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1.
Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity. 相似文献
2.
Wallis TP Huang CY Nimkar SB Young PR Gorman JJ 《The Journal of biological chemistry》2004,279(20):20729-20741
The 12 half-cystines of NS1 proteins are absolutely conserved among flaviviruses, suggesting their importance to the structure and function of these proteins. In the present study, peptides from recombinant Dengue-2 virus NS1 were produced by tryptic digestion in 100% H(2)(16)O, peptic digestion in 50% H(2)(18)O, thermolytic digestion in 50% H(2)(18)O, or combinations of these digestion conditions. Peptides were separated by size exclusion and/or reverse phase high performance liquid chromatography and examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry, matrix-assisted laser desorption ionization post-source decay, and matrix-assisted laser desorption ionization tandem mass spectrometry. Where digests were performed in 50% H(2)(18)O, isotope profiles of peptide ions aided in the identification and characterization of disulfide-linked peptides. It was possible to produce two-chain peptides containing C1/C2, C3/C4, C5/C6, and C7/C12 linkages as revealed by comparison of the peptide masses before and after reduction and by post-source decay analysis. However, the remaining four half-cystines (C8, C9, C10, and C11) were located in a three-chain peptide of which one chain contained adjacent half-cystines (C9 and C10). The linkages of C8/C10 and C9/C11 were determined by tandem mass spectrometry of an in-source decay fragment ion containing C9, C10, and C11. This disulfide bond arrangement provides the basis for further refinement of flavivirus NS1 protein structural models. 相似文献
3.
CLAVATA3 (CLV3) is a plant peptide hormone in which the proline residues are post-translationally hydroxylated and glycosylated. CLV3 plays a key role in controlling the stem cell mass in the shoot meristem of Arabidopsis thaliana. In a previous report, we identified a dodecapeptide (MCLV3) from CLV3-overexpressing Arabidopsis calli; MCLV3 was the smallest functional peptide derived from the CLV3 precursor. Here, we designed a series of MCLV3 analogs in which proline residues were substituted with proline derivatives or N-substituted glycines (peptoids). Peptoid substitution at Pro9 decreased bioactivity without affecting specific binding to the CLV1-related protein in cauliflower membrane. These findings suggest that peptoid-substituted peptides would be lead compounds for developing potential agonists and antagonists of CLV3. 相似文献
4.
Batista CV del Pozo L Zamudio FZ Contreras S Becerril B Wanke E Possani LD 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,803(1):55-66
Scorpion venom are complex mixtures of peptides, known to cause impairment of ion-channel function in biological membranes. This report describes the separation of approximately 60 different components by high performance liquid chromatography and the characterization by Edman degradation and mass spectrometry of 26 peptides from the soluble venom of the Amazonian scorpion Tityus cambridgei. One of these peptides, named Tc48a, was fully characterized. It contains 65 amino acid residues, the C-terminal residue is amidated and it affects Na(+)-channels with a K(d) of about 82 nM. Furthermore, this report shows the thermo-instability of scorpion toxins subjected to electron spray ionization-mass spectrometry (ESI-MS). When a proline residue is located near the N-terminal region of the toxin, not stabilized by disulfide bridges, artificial components are generated by the mass spectrometer conditions, due to the cleavage of the peptide bond at the proline positions. This phenomenon was confirmed by using four model proteins (variable regions of immunoglobulins) studied by ESI-MS and matrix assisted laser desorption ionization-time of flight (MALDI-TOF)/MS. 相似文献
5.
An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids. 总被引:1,自引:1,他引:0
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K. Hardeman B. Samyn J. Van der Eycken J. Van Beeumen 《Protein science : a publication of the Protein Society》1998,7(7):1593-1602
An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition. 相似文献
6.
Fernandez Ocaña M Jarvis J Parker R Bramley PM Halket JM Patel RK Neubert H 《Proteomics》2005,5(5):1209-1216
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. 相似文献
7.
《Biochimica et Biophysica Acta.Protein Structure》1981,667(2):233-241
Tryptic hydrolyzates of normal and abnormal human globin chains were analyzed with mass spectrometry, using field desorption ionization technique. All the peptides, including cote peptides, were detected as protonated molecular ions in field desorption mass spectra. Since the technique makes it possible to determine the mass number of each peptide, it is very useful for structural analysis of human hemoglobin variants, even those with electrophoretically and chromatographically silent mutations. 相似文献
8.
Schopfer LM Champion MM Tamblyn N Thompson CM Lockridge O 《Analytical biochemistry》2005,345(1):122-132
A mass spectrometry-based method was developed for selective detection of FP-biotinylated peptides in complex mixtures. Mixtures of peptides, at the low-picomole level, were analyzed by liquid chromatography and positive ion, nanospray, triple quadrupole, linear ion trap mass spectrometry. Peptides were fragmented by collision-activated dissociation in the mass spectrometer. The free FP-biotin and peptides containing FP-biotinylated serine or FP-biotinylated tyrosine yielded characteristic fragment ions at 227, 312, and 329 m/z. FP-biotinylated serine yielded an additional characteristic fragment ion at 591 m/z. Chromatographic peaks containing FP-biotinylated peptides were indicated by these diagnostic ions. Data illustrating the selectivity of the approach are presented for tryptic digests of FP-biotinylated trypsin and FP-biotinylated serum albumin. A 16-residue peptide from bovine trypsin was biotinylated on the active site serine. A 3-residue peptide from bovine albumin, YTR, was biotinylated on Tyr410. This latter result confirms that the organophosphorus binding site of albumin is a tyrosine. This method can be used to search for new biomarkers of organophosphorus agent exposure. 相似文献
9.
A simple procedure for preparing alkyl and benzyl esters of peptides is described. The procedure can provide an increase in the secondary ion yield of a factor of 25 or more in the liquid secondary ion mass spectra of hydrophilic peptides. The procedure allows rapid in situ derivatization of, e.g., collected, lyophilized HPLC fractions. No sample transfers are required and excess reagents are easily removed. Mass spectrometry of such fractions is typically required to prepare a mass map of the peptides produced by proteolytic digestion of a protein. However, small hydrophilic peptides are often not detected because their low secondary ion yield. Relative yields of MH+ ions from peptides esterified with various alcohols are compared: methanol, 2-propanol, 1-butanol, 1-hexanol, 1-octanol, and benzyl alcohol. The best combination of ion yield and ease of reagent removal is obtained with 1-hexanol. The degree of improvement depends on the specific peptide; the greatest improvement is generally observed with the most hydrophilic peptides. The procedure does not affect side-chain amides. Partial derivatization is sometimes observed with peptides containing more than one carboxyl group. Hexylation is shown to have a leveling effect on the mass spectra of peptide mixtures, allowing detection of surface-inactive peptides in the presence of surface-active ones. Benzyl alcohol is useful for derivatizing peptides that are not retained or that elute very early from reverse-phase HPLC columns. The derivatives have longer retention times and greater uv molar absorptivity and are more easily detected by subsequent mass spectrometry than the underivatized peptides. 相似文献
10.
Sensitivity and detection limit enhancements are obtained for peptides by performing high repetition rate (150 Hz) matrix-assisted laser desorption/ionization (MALDI) coupled with ion mobility-time-of-flight mass spectrometry. Absolute limits of detection (3sigma) for model peptides are on the order of 0.1 fmol of peptide deposited and represent a factor of 40-60 improvement over data obtained using typical low repetition (20 Hz) MALDI. This increase in sensitivity is demonstrated for two-dimensional MALDI-IM-TOFMS peptide mass mapping of bovine hemoglobin. 相似文献
11.
Hsp70-class molecular chaperones interact with diverse polypeptide substrates, but there is limited information on the structures of different Hsp70-peptide complexes. We have used a site-directed fluorescence labeling and quenching strategy to investigate the orientation of different peptides bound to DnaK from Escherichia coli. DnaK was selectively labeled on opposite sides of the substrate-binding domain (SBD) with the fluorescent probe bimane, and the ability of peptides containing N- or C-terminal tryptophan residues to quench bimane fluorescence was measured. Tryptophan-labeled derivatives of the model peptide NRLLLTG bound with the same forward orientation previously observed in the crystal structure of the DnaK(SBD)-NRLLLTG complex. Derivatives of this peptide containing arginine in the C-terminal rather than N-terminal region, NTLLLRG, also bound in the forward direction indicating that charged residues in the flanking regions of the peptide are not the major determinant of peptide binding orientation. We also tested peptides having proline in one (ELPLVKI) or two (ELPPVKI) central positions. Tryptophan derivatives of each of these peptides bound with a strong preference for the reverse direction relative to that observed for the NRLLLTG and NTLLLRG peptides. Computer modeling the peptides NRLLLTG and ELPPVKI in both the forward and reverse orientations into the DnaK(SBD) indicated that differential hydrogen-bonding patterns and steric constraints of the central peptide residues are likely causes for differences in their binding orientations. These findings establish that DnaK is able to bind substrates in both forward and reverse orientations and suggest that the central residues of the peptide are the major determinants of directional preference. 相似文献
12.
Collagen is one of the most important proteins containing mostly proline hydroxyproline and glycine. In collagen, approximately 33 percent of the amino acid residues are glycine and they occur at every third position, whereas remaining percentage is constituted by mainly proline or hydroxyproline and some part by alanine etc. having no definite positional placement in the chain. Thus, a study of conformation of proline and glycine containing dipeptides and tripeptides is important for understanding the conformation of collagen as a sequence of its constituent amino acids. In the present communication, we have studied spectral features of L-proline, L-prolyl-glycine (PG), L-prolyl-alanine (PA), L-glycylglycine (GG), Collagen and L-prolyl-glycyl-glycine (PGG). We have carried out detailed normal mode analysis of only PGG, because interpretation of spectra of other proline and glycine containing peptides can be treated as derivatives of this molecule. Urey-Bradley force field, which involves non-bonded interactions in the gem and cis configurations is used for calculation of normal modes. The "best-fit" set of constants are generated for PGG. 相似文献
13.
Alpha-cyano-4-hydroxycinnamic acid (α-CHCA) as a matrix facilitates the ionization of proteins and peptides in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer. The matrix itself also ionizes and so do its sodium and potassium adducts. Matrix clusters and metal ion adducts interfere with peptide ionization and peptide mass spectrum interpretation. These matrix adducts are significantly reduced with addition of ammonium monobasic phosphate or ammonium dibasic citrate to the matrix and sample deposited onto the MALDI target. The reduction of matrix adducts results in the increase of peptide intensity and signal-to-noise ratio as well as in improvement of peptide ionization for samples deposited onto the target at levels of 10 fmol or below. These improvements were particularly significant in the detection of peptides at amol levels when reduced amounts of matrix were also used. 相似文献
14.
Steven K. Drake Glen L. Hortin 《The international journal of biochemistry & cell biology》2010,42(1):174-179
Sulfation of tyrosine residues is a common post-translational modification, but detecting and quantitating this modification poses challenges due to lability of the sulfate group. The goal of our studies was to determine how best to detect and to assess the stoichiometry of this modification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Sulfated and nonsulfated forms of peptides—hirudin(55–65), caerulein, and cholecystokinin octapeptide and phosphorylated and nonphosphorylated pp60-c-src (521–533)—were analyzed using several matrices: sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DBA), and cyano-4-hydroxycinnamic acid (CHCA). Intact sulfated peptides were difficult to detect using positive ion mode; peptides were observed as desulfated ions. Phosphorylated peptide was stable and was detected in positive and negative ion modes. Detection of sulfated peptides improved with: (1) Analysis in negative ion mode, (2) Decreased laser power, (3) Matrix selection: DBA ≥ SA > CHCA. In negative ion mode, desorption/ionization of sulfated peptide was equivalent or more efficient than nonsulfated peptide, depending on conditions of analysis. Examination of a tryptic digest of α2-antiplasmin detected the single site of sulfation in negative ion mode but not in positive ion mode. We conclude that improved detection of sulfated peptides can be achieved in negative ion mode. Dual analysis in positive and negative ion modes serves as a potential means of identifying peptides with labile modifications such as sulfation and distinguishing them from phosphorylation. 相似文献
15.
Previous studies have shown that increases in surface-peptide binding affinity result in decreases in peptide matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) ion signals. The present work demonstrates that, with appropriate corrections for peptide ionization efficiency under MALDI conditions, relative surface-peptide binding affinities can be assayed using the MALDI MS methodology. Peptides with a range of pI values are allowed to interact with amine-modified and carboxylic acid-modified polymer surfaces (produced by pulsed radio-frequency plasma polymerization of allyl amine and vinyl acetic acid) in buffered solutions of neutral pH. Because of the net positive and negative charges associated with the peptides and surfaces in solution, both electrostatic and hydrophilic interactions play a role in the surface-peptide interaction. Consistent with expectations, the peptide MALDI ion signals for peptides with net negative charges in solution are smaller than those for peptides with net positive charges in solution when the peptides are allowed to interact with positively charged surfaces. A reversal of the relative peptide MALDI ion signal intensities is observed when the same peptides are allowed to interact with negatively charged surfaces. Cumulatively, the results demonstrate that even modest changes in surface-peptide interactions can be comparatively probed by MALDI mass spectrometry. 相似文献
16.
Shi Y Xiang R Crawford JK Colangelo CM Horváth C Wilkins JA 《Journal of proteome research》2004,3(1):104-111
New mass-tagging reagents for quantitative proteomics measurements have been designed using solid phase peptide synthesis technology. The solid phase mass tags have been used to accurately measure the relative amounts of cysteine-containing peptides in model peptide mixtures as well as in mixtures of tryptic digests in the femtomol range. Measurements were made using both matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and online reversed-phase capillary liquid chromatography coupled through a nanoelectrospray interface to an ion trap mass spectrometer (capillary LC/ESI-MS). Results of mass-tagging experiments obtained from these two mass spectrometry techniques and their relative advantages and disadvantages for identification and quantitation of mass tagged peptides are compared. These reagents provide a simple, rapid and cost-effective alternative to currently available mass tagging technologies. 相似文献
17.
Flensburg J Haid D Blomberg J Bielawski J Ivansson D 《Journal of biochemical and biophysical methods》2004,60(3):391-334
A new matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-ToF MS), developed specifically for the identification and characterization of proteins and peptides in proteomic investigations, is described. The mass spectrometer which can be integrated with the 2-D gel electrophoresis workflow is a bench-top instrument, enabling rapid, reliable and unattended protein identification in low-, as well as high-throughput proteomics applications. To obtain precise information on peptide sequences, the instrument utilizes a timed ion gate and a unique quadratic field reflectron (Z2 technology), allowing single-run, post-source decay (PSD) of selected peptides. In this study, the performance of the instrument in reflectron, PSD and linear mode, respectively, was investigated. The results showed that the limit of detection for a single peptide in reflectron mode was 125 amol with a signal to noise ratio exceeding 20. Average mass resolution for peptides larger than 2000 u was around 13,000 full width, half maximum (FWHM). The limit for protein identification during peptide mass fingerprinting (PMF) was 500 amol with a sequence coverage of 18%. Mass error during PMF analysis was less than 15 ppm for 17 out of 25 (68%) identified peptides. In PSD mode, a complete series of y-ions of a CAF-derivatized peptide could be obtained from 3.75 fmol of material. The average mass error of PSD-generated fragments was less than 0.14 u. Finally, in linear mode, intact proteins with molecular masses greater than 300,000 u were detected with mass errors below 0.2%. 相似文献
18.
Direct matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of human serum yielded ion signals from only a fraction of the total number of peptides and proteins expected to be in the sample. We increased the number of peptide and protein ion signals observed in the MALDI-TOF mass spectra analysis of human serum by using a prefractionation protocol based on liquid phase isoelectric focusing electrophoresis. This pre-fractionation technique facilitated the MALDI-TOF MS detection of as many as 262 different peptide and protein ion signals from human serum. The results obtained from three replicate fractionation experiments on the same serum sample indicated that 148 different peptide and protein ion signals were reproducibly detected using our isoelectric focusing and MALDI-TOF MS protocol. 相似文献
19.
Two-dimensional gel electrophoresis-separated and excised haptoglobin alpha2-chain protein spots were subjected to in-gel digestion with trypsin. Previously unassigned peptide ion signals observed in mass spectrometric fingerprinting experiments were sequenced using the matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectrometer and showed that the haptoglobin alpha-chain derivative under study was cleaved by trypsin unspecifically. Abundant cleavages occurred C-terminal to histidine residues at H23, H28, and H87. In addition, mild acidic hydrolysis leading to cleavage after aspartic acid residues at D13 was observed. The uninterpreted tandem mass spectrometry (MS/MS) spectrum of the peptide with ion signal at 2620.19 was submitted to database search and yielded the identification of the corresponding peptide sequence comprising amino acids (aa) aa65-87 from the haptoglobin alpha-chain protein. Also, the presence of a mixture of two tryptic peptides (mass to charge ratio m/z 1708.8; aa40-54, and aa99-113, respectively), that is caused by a tiny sequence variation between the two repeats in the haptoglobin alpha2-chain protein was resolved by MS/MS fragmentation using the MALDI-QIT-TOF mass spectrometer instrument. Advantageous features such as (i) easy parent ion creation, (ii) minimal sample consumption, and (iii) real collision induced dissociation conditions, were combined successfully to determine the amino acid sequences of the previously unassigned peptides. Hence, the novel mass spectrometric sequencing method applied here has proven effective for identification of distinct molecular protein structures. 相似文献