Alkaloid profiling in crude and processed Strychnos nux-vomica seeds by matrix-assisted laser desorption/ionization-time of flight mass spectrometry

Direct analysis of alkaloids in the tissues of crude and processed Strychnos nux-vomica seeds by MALDI-TOFMS was described. The alkaloid profiles of the herb drugs were obtained without the need of complicated sample preparation to avoid potential damage or change of the active components. Seed tissues that were optimally sliced to a thickness of 10-20 microm from the crude and processed Strychnos nux-vomica seeds as well as various parts of tissue such as endosperm and epidermis were analyzed on MALDI target plate after the matrix was directly applied onto the tissue surface. The obtained alkaloid profiles provided valuable information for the differentiation of crude and processed Strychnos nux-vomica seeds and for the explanation of the significantly different toxicity. Experimental results indicated that the direct MALDI-TOFMS analysis allowed rapid screening of the alkaloid components in Strychnos nux-vomica seeds.     

Use of matrix-assisted laser desorption/ionisation mass spectrometry in cancer research

Cancer significantly affects millions of people worldwide. It is possible to use proteomic techniques to aid in detection, monitoring of treatment and progression, as well as gaining an increased understanding of cancer. Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry can be utilised to detect the presence of proteins and peptides within various samples from the body, including blood, biological fluids and tumour tissue. This review aims to introduce MALDI mass spectrometry and discuss a range of applications in the field of cancer research, from quantitative to qualitative methods. Also described is MALDI imaging mass spectrometry which differs from typical sample preparation methods, as analytes are ionised directly from the tissue. Finally, presented is a brief summary of the status of biomarker discovery using blood/serum and biological fluids samples, and the implications in the clinic.     

Monitoring the neuropeptide metabolites by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The developments of bio-analytical methods for analyzing bioactive peptides are of paramount importance. Neuropeptides and their bioactive fragments play a vital role in the regulation of many biological processes and diseases. This paper presents the use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) method for monitoring neuropeptides along with their degradation products in plasma samples from cancer patients. The neuropeptides focused in this study were beta-endrophin, substance P, and bradykinin. The method involves the enzyme digestion of the neuroactive peptides followed by MALDI-MS sample preparation and subsequent acquisition of the MS spectral data. The mass spectral profile identifies most of the C-terminal and N-terminal peptides, and the mass accuracy was in the range of -1.68 to 1.46 Da with the mass spectrometer utilised. Analysis of the neuropeptide degradation patterns from the cancer patients were compared with the controls showed similar results. The study reveals that this approach can be used to identify the enzymatic digestion products of protein.     

Quantitative analysis of polypeptide pharmaceuticals by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An accurate method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been developed for quantitative analysis of calcitonin and insulin in different commercially available pharmaceutical products. Tryptic peptides derived from these polypeptides were chemically modified at their C-terminal lysine-residues with 2-methoxy-4,5-dihydro-imidazole (light tagging) as standard and deuterated 2-methoxy-4,5-dihydro-imidazole (heavy tagging) as internal standard (IS). The heavy modified tryptic peptides (4D-Lys tag), differed by four atomic mass units from the corresponding light labelled counterparts (4H-Lys tag). The normalized peak areas (the ratio between the light and heavy tagged peptides) were used to construct a standard curve to determine the concentration of the analytes. The concentrations of calcitonin and insulin content of the analyzed pharmaceutical products were accurately determined, and less than 5% error was obtained between the present method and the manufacturer specified values. It was also found that the cysteine residues in CSNLSTCVLGK from tryptic calcitonin were converted to lanthionine by the loss of one sulfhydryl group during the labelling procedure.     

Chemical profiling of Radix Paeoniae evaluated by ultra-performance liquid chromatography/photo-diode-array/quadrupole time-of-flight mass spectrometry

In this study, an ultra-performance liquid chromatography/photo-diode-array/quadrupole time-of-flight mass spectrometry (UPLC-PDA-QTOFMS) based chemical profiling method was established for rapid global quality evaluation of Radix Paeoniae. By virtue of the high resolution, high speed of UPLC and the accurate mass measurement of TOFMS, a total of 40 components including 29 monoterpene glycosides, 8 galloyl glucoses and 3 phenolic compounds were simultaneously separated within 12 min, and identified through the matching of empirical molecular formulae with those of published components in the in-house library, and were further elucidated by adjusted lower energy collision-induced dissociation (CID) mass spectra. Among forty components, five monoterpene glycoside sulfonates were identified as novel components. The established method was successfully applied to rapidly and globally compare the quality of Radix Paeoniae Alba and Radix Paeoniae Rubra, two post-harvesting handled products of Radix Paeoniae. Together with paeoniflorin sulfonate, five newly assigned monoterpene glycoside sulfonates were characteristic markers to detect non-official sulfur dioxide gas fumigated Radix Paeoniae Alba samples. It could be concluded that UPLC-PDA-QTOFMS based chemical profiling is a powerful approach for the global quality evaluation of Radix Paeoniae as well as other herbal medicines.     

Capillary electrophoresis to characterize ricin and its subunits with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been employed as highly efficient methods to characterize ricin, its subunits, and the chemically deglycosylated forms. As a CE method, sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was used because of its merit over the conventional slab gel techniques. SDS-CGE showed higher resolution capability over other analytical tools in the analysis of the ricin mixture as well as in each of its purified forms. The high resolution was considered to be a result of the presence of carbohydrates on ricin subunits, and this property was useful for identifying the native ricin or its A chain from their chemically deglycosylated forms. However, this method exhibited an overestimation of the molecular mass due to the carbohydrate moieties on ricin subunits, and the inaccuracies were observed to be dependent on the carbohydrate content of the subunits. The exact molecular masses were measured by MALDI-TOF MS, and the results were almost consistent with the expected values. This study clearly illustrates the usefulness and necessity of complementary use of two powerful analytical techniques to characterize ricin and its subunits in a various research fields such as poisoning and immunotoxin research.     

基质辅助激光解析电离飞行时间质谱检测耐甲氧西林金黄色葡萄球菌δ-毒素的应用

目的 本研究采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)检测δ-毒素,评估其在耐甲氧西林金黄色葡萄球菌(MRSA)的分型和毒力表达中的作用。方法 使用Bruker microflex MALDI-TOF仪器,采集2000~20000Da质量范围内,以正线性模式采集图谱,使用仪器配套的MALDI Biotyper 2.0软件分析MRSA菌株的原始图谱,产生的峰列表直接使用flexAnalysis、clinProTools3.0软件分析。结果 本研究中共检测83株MRSA,共有39(47.0%)株MRSA检出(3005±5)m/z信号峰,其中HA-MRSA 19(22.9%)株,CA-MRSA 20(26.5%)株,P=0.766,两者之间无显著性差异;33(39.8%)株MRSA检出(3035±5)m/z信号峰,其中HA-MRSA 9 (10.8%)株,CA-MRSA 24(28.9%)株,P=0.003,两者之间有显著性差异;11(13.%)株MRSA既未检出(3005±5)m/z信号峰也未检出(3035±5)m/z信号峰,全部为HA-MRSA菌株。spa分型中检出(3005±5)m/z信号峰,15(18.1%)株为t062型,8株为t015(9.6%)型,4株为t030(4.8%)株,P=0,有显著性差异;检出(3035±5)m/z信号峰,31(37.3%)株为t437型,2(2.4%)株t8660型,P=0,有显著性差异;(3035±5)m/z作为spa t437型特征信号ROC曲线下面积0.89,P=0。11株未检出δ-毒素,6株分离自骨关节标本,3株分离自呼吸道标本,1株分离自慢性溃疡的分泌物标本,1株分离自血液;在血平板的菌落特征,6株MRSA菌落形态发生改变,5株菌落形态正常。结论 MALDI-TOF MS使用常规方法即可快速检测MRSA的δ-毒素,其质谱峰为(3005±5)m/z和(3035±5)m/z两种;(3035±5)m/z是δ-毒素的同基因变异体(HldG10S)的质谱峰,该峰可快速鉴别spa t437型;不产生δ-毒素的菌株是agr调控系统失调的表现,与慢性感染、小菌落形成、无明显β-溶血有关。     

Amphetamine adducts of melanin intermediates demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The use of hair as a matrix for the determination of a history of drug abuse is becoming increasingly widespread. Melanin has been shown to play a key role in the incorporation of drugs in hair. The mechanism of this incorporation and the nature of the interaction remains poorly understood. Cationic drugs, such as amphetamine, are thought to be ionically bound to melanin; however, their inextricability has led to the suggestion that they may be covalently bound to a great degree. Identification of covalent adducts remains elusive due to the insoluble polymeric nature of melanin. We succeeded in identifying several such adducts by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF) analysis of the products of in vitro synthesis of melanin in the presence of amphetamine. Amphetamine was incubated with L-DOPA and mushroom tyrosinase under a stream of oxygen. After 1 h, a signal at m/z 281.1324 (n = 1, R = H) was observed. After 2 h, the major adduct mass visible in the spectrum was at m/z 470.1074. This appeared to be derived from the mono-decarboxylation of a minor adduct at m/z 514.1245 (n = 2, R = CO(2)H). A totally decarboxylated adduct was also observed at m/z 426.1448 (n = 2, R = H). These were identified as amphetamine adducts of indole quinones. Corroboration of their identity was obtained by observing the mass shifts with deuterated L-DOPA and amphetamine analogues. Accurate mass measurements using the reflectron mode of the MS showed that the smaller adduct was within 14 ppm, and the larger adducts were within 70 ppm of their theoretical monoisotopic masses. Postsource decay experiments agreed with our structural assignments.     

Analysis of protein glycation products by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The term protein glycation summarizes non-enzymatic reactions between amino groups of proteins and sugars or sugar degradation products, leading to early glycation products (intact sugar attached) and advanced glycation end-products (AGEs). Protein glycation is involved in the progression of several diseases, such as diabetes, uremia, and atherosclerosis. However, qualitative and quantitative analysis of in vitro or in vivo glycated proteins is still a challenging task. The introduction of matrix-assisted laser desorption ionization time-of-flight technique (MALDI-TOF) changed mass spectrometry (MS) into a valuable tool for biomedical analysis, because the soft ionization procedure allows the measurement of proteins up to 100 kDa. In the last few years, MALDI-TOF-MS was applied to the investigation of glycation processes: the analyses of plasma proteins from diabetic or uremic patients allowed a precise determination of the average number of sugar residues attached to serum albumin or immunoglobulins of each patient. Thus, a more individualized diagnosis of each patient was achieved by MALDI-TOF-MS than by other diagnostic tools. In a similar way, the glycation rate of hemoglobin, isolated from diabetic blood and of beta-2-microglobulin isolated from amyloid plaques from uremic patients was determined. The application of MALDI-TOF-MS for in vitro studies revealed important new insights into glycation mechanisms. Whereas the measurement of the intact proteins allows the determination of the average glycation rate, peptide mapping prior to MALDI-TOF-MS can reveal the exact structures of the glycation products and the glycation site. Furthermore, when the unmodified peptide is used as internal standard, MALDI-TOF-MS can also be used for reliable, site specific relative quantification of defined glycation products.     

Ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) for time-dependent profiling of raw and steamed Panax notoginseng

The metabolic profiles of Panax notoginseng and its associated therapeutic values are critically affected by the duration of steaming. The time-dependent steaming effect of P. notoginseng is not well-characterized and there is also no official guideline on its duration of steaming. In this paper, a UHPLC/TOFMS-based metabolomic platform was developed for the qualitative profiling of multiparametric metabolic changes of raw P. notoginseng during the steaming process. Our method was successful in discriminating the differentially processed herbs. Both the unsupervised principal component analysis (PCA) score plot (R²X = 0.664, Q² (cum) = 0.622, and PCs = 2) and the supervised partial least square-data analysis (PLS-DA) model (R²X = 0.708, R²Y = 0.461, and Q²Y = 0.271) demonstrated strong classification and clear trajectory patterns with regard to the duration of steaming. The PLS-DA model was validated for its robustness via a prediction set, confirming that the UHPLC/TOFMS metabolic profiles of the raw and differentially steamed P. notoginseng samples were highly reproducible. Based on our method, the minimum durations of steaming for the maximum production of bioactive ginsenosides such as Rg3 and Rh2 were also predicted. Our novel time-dependent metabolic profiling approach represents the paradigm shift in the quality control of P. notoginseng products.     

Sequence analysis of phosphorothioate oligonucleotides via matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Modification of the natural phosphodiester backbone of deoxyribooligonucleotides can impart increased biostability via nuclease resistance. Further, uniform incorporation of phosphorothioate linkages renders oligonucleotides highly resistant to reagents traditionally used in sequencing reactions. As a consequence, analytical tests crucial for establishing the identity of such oligonucleotide drugs are less informative. To circumvent this problem, chemical oxidation has been employed for converting the phosphorothioate to the uniform phosphodiester, thereby facilitating enzymatic degradation. Following oxidation, exonucleases which sequentially cleave individual bases from the 3′ or 5′ terminus of the oligonucleotide or base-specific cleavage chemicals were used to facilitate sequence identification of the oligonucleotide. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), previously used to sequence natural phosphodiester DNA, was then used to sequence the chemically oxidized phosphorothioate. Sequential enzymatic cleavage of desulphurized phosphorothioates in combination with MALDI analysis not only provides a viable alternative to radiolabeling as used in conventional sequencing approaches (e.g. Maxam-Gilbert), but also enables rapid sequencing of phosphorothioate oligonucleotides, for routine drug analysis.     

Nicotine and cotinine adducts of a melanin intermediate demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Pigmentation is a major factor in the incorporation of many drugs into hair. In an attempt to elucidate potential mechanisms of drug-melanin interaction, melanin was synthesized in vitro in the presence of nicotine, which we have shown to have a substantial interaction with melanin, and cotinine, a primary nicotine metabolite. L-DOPA, a precursor of eumelanin, was oxidized and oligomerized with tyrosinase. Nicotine, cotinine, and/or their deuterated analogues were added to the oligomerization reaction mixture in a 10:1 L-DOPA:drug ratio. A black precipitate formed within 60 min. Aliquots were removed from the incubation mixture at 60, 120, and 360 min. MALDI-TOF MS determinations were carried out on each sample to provide a mean and standard error for the masses of interest. Internal calibration allowed accurate mass measurement of the products. A careful comparison of the spectra of samples prepared both with and without drug indicated the presence of masses corresponding to the protonated drug, melanin oligomers, and nicotine or continine adducts of the monomeric melanin intermediate dopaquinone (DOPAQ). Additional support for the presence of drug-melanin adducts was provided by employing deuterated analogues of nicotine and L-DOPA in the reaction and observing that the masses shifted accordingly. Structures of the adducts were further confirmed by select ion gating and postsource decay analysis.     

Primary structures of proteins characterized by proteinase K digestion and matrix-assisted laser desorption/ionization mass spectrometry

To improve the sequence ions of a protein in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), proteinase K was used to digest the protein followed by MALDI-MS characterization of the peptide fragments. The primary structures of three proteins, insulin B chain, cytochrome c and lysozyme, were determined by this method. A series of peptide fragments including those differentiated by one residue can be produced from the protein by using proteinase K digestion, thus providing support to the protein sequence. The peptide fragments liberated from proteinase K proteolysis of the insulin B chain allow the protein to be partially sequenced. Furthermore, some of the residues are double or triple checked by generating a variety of fragments. The same method was used to investigate cytochrome c and lysozyme denaturated in 3 M guanidine hydrochloride. The success of the method relies on the intrinsic properties of proteinase K and accurate determination of the peptide fragments by MALDI-MS.     

High throughput screening of bradykinin-potentiating peptides in Bothrops moojeni snake venom using precursor ion mass spectrometry.

Snake venoms are known to be an extensive source of bioactive peptides. Bradykinin-potentiating peptides (BPPs) are inhibitors of the angiotensin-converting enzyme that have already been identified in the venom of many snake, scorpion, spider and batrachian species. Their most characteristic structural features are an invariable N-terminal pyroglutamate residue (pGlu or Z) and two consecutive proline residues at the C-terminus. Fragmentation of BPPs by collision-induced dissociation during electrospray tandem mass spectrometry analysis (ESI-MS/MS) generates a predominant signal at m/z 213.1 corresponding to the y-ion of the terminal Pro-Pro fragment. In addition, signals at m/z 226.1 and 240.1 that correspond to the b ions of the N-terminus pGlu-Asn and pGlu-Lys, respectively, can often be observed. Based on these structural determinants, the present work describes an original methodology for the discovery of BPPs in natural extracts using liquid chromatography coupled to ESI-MS/MS operated in precursor ion-scan mode. The venom of the Bothrops moojeni snake was used as a model and the methodology was applied for subsequent structural analysis of the identified precursors by tandem mass spectrometry on quadrupole-time-of-flight (Q-TOF) and matrix-assisted laser-desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) instruments. More than 40 peptides below 2500 Da could be detected, among them 20 were shown to belong to the BPP-like family including the related tripeptides pGlu-Lys-Trp and pGlu-Asn-Trp. A total of 15 new sequences have been identified using this approach.     

Antioxidant activity and ultra-performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry for phenolics-based fingerprinting of Rose species: Rosa damascena, Rosa bourboniana and Rosa brunonii

Roses are one of the most important groups of ornamental plants and their fruits and flowers are used in a wide variety of food, nutritional products and different traditional medicines. The antioxidant activity of methanolic extracts from fresh flowers of three rose species (Rosa damascena, Rosa bourboniana and Rosa brunonii) was evaluated by 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-radical method. The ability to scavenge DPPH radical was measured by the discoloration of the solution. The methanolic extract from R. brunonii exhibited maximum free-radical-scavenging activity (64.5 ± 0.38%) followed by R. bourboniana (51.8 ± 0.46%) and R. damascena (43.6 ± 0.25%) at 100 μg/ml. Simultaneously, ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) was used to study phenolic composition in the methanolic extracts from the fresh flowers of rose species. The phenolic constituents were further investigated by direct infusion-ESI-QTOF-MS/MS in negative ion mode. Characteristic Electrospray ionization tandem mass spectrometry (ESI-MS/MS) spectra with other diagnostic fragment ions generated by retro Diels–Alder (RDA) fragmentation pathways were recorded for the flavonoids. Distinct similarities were observed in the relative distribution of polyphenolic compounds among the three species. The dominance of quercetin, kaempferol and their glycosides was observed in all the three species.     

Structural identification by mass spectrometry of a novel antimicrobial peptide from the venom of the solitary bee Osmia rufa (Hymenoptera: Megachilidae)

The venom from the solitary bee Osmia rufa (Hymenoptera: Megachilidae) was analyzed using mass spectrometry (MS)-based techniques. Sensitive proteomic methods such as on-line LC-ESI-MS and nanoESI-MS analyses revealed more than 50 different compounds with molecular masses ranging from 400 to 4000 Da. The major component has a monoisotopic molecular mass of 1924.20 Da and its amino acid sequence was elucidated by de novo sequencing using tandem mass spectrometry and Edman degradation. This 17-residue cysteine-free peptide, named osmin, shows some similarities with the mast cell degranulation (MCD) peptide family. Free acid and C-terminally amidated osmins were chemically synthesized and tested for antimicrobial and haemolytic activities. The synthetic C-amidated peptide (native osmin) was found to be about three times more haemolytic than its free acid counterpart, but both peptides are much less lytic than melittin from social bee venom. Preliminary antimicrobial and antifungal tests indicate that both peptides are able to inhibit bacterial and fungal growth at micromolar concentrations.     

Molecular cloning of toxins expressed by the venom gland of Lasiodora sp.

The present work describes the identification of toxins expressed by the venom gland of the spider Lasiodora sp. The toxins LTx1, LTx2 and LTx3 were identified by the screening of a cDNA library. These toxins showed significant similarity at the amino acid level with spider toxins from Lasiodora parahybana,Eurypelma californicum, Brachypelma smithii, Selenocosmia huwena.     

Effects of the venom of a Mygalomorph spider (Lasiodora sp.) on the isolated rat heart.

We studied the effect of the venom of the Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae), on force generation and electrical activity in the isolated rat heart. Previous work showed that this venom is excitotoxic to excitable cells due to Na(+) channel gating modifier activity [Toxicon 39 (2001) 991]. In the isolated heart, the venom (10-100 microg bolus administration) caused a dose-dependent bradycardia, with transient cardiac arrest and rhythm disturbances. The electrocardiogram showed that the reduction of heart rate was due to sinus bradycardia, sinus arrest and partial or complete A-V block. All of the effects were reversible upon washout of the venom. The effect of the venom was potentiated by the anticholinesterase neostigmine (3.3 microM), suppressed by the muscarinic acetylcholine receptor antagonist atropine (1.4 microM), and inhibited by the vesicular acetylcholine transporter inhibitor (-)-vesamicol (10 microM). Tetrodotoxin (200 nM) did not inhibit the effect of the venom. Together, these data suggest that this Lasiodora venom evokes vesicular release of acetylcholine from parasympathetic nerve terminals by activating tetrodotoxin-resistant Na(+) channels.     

Characterisation of major peptides in 'jack jumper' ant venom by mass spectrometry.

The jack jumper ant, Myrmecia pilosula, is endemic to South-Eastern Australia, where around 2.7% of the population has a history of systemic allergic reactions (anaphylaxis) to its venom. Previous work had indicated that there were several allergenic peptides derived from the cDNA Myr p 1, the major expressed allergenic product being a 56-residue peptide (Myr p 1 57-->112, 'pilosulin 1', approximately 6052 Da). Another major allergen had been described as a 27 residue peptide derived from the cDNA Myr p 2 (Myr p 2 49-->75, 'pilosulin 2', approximately 3212 Da), possibly existing as part of a disulfide complex. As a preliminary step in detailed stability studies of a pharmaceutical product used for venom immunotherapy, LC-MS and Edman sequencing analysis of venom collected from various locations by both electrical stimulation and venom sac dissection was undertaken. More than 50 peptides in the 4-9 kDa range were detected in LC-MS analyses. A subsequence of Myr p 2 was found as part of the major peptide present in all samples; this was a bis-disulphide linked, antiparallel aligned heterodimer consisting of Myr p 2 49-->74, (des-Gly(27)-pilosulin 2, approximately 3155 Da) and a previously unreported peptide of approximately 2457 Da. Pilosulin 1 was found by a combination of tandem mass spectrometry and Edman sequencing to exist mainly, and sometimes exclusively, as a previously unreported approximately 6067 Da variant, in which the valine at residue 5 is replaced by isoleucine. A range of hydrolysis products of [Ile(5)]pilosulin 1 and pilosulin 1 were also detected in partially degraded venom. Further IgE-binding studies using these peptides are warranted and a revision of the nomenclature of allergenic components of M. pilosula venom may be required to conform with established IUIS guidelines.