterization of dissociation were directed by the size of metal ion,the sequesce of base and the steric hindrance. 相似文献
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Lindh I Hjelmqvist L Bergman T Sjövall J Griffiths WJ 《Journal of the American Society for Mass Spectrometry》2000,11(8):673-686
A series of synthetic peptides (3-15 residues), C-terminally derivatized with 4-aminonaphthalenesulfonic acid (ansa), have been analyzed on a hybrid magnetic sector-orthogonal acceleration time-of-flight tandem mass spectrometer, fitted with a nano-electrospray (nano-ES) interface. Deprotonated molecules generated by negative-ion ES were subjected to collision-induced dissociation (CID) using either methane or xenon as the collision gas, at a collision energy of 400 eV (laboratory frame of reference). As a consequence of charge localization on the sulfonate group, only C-terminal fragment ions were formed, presumably by charge-remote fragmentation mechanisms. Interpretable CID spectra were obtained from fmol amounts of the small peptides (up to 6 residues), whereas low pmol amounts were required for the larger peptides. CID spectra were also recorded of derivatized, previously noncharacterised peptides obtained by proteolysis of cytosolic hamster liver aldehyde dehydrogenase. Interpretation of these CID spectra was based on rules established for the fragmentation of the synthetic peptides. This study shows that derivatization with ansa may be useful in the de novo sequencing of peptides. 相似文献
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Samgina TY Artemenko KA Gorshkov VA Poljakov NB Lebedev AT 《Journal of the American Society for Mass Spectrometry》2008,19(4):479-487
Five natural peptides isolated from ranid skin secretions of European frog species of Rana ridibunda and Rana arvalis (molecular masses 3516, 2674, 2636, 1874, and 1810 Da) were studied by MALDI-TOF/TOF to compare two procedures of disulfide bond cleavage: (1) performic oxidation and (2) reduction/carboxamidomethylation. The processes are relevant for the elucidation of the amino acid sequence inside the seven-member cystine ring at the C-terminus. The results clearly demonstrated that oxidation of the disulfide bond led to notably higher abundances of b- and y-ions, corresponding to the C-terminal peptide bonds, than reduction/carboxamidomethylation. This conclusion is true for all five peptides studied. Besides that, the oxidation procedure is simpler than carboxamidomethylation, as it is a one-step process with no purification required. The oxidation is more reproducible. The results were similar each time the peptide was subjected to the process. It was successfully applied to all five peptides while reduction/carboxamidomethylation failed in the case of brevinin-1Ra, despite all variations of reaction conditions. 相似文献
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Jarrett D. Egertson Jimmy K. Eng Michael S. Bereman Edward J. Hsieh Gennifer E. Merrihew Michael J. MacCoss 《Journal of the American Society for Mass Spectrometry》2012,23(12):2075-2082
We report an algorithm designed for the calibration of low resolution peptide mass spectra. Our algorithm is implemented in a program called FineTune, which corrects systematic mass measurement error in 1?min, with no input required besides the mass spectra themselves. The mass measurement accuracy for a set of spectra collected on an LTQ-Velos improved 20-fold from ?C0.1776?±?0.0010?m/z to 0.0078?±?0.0006?m/z after calibration (avg?±?95?% confidence interval). The precision in mass measurement was improved due to the correction of non-linear variation in mass measurement accuracy across the m/z range. 相似文献
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《Journal of mass spectrometry : JMS》2017,52(2):78-93
Correct sequences are prerequisite for quality control of therapeutic oligonucleotides. However, there is no definitive method available for determining sequences of highly modified therapeutic RNAs, and thereby, most of the oligonucleotides have been used clinically without direct sequence determination. In this study, we developed a novel sequencing method called ‘hydrophobic tag sequencing’. Highly modified oligonucleotides are sequenced by partially digesting oligonucleotides conjugated with a 5′‐hydrophobic tag, followed by liquid chromatography–mass spectrometry analysis. 5′‐Hydrophobic tag‐printed fragments (5′‐tag degradates) can be separated in order of their molecular masses from tag‐free oligonucleotides by reversed‐phase liquid chromatography. As models for the sequencing, the anti‐VEGF aptamer (Macugen) and the highly modified 38‐mer RNA sequences were analyzed under blind conditions. Most nucleotides were identified from the molecular weight of hydrophobic 5′‐tag degradates calculated from monoisotopic mass in simple full mass data. When monoisotopic mass could not be assigned, the nucleotide was estimated using the molecular weight of the most abundant mass. The sequences of Macugen and 38‐mer RNA perfectly matched the theoretical sequences. The hydrophobic tag sequencing worked well to obtain simple full mass data, resulting in accurate and clear sequencing. The present study provides for the first time a de novo sequencing technology for highly modified RNAs and contributes to quality control of therapeutic oligonucleotides. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
16.
Angelika Semmler Reinhold Weber Michael Przybylski Valentin Wittmann 《Journal of the American Society for Mass Spectrometry》2010,21(2):215-219
An efficient approach in combinatorial chemistry is the synthesis of one-bead-one-compound peptide libraries. In contrast
to synthesis and functional screening, which is performed in a largely automated manner, structure determination has been
frequently laborious and time-consuming. Here we report an approach for de novo sequencing of peptides on single beads by
matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance (MALDI-FTICR) tandem mass spectrometry,
using a resin with a photolinker for solid-phase peptide synthesis. Upon sorting out single beads, an efficient sample preparation
on the MALDI target was developed that enables fragmentation upon irradiation of the bead-matrix mixture with the ultraviolet
(UV)-MALDI laser, with enhanced yield of sequence-specific fragment ions at increased laser energy. This approach is illustrated
by sequence determinations of two peptides from a library with sequences varying in a single amino acid; the feasibility with
tandem-MS procedures and fragment ion assignment was ascertained by sustained off-resonance irradiation/collision induced
dissociation (SORI/CID) and infrared multiphoton dissociation (IRMPD) fragmentation. 相似文献
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Christian HaaseMatthew F. Burton Stijn M. AgtenLuc Brunsveld 《Tetrahedron letters》2012,53(35):4763-4765
Unwanted trifluoroacetylation occurred at the N-terminus of prolinyl peptides during detachment from the solid phase. This was observed when the N-α-Fmoc protecting group had been removed prior to the final TFA treatment. Subtly changing the SPPS protocol and incorporating Boc- in place of the Fmoc-protected proline as the N-terminal building block efficiently suppressed this side reaction. 相似文献
18.
Hubert A. Scoble James E. Biller Klaus Biemann 《Fresenius' Journal of Analytical Chemistry》1987,327(2):239-245
Summary A graphics display-oriented method for the computer-aided interpretation of the mass spectrum of a peptide in terms of its amino acid sequence is presented. The spectrum is obtained by collision induced decomposition of the protonated molecular ion (M+H)+ of the peptide generated by fast atom bombardment in the first double focussing mass analyzer of a tandem mass spectrometer and the product ion mass spectrum (CID spectrum) recorded by the second double focussing mass analyzer. The algorithm displays all series of peaks differing by the mass of -NH-CHR-CO- for the various amino acids. This display, which is very fast and can be augmented through interrogation by the user, greatly facilitates the determination of the amino acid sequence. The method is demonstrated on the spectra of a undecapeptide of (M+H)+ m/z = 1208.2 and a tetradecapeptide of (M+H)+ m/z = 1758.9.
Bestimmung der Aminosäure-Sequenz von Peptiden durch Tandem-Massenspektrometrie mit Hilfe eines graphischen Display相似文献
19.
Applications of LC/ESI-MS/MS and UHPLC QqTOF MS for the determination of 148 pesticides in fruits and vegetables 总被引:1,自引:0,他引:1
This paper presented the applications of liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS)
and ultra-high-pressure liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC QqTOF
MS) for the determination of 148 pesticides in fruits and vegetables. Pesticides were extracted from fruits and vegetables
using a buffered QuEChERS method. Quantification was achieved using matrix-matched standard calibration curves with isotopically
labeled standards or a chemical analog as internal standards in an analytical range from 5 to 500 μg/kg. The method performance
parameters including overall recovery, intermediate precision, and measurement uncertainty were evaluated according to a statistically
designed experiment, i.e., a nested design. For LC/ESI-MS/MS, 95% of the pesticides had recoveries between 81% and 110%; 97%
had an intermediate precision ≤20%; and 95% (in fruits) or 93% (in vegetables) showed measurement uncertainty ≤40%. Compared
to LC/ESI-MS/MS, UHPLC QqTOF MS showed a relatively poor repeatability and large measurement uncertainty. About 93% (in fruits)
or 94% (in vegetables) of the pesticides had recoveries between 81% and 110%; 86% (in fruits) or 90% (in vegetables) had an
intermediate precision ≤20%; and 79% (in fruits) or 88% (in vegetables) showed measurement uncertainty ≤40%. LC/ESI-MS/MS
proved to be the first choice for quantification or pre-target analysis due to its superior sensitivity and good repeatability.
UHPLC QqTOF MS provided accurate mass measurement and isotopic patterns, and was an ideal tool for post-target screening and
confirmation. 相似文献
20.
Pfeifer T Rücknagel P Kuellertz G Schierhorn A 《Rapid communications in mass spectrometry : RCM》1999,13(5):362-369
A modification procedure for Lys-C peptides is described which simplifies the correct assignment of the amino acid sequence. Release of the C-terminal lysine from Lys-C peptides by carboxypeptidase B and subsequent N-terminal acetylation of the resulting peptides leads to predictable shifts of the C- and N-terminal fragment ions in Matrix-assisted laser desorption/ionisation time-of-flight post-source decay mass spectra and facilitates the correct assignment of mostly complete amino acid sequences for oligopeptides. The derived sequences of peptides from unknown proteins were used to search in databases for homologous protein sequences. Our method was applied to an unknown protein isolated from eggs of Drosophila melanogaster, resulting in the identification of a peptidyl prolyl cis-trans-isomerase. 相似文献