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1.
Results are presented showing the ability to obtain C-terminal sequence information from peptides by multiple stages of mass spectrometry. Under typical low-energy collision-induced dissociation conditions of quadrupole ion trap and ion cyclotron resonance mass spectrometers, lithium- and sodium-cationized peptides dissociate predominantly by reaction at the C-terminal peptide bond or an adjacent bond. For the majority of cases studied, the dominant reaction is a rearrangement process that results in the loss of the C-terminal residue and formation of a product ion that is one amino acid shorter than the original peptide ion. Using the multistage MS/MS capabilities of quadrupole ion trap and ion cyclotron resonance mass spectrometers, a subsequent stage of MS/MS can be performed to determine the identity of the new C-terminal residue. Up to eight stage of MS/MS have been performed with both quadrupole ion trap and ion cyclotron resonance mass spectrometers. In general, the same dissociation pathways are observed with both instruments, although occasionally there are significant differences in the branching ratios of competing pathways. 相似文献
2.
Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry 总被引:1,自引:0,他引:1
Matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometry is shown to be a powerful tool for the elucidation of protein modifications. Low-energy covalent bonds that originate from certain posttranslational modifications dissociate preferentially to produce characteristic mass spectrometric signatures that prove useful for the accurate, confident identification and characterization of such modifications. Because the MALDI ion trap is an authentic tandem mass spectrometer, it proves feasible to acquire secondary information to test hypotheses as to the nature and site of the putative modifications--further increasing the reliability of the tool. The method combines the advantageous features of MALDI (i.e., the ability to measure the same sample repeatedly, to measure unfractionated complex mixtures without the need for sample cleaning, and to determine peptide mixtures with subpicomole sensitivity) with the ease and the speed of the ion trap measurement. We demonstrate how the unique properties of MALDI ion trap MS can be used to address problems involving the determination of both native posttranslational modifications of proteins (e.g., disulfide mapping, glycosylation determination, and phosphorylation determination) and non-native chemical modifications of proteins (e.g., methionine oxidation and photo-cross-linking of proteins with DNA). 相似文献
3.
JR Yates SF Morgan CL Gatlin PR Griffin JK Eng 《Canadian Metallurgical Quarterly》1998,70(17):3557-3565
We report the development of a method to compare collision-induced dissociation (CID) spectra of peptides. This method employs a cross-correlation analysis of a CID spectrum to a reference spectrum and normalizes the cross-correlation score to the autocorrelation of the CID spectra. The query spectrum is compared by using both mass information and fragmentation patterns. Fragmentation patterns are compared to each other using a correlation function. To evaluate the specificity of the approach, a set of 2180 tandem mass spectra obtained from both triple-quadrupole tandem mass spectrometers (TSQ) and quadrupole ion trap mass spectrometers (LCQ) was created. Comparisons are performed between tandem mass spectra obtained on the same instrument type as well as between different instrument types. Accurate and reliable comparisons are demonstrated in both types of analyses. The scores obtained in the cross-comparison of TSQ and LCQ tandem mass spectra of the same peptide are found to be slightly lower than comparisons performed with spectra obtained on the same instrument type. The method appears insensitive to variations in day-to-day performance of the instrument, minor variations in fragment ion abundance, and instrumental differences inherent in the same instrument model. The use of this method of comparison is demonstrated for library searching and subtractive analysis of tandem mass spectra obtained during LC/MS/MS experiments. 相似文献
4.
De novo peptide sequencing in an ion trap mass spectrometer coupled on-line with a capillary HPLC using 18O labeling provides a viable alternative to the method using the combination of nanospray, 18O labeling and a quadrupole/time-of-flight mass spectrometer. Seven to sixteen amino acid residues can be sequenced from the liquid chromatography/randem mass spectrometry (LC/MS/MS) spectra. This approach combines the benefit of capillary LC and the high sensitivity of the ion trap operated in the MS/MS mode. The wide availability of the LCQ mass spectrometer makes this approach readily adaptable to the biological mass spectrometry community. 相似文献
5.
IA Kaltashov V Doroshenko RJ Cotter K Takayama N Qureshi 《Canadian Metallurgical Quarterly》1997,69(13):2317-2322
The chemical structure of nontoxic diphosphoryl lipid A from Rhodobacter sphaeroides was confirmed using a combination of LSIMS (on a two-sector mass spectrometer) and MALDI (on time-of-flight and ion trap mass spectrometers) in conjunction with tandem mass spectrometry in both positive and negative ion modes. Accurate molecular weight measurement accompanied by the analysis of fragment ion masses yielded the composition of fatty acyl groups. Tandem experiments (collisionally induced dissociation of both quasimolecular and oxonium ions) were also performed, revealing the precise location and nature of the fatty acyl groups on the disaccharide backbone. 相似文献
6.
A strength of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is its ability to analyze mixtures without separation. MALDI mass spectrometers capable of providing a linear mass calibration over a broad mass range should find wide use in these applications. This work addresses issues pertinent to mass measurement accuracy of a time-lag focusing MALDI time-of-flight instrument and presents a new approach to improving mass accuracy by using a functional wave extraction pulse, instead of a square wave, for time-lag focusing. A model is described of an ideal extraction pulse shape that provides constant total kinetic energy for all ions. If total kinetic energy is constant, then there is an exact linear correlation between ion mass and flight time raised to the second power. Using a descending wave extraction pulse, it is demonstrated that mass accuracy of better than 30 ppm using two internal calibrants and better than 70 ppm using external calibrants can be obtained over a 25 ku mass range. The practical aspects of an instrument needed to obtain consistent mass accuracy is discussed. It is found that ion flight time shows a small dependence upon laser flux; flight times increase slightly as the flux increases. But this dependence is much smaller than is observed in continuous-extraction MALDI. 相似文献
7.
Oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino) ethyl ester (ABDEAE) can be analyzed by ESI (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) and MALDI (Takao, T.; et al. Rapid Commun. Mass Spectrom. 1996, 10, 637-640) mass spectrometry. In this study, oligosaccharides derived from the enzymatic cleavage of the sugar chains of glycoproteins ribonuclease B, erythropoietin, and transferrin were subjected to ABDEAE derivatization, prior to analysis on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) for high-resolution mass measurement and a postsource decay (PSD) experiment. In the mass measurement of ABDEAE derivatives, quasi-molecular ion species have been observed in monoisotopic resolution using 2,5-dihydroxybenzoic acid as the matrix from spots that contain 50-200 fmol of sample; in the PSD analyses from the spots contained 500 fmol-1 pmol of sample, the predominant backbone ion series which covers the entire mass range for all the derivatives, the internal ion series which reflect the branched trimannosyl core structure of N-glycans, and the low m/z fingerprint ion of ABDEAE were consecutively observed, permitting structure elucidation of the oligosaccharides. Given the effectiveness of this derivatization in terms of its high sensitivity and resolution with respect to MALDI-TOF MS, current methodology is clearly applicable to the sensitive detection and accurate structural analysis of N-glycans. 相似文献
8.
In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed. 相似文献
9.
We report a study of the application of delayed extraction (DE) to infrared-wavelength matrix-assisted time-of-flight mass spectrometry (IR-MALDI-TOF-MS) of proteins. The shapes of the spectral peaks obtained with DE-IR-MALDI-MS are compared with those obtained from the same samples and matrix using continuous extraction (CE) IR-MALDI-MS. Application of DE results in significant improvements in the peak resolution, revealing spectral features (in proteins with molecular masses < 12 kDa) that were not resolved in the corresponding CE-IR-Maldi mass spectra. Particularly significant is a series of peaks on the high mass side of the protonated protein peaks that arise through replacement of protons by adventitious sodium ions in the sample. We deduced that these sodium replacement species are a significant contributor to the broad tails (and resulting peak asymmetries) that are a feature of the DE-IR-MALDI mass spectra of proteins with molecular masses > or = 17 kDa. The peak width reduction observed in IR-MALDI by DE suggests that, as in UV-MALDI, the initial velocity distribution for ions produced in the MALDI process contributes to the peak broadness in the CE mass spectra. In a systematic comparison between DE UV-MALDI and DE IR-MALDI, we determined that photochemical matrix adduction is present in UV-MALDI but absent in IR-MALDI. In addition, we find that protein ions produced by IR irradiation are less internally excited (i.e., cooler), exhibiting less fragmentation, more Na+ replacement and/or unspecified noncovalent adduction, and more heme adduction with apomyoglobin. Thus, IR-MALDI appears to be a softer means for producing gas-phase protein ions than is UV-MALDI. It will be of considerable practical interest to determine whether large protein ions produced by IR-MALDI are sufficiently cool to survive transport through reflecting TOF mass spectrometers (without loss of small neutral species such as H2O, NH3, and CO2) and the extended time periods required for detection by quadrupole ion trap and Fourier transform ion cyclotron resonance mass analyzers. 相似文献
10.
Various sugars, peptides, and lipids were analyzed on a Fourier transform mass spectrometer using laser desorption and ionization with and without the assistance of matrixes. A compact Nd:YAG laser with an output at 1.06 microns corresponding to fundamental frequency was employed. Gram-negative and Gram-positive bacteria were also subjected to laser desorption mass spectrometry. Characteristics ions of conjugated lipid, formed by attachment of alkali metal cations, endogenous to the cells, were observed. Particle/liquid matrixes (e.g., cobalt in glycerol) proved to be useful with the 1.06-micron laser. The particles absorb efficiently laser radiation in a broad wavelength range. The liquid provides the same advantages as in fast atom bombardment: increased signal-to-noise ratios and enhanced sample lifetimes. The effect of laser power on total ion current was shown to differ for samples with and without the particle/liquid matrix. The Fourier transform analyzer provides MS/MS capability for both positive and negative ions from complex mixtures. Ions desorbed externally are introduced into the cell via a quadrupole ion guide with a lower mass cutoff. Such a setup allows matrix ions to be excluded and thus provides excellent signal-to-noise ratios for lower mass range fragment ions formed inside the cell. 相似文献
11.
J Noguchi K Ishiwata S Wakabayashi T Nariai S Shumiya S Ishii H Toyama K Endo F Suzuki M Senda 《Canadian Metallurgical Quarterly》1998,39(3):498-503
A simple and effective method has been proposed in this work for combination of immunoaffinity extraction with MALDI MS. In this method, an antibody is attached to the surface of a MALDI probe tip via a thin nitrocellulose film. This allows the corresponding antigen to be selectively captured and concentrated on the probe tip from complex plasma solution for MALDI MS analysis. The whole procedure can be completed within 1 h. This combination offers several excellent performance features in the analysis of SNX-111, a therapeutic peptide. It combines the high specificity of affinity chromatography with the high sensitivity of mass spectrometry in a rapid analysis. Direct mass detection provides unambiguous determination by the observation of signals at characteristic m/z values. This method has been used successfully to determine the therapeutic peptide at relevant doses. 相似文献
12.
This review offers an introduction to the principles and generic applications of FT-ICR mass spectrometry, directed to readers with no prior experience with the technique. We are able to explain the fundamental FT-ICR phenomena from a simplified theoretical treatment of ion behavior in idealized magnetic and electric fields. The effects of trapping voltage, trap size and shape, and other nonidealities are manifested mainly as perturbations that preserve the idealized ion behavior modified by appropriate numerical correction factors. Topics include: effect of ion mass, charge, magnetic field, and trapping voltage on ion cyclotron frequency; excitation and detection of ICR signals; mass calibration; mass resolving power and mass accuracy; upper mass limit(s); dynamic range; detection limit, strategies for mass and energy selection for MSn; ion axialization, cooling, and remeasurement; and means for guiding externally formed ions into the ion trap. The relation of FT-ICR MS to other types of Fourier transform spectroscopy and to the Paul (quadrupole) ion trap is described. The article concludes with selected applications, an appendix listing accurate fundamental constants needed for ultrahigh-precision analysis, and an annotated list of selected reviews and primary source publications that describe in further detail various FT-ICR MS techniques and applications. 相似文献
13.
Electrospray ionization coupled to a quadrupole ion trap mass spectrometer is used to differentiate between the isobaric amino acids lysine and glutamine in sequence analysis of peptides. Collision-induced dissociation is used for fragmentation. Several isobaric peptides with one or more lysines or glutamines at different positions were investigated. The ambiguous amino acid either in the peptide chain or at the C- or N-terminus can be clearly identified based on specific side chain fragment ions resulting from MS3 or MS4 of B- and Y"-fragment ions. 相似文献
14.
V Redeker JY Toullec J Vinh J Rossier D Soyez 《Canadian Metallurgical Quarterly》1998,70(9):1805-1811
The combination of two sensitive and powerful analytical techniques on the same biological sample was examined: (i) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gives informative peptide profiling on complex samples such as organs or cells; (ii) immunological tools such as enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to probe for specific peptides in biological extracts or cells. The cellular expression of the two precursors of the hyperglycemic hormone (cHH) was analyzed in neurosecretory cells (30-micron diameter) from the crayfish Orconectes limosus. Neurohemal organs were used to optimize the sample preparation and to demonstrate that, after peptide fingerprinting by MALDI-TOF MS, the sample can be recovered from the MALDI plate for further immunological analysis by ELISA. It was also established that, after immunocytochemistry following 4% paraformaldehyde fixation of the organ, the stained tissue could be recovered for further MALDI-TOF MS analysis. This dual characterization was successfully scaled down to the level of a single crayfish neurosecretory cell. Direct peptide profiling by MALDI-TOF MS on a single cHH-producing cell previously identified by immunocytochemistry demonstrated that both procHH isoforms were expressed in each cell analyzed. 相似文献
15.
The ultradian clocks of eukaryotic microbes: timekeeping devices displaying a homeostasis of the period 总被引:1,自引:0,他引:1
F Kippert 《Canadian Metallurgical Quarterly》1997,14(5):469-479
Ultra-high-sensitivity, biopolymer sequencing is a goal in many fields of molecular biology, and collisionally activated decomposition electrospray mass spectrometry (CAD ES MS/MS) using a triple quadrupole mass spectrometer has become a method of choice for work in the high- to mid-femtomole range. However, when the detection of ions becomes statistical, as it may in that range, the mass assignment of fragment ions is inaccurate and either sequencing becomes impossible or ambiguities result due, for example, to the closeness in amino acid residue masses (I/L, N or K/Q, E). Some ambiguities may be resolved by synthesizing possible sequences, but this is unsatisfactory. In considering the limitations of triple quadrupole MS/MS with respect to scanning ion detection, resolution, transmission, and mass accuracy, we reasoned that a novel geometry quadrupole orthogonal acceleration time-of-flight (Q-TOF) instrument would have special merit for ultra-high-sensitivity MS/MS sequencing, and suggested its construction for this purpose some three years ago. A prototype Q-TOF has now been built by Micromass [Morris et al. (1996), Rapid Commun. Mass Spectrom. 10, 889-896], and in the first research on the instrument, including MHC antigen and filarial nematode glycoprotein studies, we demonstrate low-femtomole- and attomole-range sequencing with mass accuracy of better than 0.1 Da throughout the daughter-ion spectrum, thus removing sequencing ambiguities in some of the most challenging work demanding the highest sensitivity. 相似文献
16.
Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) were combined in an off-line arrangement to provide separation and mass analysis of peptide and protein mixtures in the attomole range. A membrane target, precoated with MALDI matrix, was used for the continuous deposition of effluent exiting from a CE device. A sample track was produced by linear movement of the target during the electrophoretic separation and this track was subsequently analyzed by MALDI/MS. The technique is effective for peptides and proteins, having limits of detection (signal-to-noise >3) of about 50 amol for neurotensin (1673 Da) and 250 amol for cytochrome c (12361 Da) and apomyoglobin (16951 Da). The electrophoretic separation achieved from the membrane target, as measured by theoretical plate numbers from the mass spectrometric data, can be as high as 80-90% of that achieved by on-line UV detection under optimal conditions, although band broadening occurs and with some loss of separation efficiency. Non-volatile buffers such as 10-50 mM phosphate can also be used in the electrophoresis process and directly deposited on the membrane. The use of post-source decay techniques is shown for peptides in the CE sample track in order to obtain sequence verification. The effectiveness of this method of integration of CE and MALDI/MS is demonstrated with both peptide and protein mixtures and with the analysis of a tryptic digest of a protein. 相似文献
17.
CG de Koster MC Duursma GJ van Rooij RM Heeren JJ Boon 《Canadian Metallurgical Quarterly》1995,9(10):957-962
Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) by external injection of matrix-assisted laser desorbed and ionized (MALDI) polymers offers good possibilities for characterization of low molecular weight homopolymers (MW range up to 10 kDa). The molecular masses of the molecular weight distribution (MWD) components of underivatized and derivatized (dimethyl, dipropyl, dibutyl and diacetyl) polyethylene glycol (PEG) 1000 and 4000 were measured by MALDI-FTICR-MS. These measurements have been performed using a commercial FTICR spectrometer with a home-built external ion source. MALDI of the samples with a 2,5-dihydroxybenzoic acid matrix in a 1000:1 matrix-to-analyte molar ratio produces sodiated molecules in a sufficient yield to trap the ions in the ICR cell. The masses of the molecular weight distribution of PEG components were measured in broad-band mode with a mass accuracy of < 5 ppm in the mass range around 1000 u and within 40 ppm accuracy around 4000 u. From these measurements, the endgroup mass of the polymer was determined by correlation of the measured component mass with the degree of polymerization. The masses of the PEG endgroups have been determined within a deviation of 3-10 millimass units for the PEG1000 derivatives and 10-100 millimass units for the PEG4000 derivatives, thus confirming the identity of the distal parts of the model compounds. 相似文献
18.
FT Mangano JP McAllister HC Jones MJ Johnson RM Kriebel 《Canadian Metallurgical Quarterly》1998,20(8):697-704
A number of different procedures have been developed for use with matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) for the analysis of non-covalent protein-protein complexes. These include use of specific matrix and laser combinations, accumulation of "first shot" spectra, modification of pH and solvent conditions during sample preparation and use of cross-linking agents to attach the monomers covalently to each other in the complex. The results have shown the techniques to be effective with some but not all complexes, although cross-linking is the most successful. The physical and chemical nature of the complex is critical and therefore a diversity of approaches is recommended for such studies. 相似文献
19.
K Hirayama R Yuji N Yamada K Kato Y Arata I Shimada 《Canadian Metallurgical Quarterly》1998,70(13):2718-2725
Complete and rapid peptide and glycopeptide mapping of a mouse monoclonal immunoglobulin (IgG2b) were carried out by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry (LC/ ESI IT-MS/MS). It was possible to obtain spectra of a minor glycopeptide at a quantity as low as 1.8 pmol. Reduced and carboxymethylated mouse antidansyl monoclonal IgG2b (RCM-IgG2b) was digested with lysyl-endopeptidase. Proteolytic peptides were subjected to capillary HPLC separation followed by analysis with an ion trap mass spectrometer. The complete amino acid sequence of the IgG2b was characterized by using LC/ ESI IT-MS/MS. The structures of 12 different types of O-linked oligosaccharides attached to Thr-221AH in the hinge region and those of three major types of N-linked oligosaccharides attached to Asn-297H have been characterized. 相似文献
20.
M Ozata M Bulur Z Beyhan A Sengül M Saglam M Turan A Corakci M Ali Gundogan 《Canadian Metallurgical Quarterly》1997,44(5):719-724
Various monosialo- and disialo-gangliosides and their derivatives were examined by delayed ion extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF MS) in the reflector mode with alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid used as the matrix. Native gangliosides were generally found to give good spectra in the negative ion mode. 2,5-Dihydroxybenzoic acid was a better matrix for gangliosides than alpha-cyano-4-hydroxycinnamic acid, because this matrix seemed to minimize loss of sialic acid and carbon dioxide of gangliosides. About 1 pmol of ganglioside was able to be detected with this matrix. When "A-series" gangliosides such as GD1a and GalNAc-GD1a gave undesirable extra peaks probably due to loss of sialic acid besides molecule-related ion peaks, the methyl-esterification of the gangliosides at the carboxyl groups of sialic acids was found to be necessary to obtain good DE MALDI-TOF mass spectra in the positive ion mode. In contrast, "B-series" gangliosides such as GD1b, GD2, and GD3 gave rise to major dehydrated molecule-related ion [M-H2O-H]- peaks in the negative ion mode without the pretreatment of methyl-esterification. The DE MALDI-TOF mass spectrometric analysis enabled us to distinguish between GD1a and GD1b, which have the same molecular weight. It was also found that not only a purified sample, but also a mixed sample of various gangliosides was amenable to the identification of them by DE MALDI-TOF MS. 相似文献