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.

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.

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.

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.

Nocardia cyriacigeorgica infection in a patient with repeated fever and CD4+T cell deficiency:A case report

BACKGROUND Nocardia pneumonia shares similar imaging and clinical features with pulmonary tuberculosis and lung neoplasms,but the treatment and anti-infective medication are completely different.Here,we report a case of pulmonary nocardiosis caused by Nocardia cyriacigeorgica(N.cyriacigeorgica),which was misdiagnosed as community-acquired pneumonia(CAP)with repeated fever.CASE SUMMARY A 55-year-old female was diagnosed with community-acquired pneumonia in the local hospital because of repeated fever and chest pain for two months.After the anti-infection treatment failed in the local hospital,the patient came to our hospital for further treatment.Enhanced computed tomography showed multiple patchy,nodular and strip-shaped high-density shadows in both lungs.A routine haematological examination was performed and showed abnormalities in CD19+B cells and CD4+T cells.Positive acid-fast bifurcating filaments and branching gram-positive rods were observed in the bronchoalveolar lavage fluid of the patient under an oil microscope,which was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry as N.cyriacigeorgica.The patient’s condition quickly improved after taking 0.96 g compound sulfamethoxazole tablets three times a day.CONCLUSION The antibiotic treatment of Nocardia pneumonia is different from that of common CAP.Attention should be given to the pathogenic examination results of patients with recurrent fever.Nocardia pneumonia is an opportunistic infection.Patients with CD4+T-cell deficiency should be aware of Nocardia infection.

Elizabethkingia miricola :A rare non-fermenter causing urinary tract infection

Elizabethkingia miricola(E.miricola) is a gram-negative non-fermentative bacterium which is rarely encountered.It is usually misidentified or considered as a contaminant in routine microbiology laboratories due to the limitations in conventional biochemical techniques.However,with the advent of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOFMS),the identification of non-fermenters has become easy and this has led to enhanced understanding of the clinical significance of these uncommonly isolated microorganisms.The genus Elizabethkingia has only two species E.meningoseptica and E.miricola.Both of these organisms are known to be multi-drug resistant and therefore,their accurate identification and antimicrobial susceptibility testing are necessary prior to the initiation of appropriate therapy.In the world literature till date,only 3 cases of sepsis caused by E.miricola have been reported.We present the first case of E.miricola association with urinary tract infection.

利用基质辅助激光解吸电离飞行时间质谱快速鉴定产气荚膜梭菌

分别采用基质辅助激光解吸电离飞行时间质谱方法(matrix-assisted laser desorption/ionization time-offlight mass spectrometry,MALDI-TOF MS)、16S rRNA基因测序、VITEK 2 COMPACT和传统生化方法(牛乳汹涌发酵实验)鉴定实验室保存的51株产气荚膜梭菌,以评估MALDI-TOF MS方法是否适用于产气荚膜梭菌的快速鉴定。MALDI-TOF MS方法将51株菌均鉴定为产气荚膜梭菌且鉴定分值在2. 300以上,达到鉴定到种的要求。16S rRNA基因序列比对结果与MALDI-TOF MS方法结果一致。VITEK 2 COMPACT将49株菌鉴定为产气荚膜梭菌,2株菌未能成功鉴定。47株菌的牛乳汹涌发酵实验呈阳性,4株菌为牛乳汹涌发酵实验阴性,且这4株菌经其他3种方法均鉴定为产气荚膜梭菌。结果表明,无论是牛乳汹涌发酵实验还是VITEK 2 COMPACT,在检测产气荚膜梭菌时都有可能发生漏检。相比牛乳汹涌发酵实验、VITEK 2 COMPACT和16S rRNA测序方法,MALDI-TOF MS方法更加快速、准确和低成本,适用于产气荚膜梭菌的快速鉴定。

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.

Lipopeptide Antibiotics Produced by the Engineered Strain Bacillus subtilis GEB3 and Detection of Its Bioactivity

MALDI-TOF-MS technology was used for identification of lipopeptide antibiotics producedby GEB3 strain, a derivative of Bacillus subtilis 168 which was transformed by lpaB3gene. The result showed GEB3 only produced lipopeptide antibiotic surfactin. The analysisby LC-MS demonstrated that GEB3 produced standard surfactin isoforms with side chainlengths of 13,14 and 15 carbon atoms. The bioactivity detection of surfactin indicatedthat the surfactin produced by GEB3 had inhibition effect on plant pathogens Rhizoctoniasolani and Pyricularia oryzae.

C_(60)衍生物的MALDI-TOFMS分析

C60衍生物在超导、非线性光学、催化、材料和生物活性等方面有巨大的潜在应用价值.C60衍生物大多为固体,蒸汽压较低,采用需要加热才能够使样品气化电离的质谱或"硬"电离质谱方法进行测定,易造成C60衍生物分解并释放出配体.……

Insights into glycan biosynthesis in chemically-induced hepatocellular carcinoma in rats: A glycomic analysis

AIM： To evaluate the qualitative and quantitativechanges in N-linked glycosylation, which occurredin association with diethyl nitrosamine-inducedhepatocellular carcinoma （HCC） in rodents.METHODS： Liver tissues of （1） normal （non-tumorbearing）rats; and （2） tumor-bearing rats; were collectedand were used for histological and GlycanMap？ analyses.Briefly, GlycanMap？ analysis is a high-throughputassay that provides a structural and quantitativereadout of protein-associated glycans using a unique,automated 96-well assay technology coupled tomatrix-assisted laser desorption/ionization time-offlightmass spectrometry and custom bioinformatics.Histopathological studies were carried out to ensure thedevelopment of HCC in the tested animals.RESULTS： The N-glycomic analysis revealed 5glycans; Glc1Man9GlcNAc2, Gal2Man3GlcNac4Fuc1Neu1,Man4G l c N a c 2, G a l 2Man3G l c N a c 4Neu3OAc 3, andMan3GlcNac5Fuc1, which showed significant changesin rat HCC tissues when compared with normal livertissues. Four glycans were increased （P 〈 0.05） andGlc1Man9GlcNAc2 was decreased （5.89 ± 0.45 vs 3.54± 0.21, P 〈 0.01） in HCC tissues compared to normal liver tissues. An increase （66.5 ± 1.05 vs 62.7 ± 1.1,P 〈 0.05） in high-mannose structures in HCC rats wasobserved compared to normal rats. Importantly, HCCrats showed an increase （P 〈 0.05） in both tumorassociatedcarbohydrates and in branched glycans. Thechanges in glycans correlated well with glycan flowchanges reported in the glycan biosynthetic pathway,which indicates the importance of enzyme activitiesinvolved in glycan synthesis at different subcellularlocalizations.CONCLUSION： The reported HCC-associated changesin glycan flow and subcellular localization explain theincrease in high mannose glycans and siayl Lewisglycans common in HCC liver tissues.

Radial Basis Function Networks Applied in Bacterial Classification Based on MALDI-TOF-MS

The radial basis function networks were applied to bacterial classification based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) data. The classification of bacteria cultured at different time was discussed and the effect of the network parameters on the classification was investigated. The cross-validation method was used to test the trained networks. The correctness of the classification of different bacteria investigated changes in a wide range from 61.5% to 92.8%. Owing to the complexity of biological effects in bacterial growth, the more rigid control of bacterial culture conditions seems to be a critical factor for improving the rate of correctness for bacterial classification.

Maternal peripartum bacteremia caused by intrauterine infection with Comamonas kerstersii:A case report

BACKGROUND Comamonas kerstersii(C.kerstersii)infections have considered as non-pathogenic to humans,however due to new techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF-MS),more cases have been identified.CASE SUMMARY This is the first report of a maternal patient with a C.kerstersii bacteremia following caesarean section.Due to the severity of the patient’s condition;high fever and rapidly progressing organ damage,the patient was transferred to the intensive care unit.C.kerstersii was detected by metagenomic next-generation sequencing testing.Based on the drug sensitivity test,appropriate antibiotic treatment was given and the patient recovered fully.CONCLUSION This case report confirms that the detection via MALDI-TOF-MS and metagenomic next-generation sequencing testing provides a reliable basis for the diagnosis of this rare bacterial infection.

Obligate aerobic, gram-positive, weak acid-fast, nonmotile bacilli, Tsukamurella tyrosinosolvens: Minireview of a rare opportunistic pathogen

Tsukamurella species are obligate aerobic,gram-positive,weak acid-fast,nonmotile bacilli.They are found in various environments,such as soil,water,sludge,and petroleum reservoir wastewater,and belong to the order Actinomycetales.In 2016,there was a reclassification of species within the genus Tsukamurella,merging the species Tsukamurella tyrosinosolvens(T.tyrosinosolvens)and Tsukamurella carboxydivorans.Tsukamurella species are clinically considered to be a rare opportunistic pathogen,because most reported cases have been related to bacteremia and intravascular prosthetic devices and immunosuppression.To date,it has been isolated only from human specimens,and has always been associated with clinical disease;human infections are very rare.Reported infections have included pneumonia,brain abscesses,catheter-related bloodstream infections,ocular infections,bacteremia,and sepsis presenting with septic pulmonary emboli in patients who are immunocompromised.To date,there is no commercially available test for identification.On the other hand,sequence-based identification,including matrix-assisted laser desorption ionization time-of-flight mass spectrometry,is an alternative method for identifying clinical isolates that are either slow growers or difficult to identify through biochemical profiling.The golden standards for diagnosis and optimal management still remain to be determined.However,newer molecular biological techniques can provide accurate identification,and contribute to the appropriate selection of definitive therapy for infections caused by this organism.Combinations of several antimicrobial agents have been proposed for treatment,though the length of treatment for infections has yet to be determined,and should be individualized according to clinical response.Immunocompromised patients often experience severe cases due to infection,and life-threatening T.tyrosinosolvens events associated with dissemination and/or failure of source control have occurred.Favorable prognoses can be achieved through earlier identification of the cause of infection,as well as successful management,including appropriate antibiotic therapy together with source control.Further analyses of similar cases are required to establish the most adequate diagnostic methods and treatment regimens for infections.

Comparision of FAB and MALDI Mass Spectrometry of Ginsenosides

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Multiple skin abscesses associated with bacteremia caused by Burkholderia gladioli:A case report

BACKGROUND Burkholderia gladioli(B.gladioli)is regarded as a rare opportunistic pathogen.Only a few patients with abscesses caused by B.gladioli infections have been reported,and these are usually abscesses at the incision caused by traumatic surgery.CASE SUMMARY A 74-year-old male patient with abscesses and pain throughout his body for 1 mo was admitted to our hospital.Some of the abscesses had ruptured with purulent secretions on admission.Color Doppler ultrasound examination of the body surface masses showed mixed masses 75 mm×19 mm,58 mm×17 mm,17 mm×7 mm,and 33 mm×17 mm in size in the muscle tissues of both the right and left forearms,the posterior area of the right knee and the left leg,respectively.Abscess secretions and blood cultures grew B.gladioli.The following 3 methods were used to jointly identify the bacterium:an automatic microbial identification system,matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,and full-length 16 S rDNA sequencing.After 27 d of treatment with meropenem,etimicin,trimethoprim-sulfamethoxazole and other antibiotics,most of his skin abscesses were flat and he was discharged without any symptoms.CONCLUSION This is the first reported case of multiple skin abscesses associated with bacteremia caused by B.gladioli.Our study provides important reference values for the clinical diagnosis and treatment of B.gladioli infections.