Rapid Profiling of Alkaloids in Several Medicinal Herbs by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

A simple method was developed for rapid and direct profiling of alkaloids in medical herbs via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF MS）. The dry herbs were first ground to powder and passed through a stainless steel sieve, mixed with the matrix solution to form a homogeneous suspension, which was then directly applied to MALDI analysis. Several matrices were investigated and 2,5- dihydroxybenzoic acid（DHB） was chosen as the optimized one, and the particle with small size was found to favor the analysis. Using this method, the profiles of alkaloids in several medical herbs were readily obtained, and the toxicities of crude and processed Radix Aconiti Lateralis Preparata were compared via the relative intensities of the peaks of the corresponding toxic components shown in their MALDI spectra. This method therefore provides a rapid and reliable protocol for obtaining profiles of alkaloids in medical herbs by using MALDI-TOF MS.

Analysis of Single Nucleotide Polymorphism in Human Paraoxonase 1 Gene(Q192R) with Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Introduction Single nucleotide polymorphisms （SNPs） are the most abundant DNA markers in the human genome occurring at a frequency of one in every 500--1000 nucleotides. A variety of methods have been used for the analysis of single nucleotide polymorphisms, including restriction fragment length polymorphism （RFLP）, direct sequencing by using laser-induced fluorescence detectionTM, fluorescence energy transfer, MALDI-TOF MS combined with primer extension or invasive cleavage, and fluorescence polarization. During the past two decades, mass spectrometry has become a very popular tool in the analysis of biomolecules and is perfectly suited to the analysis of single nucleotide polymorphisms （SNPs） due to its speed, low cost, and accuracy. In this work, we used MALDI TOF mass spectrometry to detect the fragments of restriction endonuclease hydrolysis of PCR products flanking a SNP located at paraoxonase 1（Q192R）. Compared with electrophoresis, this method requires less time of analysis and possess a higher accuracy.

A novel sample preparation method of matrix-assisted laser desorption/ionization time of flight mass spectrometry for polystyrene

A novel sample preparation method of matrix-assisted laser desorption/ionization mass spectrometry for polystyrene was reported. Compared to the conventional dried-droplet method, the efficiency of ionization and signal intensity of mass spectra were improved. The mechanism was also analyzed.

MicrobMatcher: a microbial comparison software based on matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry

Matrix-assisted Laser Desorption/Ionization with Time-of-flight Mass Spectrometry (MALDI-TOFMS) was investigated as a method for the rapid identifica-tion of species. Current demand in microbial identi-fication is how to compare unknown strains to the known one quickly, semi-automatically and accurately. In this paper, we present a software tool that allows flexibly microbial matching in a user-friendly way, by letting the users to customize comparison parameters including: in vitro transcription enzyme, mass tolerance,minimum fragment length, intensity threshold and corresponding weights. We provide three spectral scoring functions to compute the affin-ity between the species. Therefore, the precision of microbial comparison increases. To test and verify this tool, we employed experimental spectral data based on MALDI-TOFMS and the gene sequences of E.coli and Salmonella. This software is written in Java for cross-platform intention.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry traces the geographical source of Biomphalaria pfeifferi and Bulinus forskali,involved in schistosomiasis transmission

Background Freshwater snails of the genera Bulinus spp.,Biomphalaria spp.,and Oncomelania spp.are the main intermediate hosts of human and animal schistosomiasis.Identification of these snails has long been based on mor-phological and/or genomic criteria,which have their limitations.These limitations include a lack of precision for the morphological tool and cost and time for the DNA-based approach.Recently,Matrix-Assisted Laser Desorp-tion/lonization Time-Of-Flight(MALDI-TOF)mass spectrometry,a new tool used which is routinely in clinical microbi-ology,has emerged in the field of malacology for the identification of freshwater snails.This study aimed to evaluate the ability of MALDI-TOF MS to identify Biomphalaria pfeifferi and Bulinus forskali snail populations according to their geographicalorigin.Methods This study was conducted on 101 Bi.pfeifferi and 81 Bu.forskali snails collected in three distinct geo-graphical areas of Senegal(the North-East,South-East and central part of the country),and supplemented with wild and laboratory strains.Specimens which had previously been morphologically described were identified by MALDl-TOF MS[identification log score values(LSV)≥1.7],after an initial blind test using the pre-existing database.After DNA-based identification,new reference spectra of Bi.pfeiferi(n=10)and Bu.forskali(n=5)from the geographical areas were added to the MALDI-TOF spectral database.The final blind test against this updated database was per-formed to assess identification at the geographic source level.Results MALDI-TOF MS correctly identified 92.1%of 101 Bi.pfeifferi snails and 98.8%of 81 Bu.forskali snails.At the final blind test,88%of 166 specimens were correctly identified according to both their species and sampling site,with LSVs ranging from 1.74 to 2.70.The geographical source was adequately identified in 90.1%of 91 Bi.pfeifferi and 85.3%of 75 Bu.forskalii samples.Conclusions Our findings demonstrate that MALDI-TOF MS can identify and differentiate snail populations according to geographical origin.It outperforms the current DNA-based approaches in discriminating laboratory from wild strains.This inexpensive high-throughput approach is likely to further revolutionise epidemiological studies in areas which are endemic for schistosomiasis.

The isolation and identification of apolipoprotein C-I in hormone-refractory prostate cancer using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry

Androgens play a central role in prostate cancer pathogenesis, and hence most of the patients respond to androgen deprivation therapies. However, patients tend to relapse with aggressive prostate cancer, which has been termed as hormone refractory. To identify the proteins that mediate progression to the hormone-refractory state, we used protein-chip technology for mass profiling of patients＇ sera. This study included 16 patients with metastatic hormone-refractory prostate cancer who were initially treated with androgen deprivation therapy. Serum samples were collected from each patient at five time points： point A, pre-treatment; point B, at the nadir of the prostate- specific antigen （PSA） level; point C, PSA failure; point D, the early hormone-refractory phase; and point E, the late hormone-refractory phase. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, we performed protein mass profiling of the patients＇ sera and identified a 6 640-Da peak that increased with disease progression. Target proteins were partially purified, and by amino acid sequencing the peak was identified as a fragment of apolipoprotein C-I （ApoC-I）. Serum ApoC-I protein levels increased with disease progression. On immunohistochemical analysis, the ApoC-i protein was found localized to the cytoplasm of the hormone-refractory cancer cells. In this study, we showed an increase in serum ApoC-I protein levels in prostate cancer patients during their progression to the hormone-refractory state, which suggests that ApoC-I protein is related to progression of prostate cancer. However, as the exact role of ApoC-I in prostate cancer pathogenesis is unclear, further research is required.

Detection of nasopharyngeal carcinoma using surface-enhanced laser desorption and ionization mass spectrometry profiles of the serum proteome

Background and Objective: Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult due to the insufficient specificity of the conventional examination method. This study was to investigate potential and consistent biomarkers for NPC, particularly for early detection of NPC. Methods: A proteomic pattern was identified in a training set (134 NPC patients and 73 control individuals) using the surface-enhanced laser desorption and ionization-mass spectrometry (SELDI-MS), and used to screen the test set (44 NPC patients and 25 control individuals) to determine the screening accuracy. To confirm the accuracy, it was used to test another group of 52 NPC patients and 32 healthy individuals at 6 months later. Results: Eight proteomic biomarkers with top-scored peak mass/charge ratios (m/z) of 8605 Da, 5320 Da, 5355 Da, 5380 Da, 5336 Da, 2791 Da, 7154 Da, and 9366 Da were selected as the potential biomarkers of NPC with a sensitivity of 90.9% (40/44) and a specificity of 92.0% (23/25). The performance was better than the current diagnostic method by using the Epstein-Barr virus (EBV) capsid antigen IgA antibodies (VCA/IgA). Similar sensitivity (88.5%) and specificity (90.6%) were achieved in another group of 84 samples. Conclusion: SELDI-MS profiling might be a potential tool to identify patients with NPC, particularly at early clinical stages.

Two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry for detection of protein expression profiles in the hippocampus following closed brain injury

Gene expression profile changes in brain regions following traumatic brain injury at the gene level cannot sufficiently elucidate gene expression time, expression amount, protein post-translational processing or modification. Therefore, it is necessary to quantitatively analyze the gene expression profile using proteomic techniques. In the present study, we established a rat model of closed brain injury using Marmarou＇s weight-drop device, and investigated hippocampal differential protein expression using two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry. A total of 364 protein peaks were detected on weak cation exchange-2 protein chips, including 37 differential protein peaks. 345 protein peaks were detected on immobilized metal affinity capture arrays-Cu, including 12 differential protein peaks Further examination of these differential proteins revealed that glucose-regulated protein and proteasome subunit alpha type 3 expression were significantly upregulated post-injury. These results indicate that brain injury can alter protein expression in the hippocampus, and that glucose-regulated protein and proteasome subunit alpha type 3 are closely associated with the occurrence and development of traumatic brain injury.

Plasmonic polydopamine-modified TiO_(2)nanotube substrates for surface-assisted laser desorption/ionization mass spectrometry imaging

Mass spectrometry imaging(MSI)has made the spatio-chemical characterization of a broad range of small-molecule metabolites within biological tissues possible.However,available matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS)suffers from severe background interferences in low-mass ranges and inhomogeneous matrix deposition.Thus,surface-assisted LDI-MS(SALDI-MS)has been an attractive alternative for high-sensitivity detection and imaging of small biomolecules.In this study,we construct a new composite substrate,hydrophobic polydopamine(hPDA)-modified TiO_(2)nanotube(TDNT)coated with plasmonic gold nanoparticle(AuNP-hPDA-TDNT),as a dual-polarity SALDI substrate using an easy and cost-effective fabrication approach.Benefitting from the synergistic effects of TDNT semiconductor and plasmonic PDA modification,this SALDI substrate exhibits superior performance for dual-polarity detection of a vast diversity of small molecules.Highly reduced background interferences,lower detection limits,and spot-to-spot repeatability can be achieved using AuNP-hPDA-TDNT substrates.Due to its unique imprinting performance,various metabolites and lipids can be visualized within jatropha integerrima petals,ginkgo leaves,strawberry fruits,and latent fingerprints.More valuably,the universality of this matrix-free substrate is demonstrated for mapping spatial distribution of lipids within mouse brain tissue sections.Considered together,this AuNP-hPDA-TDNT material is expected to be a promising SALDI substrate in various fields,especially in nanomaterial development and life sciences.

Detection of lung adenocarcinoma using magnetic beads based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry serum protein profiling

Background Recently, due to the rapid development of proteomic techniques, great advance has been made in many scientific fields. We aimed to use magnetic beads （liquid chip） based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） technology to screen distinctive biomarkers for lung adenocarcinoma （adCA）, and to establish the diagnostic protein profiles. Methods Using weak cation exchange magnetic beads （MB-WCX） to isolate and purify low molecular weight proteins from sera of 35 lung adCA, 46 benign lung diseases （BLDs） and 44 healthy individuals. The resulting spectra gained by anchor chip-MALDI-TOF-MS were analyzed by ClinProTools and a pattern recognition genetic algorithm （GA）. Results In the working mass range of 800-10 000 Da, 99 distinctive peaks were resolved in lung adCA versus BLDs, while 101 peaks were resolved in lung adCA versus healthy persons. The profile gained by GA that could distinguish adCA from BLDs was comprised of 4053.88, 4209.57 and 3883.33 Da with sensitivity of 80%, specificity of 93%, while that could separate adCA from healthy control was comprised of 2951.83 Da and 4209.73 Da with sensitivity of 94%, specificity of 95%. The sensitivity provided by carcinoembryonic antigen （CEA） in this experiment was significantly lower than our discriminatory profiles （P 〈0.005）. We further identified a eukaryotic peptide chain release factor GTP-binding subunit （eRF3b） （4209 Da） and a complement C3f （1865 Da） that may serve as candidate biomarkers for lung adCA. Conclusion Magnetic beads based MALDI-TOF-MS technology can rapidly and effectively screen distinctive proteins/polypeptides from sera of lung adCA patients and controls, which has potential value for establishing a new diagnostic method for lung adCA.

Serum proteomic profiling for autism using magnetic bead-assisted matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a pilot study

Background The pathogenesis of autism spectrum disorders remains elusive and currently there are no diagnostic or pre-dictive biomarkers in autism available. Proteomic profiling has been used in a wide range of neurodevelopmental disorder studies, which could produce deeper perceptions of the molecular bases behind certain disease and potentially becomes useful in discovering biomarkers in autism spectrum disorders. Methods Serum samples were collected from autistic children about 3 years old in age (n = 32) and healthy controls (n = 20) in similar age and gender. The samples were identified specific proteins that are diff erentially expressed by magnetic bead-based pre-fractionation and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS). Results Eight protein peaks were significantly different in autistic children from the healthy controls (P < 0.0001). The two peaks with the most significant diff erences were 6428 and 7758 Da in size. Conclusion According to diff erences in serum protein profiles between the autistic children and healthy controls, this study identified a set of diff erentially expressed proteins those are significant for further evaluation and might function as biomark-ers in autism.

Studies on extracting solutions of endohedral rare-earth metallofullerenes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Thirteen extracting solutions of rare-earth metallofullerenes containing La,Ce,Pr,Nd Sm,Eu,Gd,Tb,Dy,Ho,Er,Tm and Yb respectively have been investigated by means of matrix-assisted laser desorpuon/ ionization time-of-flight mass spectrometry.The influences of the positive-ion/negative-ion mode,laser intensity,ma trix and mass discrimination to the analytical results are studied,based on which the optimal analytical conditions have been determined.The results show that the extracting solutions contain large quantities of rare-earth metallofullerenes besides empty fullerenes.On the basis of comparing their relative intensities,the different structure stabilities and solubilities of metallofullerenes with different rare-earth metals encapsulated into the fullerene cages,as well as some possible reasons to those differences,are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry in the genetic basis of systemic lupus erythematosus and the complicating kidney injury

Background It is necessary to develop some innovative methods to reveal and discover the novel （SLE）-related protein molecules. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI- TOF MS） was employed to detect the differential expression of serum polypeptides in the patients with systemic lupus erythematosus （SLE） presenting with a family history or complicating with kidney injury so as to identify the proteins associated with the genetic factors and kidney injury in SLE. Methods The subjects recruited were divided into four groups, that is, a group of SLE patients with both family history and kidney injury, a group of SLE patients with only kidney injury but no family history, a group of SLE patients with neither family history nor kidney injury, and a control group consisting of healthy volunteers. By adopting MALDI-TOF MS analysis, the serum samples obtained from the three groups of SLE patients were examined and compared with those from the control group; the categorized peptide fingerprint profile was established via the biological data collected from the samples. Results The expression of protein with a mlz of 4207 Da increased significantly in SLE patients; the protein with a ml z of 2658 Da was expressed in all SLE patients; three proteins （with mlz of 1465, 5332, and 5900 Da respectively） were expressed in the SLE patients complicated with kidney injury and the protein with a mlz of 1943 Da was expressed in SLE patients with family history. Conclusion A number of differential proteins were successfully detected and identified through MALDI-TOF MS detection and these proteins may be associated with the genetic basis of SLE and the complicating kidney injury.

Detection of Sialylated N-Linked Glycans by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Native and methyl-esterified sialylated glycans were analyzed with 2,4,6-trihydroxyacetophenone（THAP）and 2,5-dihydroxybenzoic acid（DHB）as matrix by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer（MALDI-TOF MS）.High quality negative-ion spectra of commercial sialylated glycan were obtained with THAP as matrix.Detection limit of the glycan was less than 0.1 pmol.After methyl esterification of sialic acid（SA）residue,sialylated glycans were detected sensitively in the positive-ion mode using DHB as matrix.Neutral and sialylated glycans from the mixture of asialofetuin and fetuin were methylesterified and simultaneously recognized in one manipulation.Methyl esterification of SA residue offers a convenient and sensitive way to identify the structure of N-linked glycans for glycan profiling.

MALDI coupled with laser-postionization and trapped ion mobility spectrometry contribute to the enhanced detection of lipids in cancer cell spheroids

Cancer cell spheroids(CCS) are a valuable three-dimensional cell model in cancer studies because they could replicate numerous characteristics of solid tumors. Increasing researches have used matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) to investigate the spatial distribution of endogenous compounds(e.g., lipids) in CCS. However, only limited lipid species can be detected owing to a low ion yield by using MALDI. Besides, it is still challenging to fully characterize the structural diversity of lipids due to the existence of isomeric/isobaric species. Here, we carried out the initial application of MALDI coupled with laser-postionization(MALDI-2) and trapped ion mobility spectrometry(TIMS) imaging in HCT116 colon CCS to address these challenges. We demonstrated that MALDI-2 is capable of detecting more number and classes of lipids in HCT116 colon CCS with higher signal intensities than MALDI. TIMS could successfully separate numerous isobaric/isomeric species of lipids in CCS. Interestingly, we found that some isomeric/isobaric species have totally different spatial distributions in colon CCS. Further MS/MS imaging analysis was employed to determine the compositions of fatty acid chains for isomeric species by examining disparities in signal intensities and spatial distributions of product ions. This work stresses the robust ability of TIMS and MALDI-2 imaging in analyzing endogenous lipids in CCS, which could potentially become powerful tools for future cancer studies.

Identification of serum biomarkers for diagnosing stage Ⅰ lung adenocarcinoma by MALDI-TOF mass spectrometry

Objective To identify specific biomarkers that could improve early diagnosis of lung adenocarcinoma using matrix-assisted laser desorption/ionization (MALDI) technology. Methods Serum samples were isolated from 17 patients with stage Ⅰ lung adenocarcinoma and 17 age-and sex-matched healthy controls,and the serum proteomic profiles were obtained by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Results Compared with healthy control group,two highly expressed potential biomarkers were identified with the relative molecular weights of 6 631.64 Da and 4 964.21 Da. The two best novel protein peaks were automatically chosen for the system training and the development of the constructed model. The constructed model was then used to test an independent set of masked serum samples from 15 lung adenocarcinoma patients and 22 healthy individuals. The analysis yielded a sensitivity of 93.3%,and a specificity of 95.5%. Conclusion These results suggest that MALDI-TOF-MS ProteinChip technology is a quick,convenient,and high-output analyzing method that is capable of selecting several relatively potential biomarkers from the serum of lung adenocarcinoma patients and may have a clinical value in the future,and will provide clues to identifying new serologic biomarkers of lung adenocarcinoma.

Establishment and Preliminary Application of Two dimensional Electrophoresis and Mass Spectrometry in Ovary Study

Objective To apply two-dimensional electrophoresis and mass spectrometry in the ovary proteome researchMethods Protein extractions from mouse ovaries were run in IPGphor isoelectric focus system with 11 cm and 24 cm IPG strips respectively (pH 3～10, 0.3 mm thick), then the protein spots were identified by mass spectrometry.Results The ovary protein exactions separated by two-dimensional electrophoresis have got high resolution, and identifing protein by mass spectrometry was highly efficient and facilitly. These two techniques should facilitate further investigation of female reproduction proteome research.Conclusion These two rapid high resolutions and efficient techniques have a variety of applications foreground in female reproduction proteome pattern research.

A Plasmonic Mass Spectrometry Approach for Detection of Small Nutrients and Toxins

Nutriology relies on advanced analytical tools to study the molecular compositions of food and provide key information on sample quality/safety. Small nutrients detection is challenging due to the high diversity and broad dynamic range of molecules in food samples, and a further issue is to track low abundance toxins. Herein, we developed a novel plasmonic matrix-assisted laser desorption/ionization mass spectrometry(MALDI MS)approach to detect small nutrients and toxins in complex biological emulsion samples. Silver nanoshells(SiO_2@-Ag) with optimized structures were used as matrices andachieved direct analysis of ~ 6 n L of human breast milk without any enrichment or separation. We performed identification and quantitation of small nutrients and toxins with limit-of-detection down to 0.4 pmol(for melamine) and reaction time shortened to minutes, which is superior to the conventional biochemical method currently in use. The developed approach contributes to the near-future application of MALDI MS in a broad field and personalized design of plasmonic materials for real-case bio-analysis.

Surface-assisted laser desorption/ionization mass spectrometry for drug and metabolite analysis

Surface-assisted laser desorption/ionization mass spectrometry(SALDI-MS)uses inorganic nanomaterials as matrixes to facilitate desorption and ionization of analytes.Compared with the traditional matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS)technique,SALDI-MS has the advantages of less interference in the low mass range,better reproducibility and higher salt tolerance.It is highly suitable for the analysis of small molecule compounds.In recent years,researchers have developed a range of nanomaterials that are successfully applied to the field of small molecule drug and metabolite analysis including drug screening and quantification,drug delivery,metabolite profiling,biomarker discovery and so forth.This review summarizes the latest progress of SALDI-MS matrix materials such as metal-based,carbon-based,silica-based nanomaterials and organic framework nanomaterials and their applications.In addition,our perspective of SALDI-MS technology is also discussed for further advancement.

Recent Advances in Surface-Assisted Laser Desorption/Ionization Mass Spectrometry and Its Imaging for Small Molecules

Mass spectrometry imaging(MSI)has provided a new perspective on acquiring spatial information of multiple molecules in various samples.Among the different ionization methods,matrix-assisted laser desorption/ionization(MALDI)has been widely utilized for detecting macromolecules,with difficulty for small molecules(m/z<700 Da)due to the matrix interference or ionization suppression.In the past two decades,surface-assisted laser desorption/ionization mass spectrometry(SALDI-MS)gives rise to lots of attention on account of its unique performances,especially in untargeted analysis of small molecules.Selecting an appropriate substrate is a precondition for SALDI-MS and offers the possibility for SALDI-MS imaging(SALDI-MSI).In the last 5 years,different kinds of nanomaterials have been widely explored as substrates including metal/metal oxide-based,carbon-based,silicon-based,metal-organic frameworks-based,covalent organic frameworksbased substrates,with growing interests on composite materials and nanomaterials with homogeneous film structure.This review highlights recent advances of various nanomaterials as SALDI substrates,and their emerging imaging applications in botanic,forensic,metabolic and pathological fields.Finally,the merits and limitations of SALDI-MS are sketched out and some recommendations of this technique and its imaging are proposed.