共查询到20条相似文献,搜索用时 15 毫秒
1.
Creaser CS Green PS Kilby PM Ratcliffe L 《Rapid communications in mass spectrometry : RCM》2006,20(5):829-836
The atmospheric-pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI-QIT) analysis of tryptic peptides is reported following capillary liquid chromatographic (LC) separation and direct analysis of a protein digest. Peptide fragments were identified by peptide mass fingerprinting from mass spectrometric data and sequence analysis obtained by tandem mass spectrometry of the principal mass spectral peaks using a data-dependent scanning protocol. These data were compared with those from mass spectrometric analysis using capillary LC/MALDI-time-of-flight (TOF) and capillary LC/electrospray ionisation (ESI)-quadrupole TOF. For all three configurations the resulting data were searched against the MSDB database, using MASCOT and the sequence coverage compared for each technique. Complementary data were obtained using the three techniques. 相似文献
2.
Cohen AM Mansour AA Banoub JH 《Rapid communications in mass spectrometry : RCM》2005,19(17):2454-2460
Vitellogenin is a protein produced by the liver of oviparous animals in response to circulating estrogens. The amino acid sequence of vitellogenin from Atlantic cod (Gadus morhua) has not yet been determined. In this study Atlantic cod vitellogenin was characterized using a 'bottom-up' mass spectrometric approach. Vitellogenin synthesis was induced 'in vivo' with beta-estradiol, and subjected to trypsin digestion for characterization by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry. A peptide mass fingerprint was obtained and 'de novo' sequencing of the most abundant tryptic peptides was performed by low-energy collision-induced dissociation tandem mass spectrometry. As a result of these experiments, the sequences of various tryptic peptides have been elucidated. The database search has shown that Atlantic cod vitellogenin shares a series of common peptides with the two different known vitellogenin sequences of haddock, a closely related species. These findings allow us to propose that Atlantic cod might also co-express at least two distinct forms of vitellogenin. 相似文献
3.
Insoluble or low solubility organometallic and coordination compounds have been characterised by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, with solvent-free sample preparation being the key step toward successful analysis. 相似文献
4.
A refined sample preparation procedure for matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) was developed for the evaluation of the degree of substitution (DS) in partially depolymerised carboxymethyl cellulose (CMC). By adding ammonium sulphate to the sample mixture prior to the analysis, good quality mass spectra could be acquired. The usual time-consuming search for 'sweet-spots' at the crystalline rim of the MALDI target spot was also avoided. This quality improvement made it possible to investigate whether various positions on the target spot generated mass spectra in which the measured DS varied. The accuracy and reproducibility of the sample preparation procedure were tested by applying it on three commercial CMCs. The study shows that the DS values that were calculated from the spectra acquired from the centre region of the MALDI target spot were in better agreement with the DS provided by the supplier than were the values obtained from the large crystals at the target spot rim. This observation could be one reasonable explanation for the higher DS values reported in other publications. By applying our refined MALDI sample preparation procedure DS values that were in good agreement with the values provided by the manufacturer could be obtained. This indicates that MALDI-TOFMS of partially depolymerised CMCs can be used for an estimation of the DS as a complement to the more established methods, e.g. NMR, titrimetry, and chromatographic techniques. 相似文献
5.
A novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight (MALDI QIT ToF) mass spectrometer has been used to analyse high mass peptide ions exceeding 2000 Da. Human adrenocorticotropic hormone (fragment 18-39) and oxidised bovine insulin chain B were utilised to evaluate the performance of the instrument both in MS and in MS/MS mode. Its ability to efficiently isolate ions and to fragment them using collisionally activated decomposition (CAD) has been demonstrated using mixtures diluted to the low-femtomole level on target. Additionally, multiple stage mass spectrometry (MS/MS/MS) provides a second-generation product ion spectrum in which new fragment ions are detected and new stretches of amino acids are identified. 相似文献
6.
7.
Ma Y Lu Y Zeng H Ron D Mo W Neubert TA 《Rapid communications in mass spectrometry : RCM》2001,15(18):1693-1700
A two-step mass spectrometric method for characterization of phosphopeptides from peptide mixtures is presented. In the first step, phosphopeptide candidates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) based on their higher relative intensities in negative ion MALDI spectra than in positive ion MALDI spectra. The detection limit for this step was found to be 18 femtomoles or lower in the case of unfractionated in-solution digests of a model phosphoprotein, beta-casein. In the second step, nanoelectrospray tandem mass (nES-MS/MS) spectra of doubly or triply charged precursor ions of these candidate phosphopeptides were obtained using a quadrupole time-of-flight (Q-TOF) mass spectrometer. This step provided information about the phosphorylated residues, and ruled out nonphosphorylated candidates, for these peptides. After [(32)P] labeling and reverse-phase high-performance liquid chromatography (RP-HPLC) to simplify the mixtures and to monitor the efficiency of phosphopeptide identification, we used this method to identify multiple autophosphorylation sites on the PKR-like endoplasmic reticulum kinase (PERK), a recently discovered mammalian stress-response protein. 相似文献
8.
Creaser CS Reynolds JC Hoteling AJ Nichols WF Owens KG 《European journal of mass spectrometry (Chichester, England)》2003,9(1):33-44
Atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI/QIT) mass spectrometry has been investigated for the analysis of polyethylene glycol (PEG 1500) and a hyperbranched polymer (polyglycidol) in the presence of alkali-metal salts. Mass spectra of PEG 1500 obtained at atmospheric pressure showed dimetallated matrix/analyte adducts, in addition to the expected alkali-metal/PEG ions, for all matrix/alkali-metal salt combinations. The relative intensities of the desorbed ions were dependent on the matrix, the alkali-metal salt added to aid cationisation and the ion trap interface conditions [capillary temperature, in-source collisionally-induced dissociation (CID)]. These data indicate that the adducts are rapidly stabilised by collisional cooling enabling them to be transferred into the ion trap. Experiments using identical sample preparation conditions were carried out on a vacuum MALDI time-of-flight (ToF) mass spectrometer. In all cases, vacuum MALDI-ToF spectra showed only alkali-metal/PEG ions and no matrix/analyte adducts. The tandem mass spectrometry (MS/MS) capability of the ion trap has been demonstrated for a lithiated polyglycol yielding a rich fragment-ion spectrum. Analysis of the hyperbranched polymer polyglycidol by AP-MALDI/QIT reveals the characteristic ion series for these polymers as also observed under vacuum MALDI-ToF conditions. 相似文献
9.
Kaur-Atwal G Weston DJ Green PS Crosland S Bonner PL Creaser CS 《Rapid communications in mass spectrometry : RCM》2007,21(7):1131-1138
A novel method is reported for rapid protein identification by the analysis of tryptic peptides using desorption electrospray ionisation (DESI) coupled with hyphenated ion mobility spectrometry and quadrupole time-of-flight mass spectrometry (IMS/Q-ToF-MS). Confident protein identification is demonstrated for the analysis of tryptically digested bovine serum albumin (BSA), with no sample pre-treatment or clean-up. Electrophoretic ion mobility separation of ions generated by DESI allowed examination of charge-state and mobility distributions for tryptic peptide mixtures. Selective interrogation of singly charged ions allowed isobaric peptide responses to be distinguished, along with a reduction in spectral noise. The mobility-selected singly charged peptide responses were presented as a pseudo-peptide mass fingerprint (p-PMF) for protein database searching. Comparative data are shown for electrospray ionisation (ESI) of the BSA digest, without sample clean-up, from which confident protein identification could not be made. Implications for the robustness of the DESI method, together with potential insights into mechanisms for DESI of proteolytic digests, are discussed. 相似文献
10.
Monti SM Gallo M Ferracane R Borrelli RC Ritieni A Greco ML Graniti A Fogliano V 《Rapid communications in mass spectrometry : RCM》2001,15(8):623-628
Strains of certain plant pathogenic bacteria, in particular several pathovars of Pseudomonas syringae, are known to produce cyclic lipodepsipeptides (LDPs) endowed with peculiar structural features and noticeable biological activities. In this study, a mass spectrometry procedure is proposed for screening LDP-producing bacterial strains and for identifying and assessing individual LDPs. After matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) screening of thirteen P. syringae strains for LDP production, the extracts from culture filtrates of eight positive strains were subjected to electrospray mass spectrometry for the identification of LDPs. Five strains were found to produce two forms of syringomycins (SR-E and SR-G) and two forms of syringopeptin 25 (SP25A and SP25B); two strains produced SR-E, SR-G and a new form of SP22; one strain produced syringotoxin (ST) and syringostatin A (SS-A) in addition to SP25A and SP25B. The yield in culture of two major LPDs: SR-G (3.2-13.8 mg x L(-1)) and SP25A (41.6-231.5 mg x L(-1)) was assessed by and high-performance liquid chromatography with electrospray mass spectrometry (HPLC/ESI-MS) in both scan and single ion monitoring (SIM) modes. Results of this investigation showed that the mass spectrometry protocol developed here is a precise and reliable method for screening bacterial strains for LDP production and for assessing the amount of each metabolite under various culture conditions. This could be of practical value in view of potential applications, e.g. biocontrol of post-harvest fungal diseases. 相似文献
11.
Lin M Campbell JM Mueller DR Wirth U 《Rapid communications in mass spectrometry : RCM》2003,17(16):1809-1814
Direct tandem mass spectrometric (MS/MS) analysis of small, singly charged protein ions by tandem time-of-flight mass spectrometry (TOFMS) is demonstrated for proteins up to a molecular mass of 12 kDa. The MALDI-generated singly charged precursor ions predominantly yield product ions resulting from metastable fragmentation at aspartyl and prolyl residues. Additional series of C-terminal sequence ions provide in some cases sufficient information for protein identification. The amount of sample required to obtain good quality spectra is in the high femtomolar to low picomolar range. Within this range, MALDI-MS/MS using TOF/TOF trade mark ion optics now provides the opportunity for direct protein identification and partial characterization without prior enzymatic hydrolysis. 相似文献
12.
Warburton KE Clench MR Ford MJ White J Rimmer DA Carolan VA 《European journal of mass spectrometry (Chichester, England)》2005,11(6):565-574
Isocyanates are an important class of compounds in occupational hygiene monitoring due mainly to their behaviour as respiratory sensitisers. Here, we demonstrate the application of matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and MALDI tandem mass spectrometry (MS-MS) to the analysis of derivatised isocyanate monomers and prepolymers. The aim of the work has been to gauge the selectivity obtainable from the direct analysis of isocyanate mixtures without prior separation. Monomeric and prepolymeric isocyanate mixtures were analysed as their 1-(2-methoxyphenyl) piperazine derivatives and the potential of MALDI time-of-flight (ToF)-MS for an NCO monitoring program was assessed. The results obtained demonstrated the possibility of direct mixture analysis by this method. MALDI-MS-MS was used for the elucidation of fragment structures in the prepolymer samples. The developed methodology was then applied to the analysis of swabs from an occupational hygiene monitoring scheme and enabled the identification of the isocyanate species detected. 相似文献
13.
A method earlier developed for the mass spectrometric (MS) identification of tetanus toxin (TTx) was applied to botulinum toxins type A and B (BTxA and BTxB). Botulinum toxins are extremely neurotoxic bacterial toxins, likely to be used as biological warfare agent. Biologically active BTxA and BTxB are comprised of a protein complex of the respective neurotoxins with specific haemagglutinins (HAs) and non-toxic non-haemagglutinins (NTNHs). These protein complexes are also observed in mass spectrometric identification. The particular BTxA complex, from Clostridium botulinum strain 62A, almost completely matched database data derived from genetic sequences known for this strain. Although no such database information was available for BTxB, from C. botulinum strain okra, all protein sequences from the complex except that of HA-70 were found to match proteins known from other type B strains. It was found that matrix-assisted laser desorption ionisation MS provides provisional identification from trypsin digest peptide maps and that liquid chromatography electrospray (tandem) mass spectrometry affords unequivocal identification from amino acid sequence information of digest peptides obtained in trypsin or pepsin digestion. 相似文献
14.
One problem of matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry is the moderate mass accuracy that typically can be obtained in routine applications, Here we report improved mass accuracy for peptides, even when low amounts and complex peptide mixtures are used. A new procedure for preparing matrix surfaces is used, and there is no need to mix the matrix with the sample or to add internal standards. Examples are shown with a mass accuracy better than 50 ppm in a peptide mixture. Peptide mapping as well as sequencing by creating “ragged ends” or “ladder sequencing” should benefit especially from the improved mass accuracy. 相似文献
15.
We have studied the fragmentation behaviour of short, singly protonated oligoribonucleotides on a MALDI Qq-TOF instrument with the aim of using this instrumental set-up to characterise modifications of RNA molecules. Individual ion species from enzymatically generated mixtures were isolated in one quadrupole and subjected to collision-induced dissociation in a second quadrupole followed by separation of the resulting product ions in an orthogonal time-of-flight mass analyser. Complex spectra were generally observed with nearly all types of cleavages along the phosphodiester backbone and of the N-glycosidic bonds (and combinations of these) occurring, albeit at different relative intensities. The most labile part of the backbone was found to be the 5'-P-O bond, resulting in c- and y-ions. Loss of neutral cytosine and guanine occurred equally often, whereas neutral loss of adenosine was less prevalent. Loss of uracil, either neutral or charged species, was not observed. Because the fragmentation pattern observed here is significantly different from what has been reported for singly protonated oligodeoxyribonucleotides, we suggest that the 2'-substituent in the sugar plays a central role in the fragmentation mechanisms of nucleic acids. Finally, we used the acquired knowledge about oligoribonucleotide fragmentation to characterise an in vivo methylated oligoribonucleotide by tandem mass spectrometry. 相似文献
16.
Onnerfjord P Ekström S Bergquist J Nilsson J Laurell T Marko-Varga G 《Rapid communications in mass spectrometry : RCM》1999,13(5):315-322
This work presents a simple method for obtaining homogeneous sample surfaces in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the automated analysis of peptides and proteins. The sample preparation method is based on applying the sample/matrix mixture onto a pre-deposited highly diluted matrix spot. The pre-deposited crystals act as seeds for the new sample containing crystals which become much smaller in size and more evenly distributed than with conventional methods. This 'seed-layer' method was developed, optimised and compared with the dried-droplet method using peptides and proteins in the 1000-20,000 Da range. The seed-layer method increases the surface homogeneity, spot to spot reproducibility and sample washability as compared with the commonly used dried-droplet method. This methodology is applicable to alpha-cyanohydroxycinnamic acid, sinapinic acid and ferulic acid, which all form homogeneous crystal surfaces. Within-spot variation and between-spot variation was investigated using statistics at a 95% confidence level (n = 36). The statistical values were generated from more than 5000 data points collected from 500 spectra. More than 90% of the sample locations results in high intensity spectra with relatively low standard deviations (RSDs). Typically obtained data showed an RSD of 19-35% within a sample spot as well as in-between spots for proteins, and an RSD of < or = 50% for peptides. Linear calibration curves were obtained within one order of magnitude using internal calibration with a point-RSD of 3% (n = 10). The sample homogeneity allows mass spectra (average of 16 laser shots) to be obtained on each individual sample within 15 sec, whereby a 100 spot target plate can be run in 25 min. High density target plates using the seed-layer method were prepared by spotting approximately 100 picoliter droplets onto the target, resulting in sample spots < or = 500 microns in diameter using a flow-through piezo-electric micro-dispenser. By using this automated sample preparation step lower standard deviations are obtained in comparison to manually prepared samples. 相似文献
17.
Naumann I Darsow KH Walter C Lange HA Buchholz R 《Rapid communications in mass spectrometry : RCM》2007,21(19):3185-3192
Sulfoglycolipids, isolated from different phototrophic organisms, particularly plants and algae, have already been identified as bioactive compounds. In addition to their antiviral activity their influence on the immune response in mammalian cells is the focus of many studies. For the first time it has been possible to investigate purified sulfoquinovosyldiacylglycerols (SQDGs) from the microalga Porphyridium purpureum by matrix-assisted laser desorption/ionisation (MALDI) in the negative ion reflectron mode. Thereby, different solid and ionic liquid matrices have been tested to improve signal intensity during the laser ionisation. By using the MALDI Trap time-of-flight (ToF) multiple-stage (MS(n)) hybrid mass spectrometer the fatty acid compositions of the SQDGs were analysed by MS, and confirmed by MS(2) and MS(3) experiments. Thereby, hexadecanoic acid (C16:0), octadecadienoic acid (C18:2), eicosatetraenoic acid (C20:4), and eicosapentaenoic acid (C20:5) were detected in the purified fraction of SQDGs. The localisation of hexadecanoic acid (C16:0) at the sn-2 position, and unsaturated fatty acids at the sn-1 position of the SQDGs, determined by specific enzymatic hydrolysis, marks a procaryotic biosynthesis of SQDGs in the eucaryotic alga cells. 相似文献
18.
Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry 总被引:5,自引:0,他引:5
Bennett KL Stensballe A Podtelejnikov AV Moniatte M Jensen ON 《Journal of mass spectrometry : JMS》2002,37(2):179-190
A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins. 相似文献
19.
Andalò C Bocchini P Pozzi R Galletti GC 《Rapid communications in mass spectrometry : RCM》2001,15(9):665-669
Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) has been investigated as a tool for accurate determination of the molecular mass of synthetic analogues of prazosine, a molecule used for the treatment of hypertension. Samples were dissolved in methanol, mixed with mass calibration standards, and crystallised on the target with alpha-cyano-4-hydroxycinnamic acid as matrix. Acquisition of spectra was rapidly completed in reflectron mode, allowing high resolution (6000-10000) and sensitive (about 1-10 pmol of sample on target) determination of the synthetic products. The results show the effectiveness of MALDI-TOFMS for accurate mass determination of these fairly large molecules, which are otherwise difficult to analyse by other high-resolution mass spectrometric techniques. 相似文献
20.
Solid-phase derivatization of tryptic peptides for rapid protein identification by matrix-assisted laser desorption/ionization mass spectrometry 总被引:1,自引:0,他引:1
Keough T Lacey MP Youngquist RS 《Rapid communications in mass spectrometry : RCM》2002,16(11):1003-1015
Solid-phase sulfonation of tryptic peptides adsorbed to C18 muZipTips has been carried out to facilitate de novo sequencing with mass spectrometry. Peptides are reacted with the sulfonation reagent while they are still adsorbed to the solid phase. Excess reagent passes through the ZipTip to waste. Washing the products before subsequent elution from the mini-column also affords sample cleanup prior to analysis. Near quantitative N-terminal sulfonation can be achieved reliably at room temperature in only a few seconds. The method has been applied successfully to model peptides and to solution or in-gel digests of proteins. Current sequencing limits are about 100 fmol of protein. Multiplexed sample sulfonation reactions have been carried out with a manual 8-position micropipettor or using centrifugal force to reliably pass reagents and wash solutions over sample-loaded ZipTips. With multiplexing, overall preparation times have been reduced to about 1 min per sample. The solid-phase format facilitates efficient use of precious digest samples by enabling them to be recovered from the matrix-assisted laser desorption/ionization (MALDI) sample stage after mass fingerprinting, derivatized and re-analyzed by MALDI postsource decay mass spectrometry. 相似文献