Forward selection radial basis function networks applied to bacterial classification based on MALDI-TOF-MS

Forward selection improved radial basis function (RBF) network was applied to bacterial classification based on the data obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The classification of each bacterium cultured at different time was discussed and the effect of parameters of the RBF network was investigated. The new method involves forward selection to prevent overfitting and generalized cross-validation (GCV) was used as model selection criterion (MSC). The original data was compressed by using wavelet transformation to speed up the network training and reduce the number of variables of the original MS data. The data was normalized prior training and testing a network to define the area the neural network to be trained in, accelerate the training rate, and reduce the range the parameters to be selected in. The one-out-of-n method was used to split the data set of p samples into a training set of size p−1 and a test set of size 1. With the improved method, the classification correctness for the five bacteria discussed in the present paper are 87.5, 69.2, 80, 92.3, and 92.8%, respectively.     

Fe3O4/TiO2 纳米材料靶上脱盐的基质辅助激光解析/电离质谱新方法研究

建立了一种简便、高效的靶上脱盐新方法。利用Fe3O4/TiO2磁性纳米材料对肽段的吸附作用,将其作为载体用于靶上肽段富集和脱盐。在对纳米材料使用量、浸洗条件进行优化的基础上,成功地鉴定了溶于10mol/L尿素溶液的100fmol的肌红蛋白样品,也对溶于3mol/L尿素溶液中的10fmol的肌红蛋白样品进行了成功地分析鉴定。通过对肌红蛋白样品进行预处理和质谱分析重复实验,表明该方法重现性好,且简便、高效,所需时间短,一次可同时处理多个样品,易于实现通量化,为有效地解决目前蛋白质组分析中所面临的基质辅助激光解析/离子化飞行时间质谱耐盐性差的问题提供了一种新的手段。     

用基质辅助激光解吸电离飞行时间质谱法测定钙调蛋白的纯度与分子量

采用基质辅助激光解吸电离飞行时间质谱法测定了钙调蛋白的纯度与分子量,并对所得的结果进行了讨论,实验结果表明本方法具有灵敏度高,分析速度快,重复性好,信息直观等特点,是其他传统测定蛋白质分子量的方法无法比拟的。     

基体辅助激光解吸电离飞行时间质谱用于寡糖的分析

将基体辅助激光解吸电离飞行时间质谱这种新兴的质谱方法用于植物中寡糖的分析。比较了不同的样品制备方法和检测方法对分析结果的影响,给出各寡糖样品的分子量分布,单体和端基基团的分子量。     

Rapid fingerprinting of red wines by MALDI mass spectrometry

Here we report a simple and fast method for wine fingerprinting based on direct matrix-assisted laser desorption/ionization (MALDI) mass spectrometry analysis of different red wine samples, useful for batch-to-batch analysis and for the detection of key compounds even in trace amounts which may vary from vintage to vintage, and from one treatment to another one. A series of 20 samples from different wines were subjected to MALDI mass spectrometry. We found that 2,5-dihydroxybenzoic acid is far superior with respect to all the matrices tested To the best of our knowledge this is the first application of an effective wine profiling not limited to detection of anthocyanins. More than 80 molecular species were detected. Moreover, qualitative and quantitative differences were observed, owing to the nature and relative abundance of different chemical compounds among the wines.     

Online Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry with In situ Mixing

Online matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is reported with in situ matrix/analyte aerosol mixing. The analyte and matrix were dissolved in separate solvents and pneumatically atomized into particles. Continuous flows of analyte particles and matrix droplets in nitrogen were mixed in a tee. The resulting particles were sampled using a nozzle, focused into a beam with an aerodynamic lens system, and vertically deposited continuously on a movable target. The matrix/analyte mixture was desorbed/ionized using a 266 nanometers pulsed laser at an incident angle of 45°. The nascent ions were analyzed using reflectron TOF MS. The performance of online MALDI-TOF MS was evaluated by the analysis of palmityl palmitate with the lithium salt of 2, 4-dihydroxybenzoic acid as the matrix. Strong and stable MALDI signals of palmityl palmitate were obtained. The matrix solvent mixture and the analyte concentration were optimized and the results demonstrate the development of an alternative for online MALDI analysis.     

Improved peptide mass fingerprinting matches via optimized sample preparation in MALDI mass spectrometry

Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides resulting from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly charged ions, along with its relative insensitivity to salts and buffers, matrix-assisted laser desorption and ionization (MALDI)-time-of-flight mass spectrometry (TOFMS) is the MS method of choice for PMF. The qualitative features of the mass spectrum can be selectively tuned by employing different methods to prepare the protein digest and matrix for MALDI-TOFMS. The selective tuning of MALDI mass spectra in order to optimize PMF is addressed here. Bovine serum albumin, carbonic anhydrase, cytochrome c, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin and then analyzed by MALDI-TOFMS. 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) were prepared using six different sample preparation methods: dried droplet, application of protein digest on MALDI plate followed by addition of matrix, dried droplet with vacuum drying, overlayer, sandwich, and dried droplet with heating. Improved results were obtained for the matrix alpha-cyano-4-hydroxycinnamic acid using a modification of the died droplet method in which the MALDI plate was heated to 80 °C prior to matrix application, which is supported by observations from scanning electron microscopy. Although each protein was found to have a different optimum sample preparation method for PMF, in general higher sequence coverage for PMF was obtained using DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest was convolved together.     

Optimization of a modified aerospray deposition device for the preparation of samples for quantitative analysis by MALDI-TOFMS

A modified aerospray apparatus was used to prepare a thin layer sample of matrix and analyte for quantitative analysis by MALDI-TOFMS. The apparatus consists of a set of coaxial tubing; the liquid sample is forced by a syringe pump through the inner capillary and it is nebulized by a flow of gas through the outer capillary. The small droplets of sample exiting the device are deposited onto a rotating plate, which serves as the sample surface for a time-of-flight mass spectrometer. An optimization was carried out after initial experiments with the device resulted in poorer than expected reproducibility of analyte signal. A two-level plus center point factorial experiment was performed investigating several factors, including the inner capillary internal diameter, gas pressure, liquid flow, spray distance, and time. After optimization the within-sample reproducibility of the analyte signal improved 3-fold, while the sample-to-sample reproducibility improved 4.5-fold.     

Detection of Cholangiocarcinoma with Fourier Transform Infrared Spectroscopy and Radial Basis Function Neural Network Classification

The aim of this study was to explore the possibility of applying Fourier transform infrared(FTIR) spectroscopy as a medical diagnostic tool based on a neural network classifier for detecting and classifying cholangiocarcinoma. A total of 51 cases of bile duct tissues were obtained and later characterized by FTIR spectroscopy prior to pathological diagnosis. The criteria for classification included 30 parameters for each FTIR spectra, including peak position(P), intensity(I) and full width at half-maximum(FWHM), were measured, calculated and subsequently compared against the normal and cancer groups. The FTIR spectra were classified by the radial basis function(RBF) network model. For establishing the RBF, 23 cases were used to train the RBF classifier, and 28 cases were applied to validate the model. Using the RFB model, nine parameters were observed to be pronouncedly different between cancerous and normal tissue, including I₁₆₄₀, I₁₅₅₀, I₁₄₆₀, I₁₄₀₀, I₁₂₅₀, I₁₁₂₀, I₁₀₈₀, I₁₀₄₀ and P₁₀₄₀. In the RBF training classification, the accuracy, sensitivity, and specificity of diagnosis were 82.6%, 80.0%, and 84.6%, respectively. While validating the classification, the accuracy, sensitivity, and specificity of diagnosis were 78.6%, 75.0%, and 81.2%, respectively. The results suggest that FTIR spectroscopy combined with neural network classifier could be applied as a medical diagnostic tool in cholangiocarcinoma diagnosis.     

Determination of self-exchange rate of alkanethiolates in self-assembled monolayers on gold using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In this paper, we describe a new method for determining the exchange rates of alkanethiolates in self-assembled monolayers (SAMs) on gold using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the compositions of the alkanethiolate in SAMs rapidly and directly. In particular, to investigate the self-exchange of alkanethiols, we prepared a deuterated alkanethiol that has the same molecular properties as the non-deuterated alkanethiol but a different molecular weight. SAMs consisting of deuterated alkanethiolates were immersed in a solution of the non-deuterated alkanethiol, and the influences of the immersion time, temperature, concentration, and solvent on the self-exchange rates were investigated. Furthermore, we assessed the exchange rates among alkanethiols with different carbon chain lengths and different size of ethylene glycol units. In addition, we performed molecular dynamics simulations using a model SAM system in order to understand the molecular mechanism of the exchange process.     

Coffee-ring effects in laser desorption/ionization mass spectrometry

This report focuses on the heterogeneous distribution of small molecules (e.g. metabolites) within dry deposits of suspensions and solutions of inorganic and organic compounds with implications for chemical analysis of small molecules by laser desorption/ionization (LDI) mass spectrometry (MS). Taking advantage of the imaging capabilities of a modern mass spectrometer, we have investigated the occurrence of “coffee rings” in matrix-assisted laser desorption/ionization (MALDI) and surface-assisted laser desorption/ionization (SALDI) sample spots. It is seen that the “coffee-ring effect” in MALDI/SALDI samples can be both beneficial and disadvantageous. For example, formation of the coffee rings gives rise to heterogeneous distribution of analytes and matrices, thus compromising analytical performance and reproducibility of the mass spectrometric analysis. On the other hand, the coffee-ring effect can also be advantageous because it enables partial separation of analytes from some of the interfering molecules present in the sample. We report a “hidden coffee-ring effect” where under certain conditions the sample/matrix deposit appears relatively homogeneous when inspected by optical microscopy. Even in such cases, hidden coffee rings can still be found by implementing the MALDI-MS imaging technique. We have also found that to some extent, the coffee-ring effect can be suppressed during SALDI sample preparation.     

Combination of extraction tip and MALDI-TOF-MS for efficient separation and analysis of cysteine-containing peptides

In our work,a new extraction tip with gold-modified polymer is developed.The simple,self-made and extremely economical tips were successfully applied to capture cysteine-containing peptides.The loading capacity of a tip(column bed:0.3 mm diameter,5 mm length)is 2–4μg peptides.We can make one tip in 30 s and each costs less than 0.1 cent.The use of these tips can achieve a stable analysis with less background interference,even for 10 ng target peptides.Compared with other separation techniques,our method can save much time and energy while providing a means to selectively capture cysteine-containing peptides from complex analyte due to the strong interaction.All results showed that our new extraction tips have minimal cost and perfect selectivity;thus they have great potential in sample pretreatment systems for proteomics.     

长白山白眉蝮蛇蛇毒酶的基质辅助激光解吸质谱分析

用基质辅助激光解吸飞行时间质谱法对长白山眉蝮蛇蛇毒所含４种主要酶：磷脂酶Ａ２,精氨酸酯酶,纤溶酶及Ｌ－氨基酸氧化酶进行了纯度鉴定和分子量测定,结果表明ＭＡＬＤＩ－ＴＯ－ＦＭＸ具有灵敏度高,分辨能力强,分析时间短及样品用量少等优点。用ＭＡＬＤＩ－ＴＯＦＭＳ法分析蛇毒酶的纯度和分子量简捷,快速且重现性好,是ＳＤＳ聚丙烯酰胺凝胶电泳所无法比拟的。     

Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry

Characterization of membrane proteins remains an analytical challenge because of difficulties associated with tedious isolation and purification. This study presents the utility of the polyvinylidene difluoride (PVDF) membrane for direct sub-proteome profiling and membrane protein characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The hydrophobic adsorption of protein, particularly membrane proteins, on the PVDF surface enables efficient on-PVDF washing to remove high concentrations of detergents and salts, such as up to 5% sodium dodecyl sulfate (SDS). The enhanced spectrum quality for MALDI detection is particularly notable for high molecular weight proteins. By using on-PVDF washing prior to MALDI detection, we obtained protein profiles of the detergent-containing and detergent-insoluble membrane fractions from Methylococcus capsulatus (Bath). Similar improvements of signal-to-noise ratios were shown on the MALDI spectra for proteins electroblotted from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) onto the PVDF membrane. We have applied this strategy to obtain intact molecular weights of the particulate methane monooxygenase (pMMO) composed of three intrinsic membrane-bound proteins, PmoA, PmoB, and PmoC. Together with peptide sequencing by tandem mass spectrometry, post-translational modifications including N-terminal acetylation of PmoA and PmoC and alternative C-terminal truncation of PmoB were identified. The above results show that PVDF-aided MALDI-MS can be an effective approach for profiling and characterization of membrane proteins.     

MALDI-TOF-MS测定溶菌酶的相对分子质量中在线纯化技术的应用

用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)测定溶菌酶的相对分子质量,采用硝酸纤维素膜作为溶菌酶的固相载体,在质谱仪上进行在线纯化溶菌酶样品,在测试前尽可能除去样品中的添加剂,以消除一些盐和蛋白质变性剂抑制样品峰的情形;此法简单、快速,并可明显增强离子峰的强度,提高测试的灵敏度。     

径向基神经网络奥斯特杨方波伏安法同时测定铬和锌

径向基函数神经网络(RBFN)和核心偏最小二乘法(KPLS)用于分析重叠的Cr（Ⅲ）和Zn(Ⅱ）的奥斯特杨(Osteryoung)方波伏安图，程序SPRBFN和SPKPLS被设计用于全部计算。在RBFN方法中，普通高斯函数可用作隐藏层非线性转移函数。由于其局部性质，RBFN能被快速训练，避免陷入局部最小。对两个方法预测能力的研究结果显示其所有组分的相对预测标准偏差(RSEP)分别为0．677％和13．0％。因此，RBFN方法较之K眦方法可提供更为精确的结果，而且在解决局部最小，改进收敛速率方面也不失为一个重要的工具。     

基质辅助激光解吸附电离飞行时间质谱分析化学合成酵母转录活化因子

基质辅助激光解吸附电离飞行时间质谱是近年来发展很快的一种新颖的质谱分析仪，尤其适用于生物大分子的分子量测定，本文介绍它在确证化学合成酵母转录活化因子Ａｃ（Ｑ＾２３６，Ｑ＾２４２），ＧＣＮ４（２２６－２５２）－ＧＧＣ－ＮＨ２的一级结构中的重要作用。     

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.     

Limited proteolysis combined with isotope labeling and quantitative LC-MALDI MS for monitoring protein conformational changes: a study on calcium-binding sites of cardiac Troponin C

Studies of protein-protein and protein-ligand interactions are important for understanding biological functions of proteins. A new technique based on the partial proteolysis of proteins combined with quantitative mass spectrometry is developed as a means of tracking structural changes after the formation of a protein-ligand complex. In this technique, a protein of interest with and without the binding of a ligand is digested with an enzyme to generate a set of peptides, followed by separation of the peptides by liquid chromatography. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is used to identify chromatographically separated peptides, and locate their sequence alignments in the parent protein. Using an isotopically labeled protein as a sample against an unlabeled protein standard, quantitative information can be gathered. This overcomes the inherent lack of quantitative capability of MALDI MS. The utility of the technique to investigate protein-ligand interactions is demonstrated in a model system involving calcium binding to cardiac Troponin C (cTnC). Using this technique, the general location of the three calcium-binding sites of cTnC can be determined by using several different enzymes to generate overlapping peptide maps of cTnC.