Superhydrophobic Properties of Nanostructured–Microstructured Porous Silicon for Improved Surface-Based Bioanalysis

Wettability is a fundamental property of a solid surface, which plays important roles in many industrial applications. The possibility to create well-controlled nonwetting states on silicon surfaces without photolithography-based processing can bring many advantages in the biotechnology and microfluidics areas. In this paper, superhydrophobic properties of macroporous–nanoporous structured silicon are reported. The superhydrophobic porous silicon layers are prepared by electrochemical etching of bulk crystalline silicon wafers. Altered anodization conditions provide surfaces with varying pore morphologies, yielding different wetting properties, ranging from highly wetting (nanoporous morphologies) to water-repellent surfaces (macroporous morphologies). Subsequent surface modification with a fluorocarbon coupling agent can further improve nonwetting properties and stabilize the surface for a long term. Contact angles as high as 176° were achieved on macroporous silicon and superhydrophobicity was maintained for several months without degradation. The porous surfaces have proven to be a very attractive substrate for protein microarrays. Fluorescence-based assay of immunoglobulin G in plasma is reported with a limit of detection of 1 pM, a spot size of 50 μm, and an array density of 15,625 spots per square centimeter. Macroporous surfaces have also been developed for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) applications, where the intrinsic hydrophobic surface properties confine the deposited sample to MALDI spots of less than 200 μm with well-defined MALDI crystals, providing a high-sensitivity readout. Furthermore, a superhydrophobic MALDI-TOF MS target anchor chip composed of nonporous anchor points surrounded by superhydrophobic porous areas for sample deposition and on anchor point confinement is reported. Such anchor chips allowed localized crystallization of large sample volumes (5 μL) improving the hydrophobic spot confinement strategy in terms of final MALDI crystal localization and readout sensitivity.     

Dual polarity accurate mass calibration for electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry using maltooligosaccharides

In view of the fact that memory effects associated with instrument calibration hinder the use of many mass-to-charge (m/z) ratios and tuning standards, identification of robust, comprehensive, inexpensive, and memory-free calibration standards is of particular interest to the mass spectrometry community. Glucose and its isomers are known to have a residue mass of 162.05282 Da; therefore, both linear and branched forms of polyhexose oligosaccharides possess well-defined masses, making them ideal candidates for mass calibration. Using a wide range of maltooligosaccharides (MOSs) derived from commercially available beers, ions with m/z ratios from approximately 500 to 2500 Da or more have been observed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and time-of-flight mass spectrometry (TOF-MS). The MOS mixtures were further characterized using infrared multiphoton dissociation (IRMPD) and nano-liquid chromatography/mass spectrometry (nano-LC/MS). In addition to providing well-defined series of positive and negative calibrant ions using either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), the MOSs are not encumbered by memory effects and, thus, are well-suited mass calibration and instrument tuning standards for carbohydrate analysis.     

Desorption/ionization on silicon for small molecules:a promising alternative to MALDI TOF

A method has been developed for laser desorption/ionization of catecholamines from porous silicon. This methodology is particularly attractive for analysis of small molecules. MALDI TOF mass spectrometry, although a very sensitive technique, utilizes matrices that need to be mixed with the sample prior to their analysis. Each matrix produces its own background, particularly in the low-molecular mass region. Therefore, detection and identification of molecules below 400 Da can be difficult. Desorption/ionization of samples deposited on porous silicon does not require addition of a matrix, thus, spectra in the low-molecular mass region can be clearly readable. Here, we describe a method for the analysis of catecholamines. While MALDI TOF is superior for proteomics/peptidomics, desorption/ionization from porous silicon can extend the operating range of a mass spectrometer for studies on metabolomics (small organic molecules and their metabolites, such as chemical neurotransmitters, prostaglandins, steroids, etc.).     

Selective metabolite and peptide capture/mass detection using fluorous affinity tags

A new and general methodology is described for the targeted enrichment and subsequent direct mass spectrometric characterization of sample subsets bearing various chemical functionalities from highly complex mixtures of biological origin. Specifically, sample components containing a chemical moiety of interest are first selectively labeled with perfluoroalkyl groups, and the entire sample is then applied to a perfluoroalkyl-silylated porous silicon (pSi) surface. Due to the unique hydrophobic and lipophobic nature of the perfluorinated tags, unlabeled sample components are readily removed using simple surface washes, and the enriched sample fraction can then directly be analyzed by desorption/ionization on silicon mass spectrometry (DIOS-MS). Importantly, this fluorous-based enrichment methodology provides a single platform that is equally applicable to both peptide as well as small molecule focused applications. The utility of this technique is demonstrated by the enrichment and mass spectrometric analysis of both various peptide subsets from protein digests as well as amino acids from serum.     

Characterization of antibody-antigen interactions: comparison between surface plasmon resonance measurements and high-mass matrix-assisted laser desorption/ionization mass spectrometry

The interaction between the bovine prion protein (bPrP) and a monoclonal antibody, 1E5, was studied with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and surface plasmon resonance (SPR). In the case of MS a cross-linking stabilization was used prior to the analysis, whereas for SPR the antibody was immobilized and bPrP was injected. We compared the determination of parameters such as the epitope, the kinetics and binding strength, and the capacity of the antigen to bind two different antibodies. The two methods are highly complementary. SPR measurements require a lower amount of sample but are more time-consuming due to all of the necessary side steps (e.g., immobilization, regeneration). High-mass MALDI MS needs a higher overall amount of sample and cannot give direct access to the kinetic constants, but the analysis is faster and easier compared with SPR.     

Amidation of methyl-esterified oligogalacturonides: examination of the reaction products using MALDI-TOF MS

Amidation of methyl-esterified oligogalacturonides (oligoGalA) was studied to produce partly and fully amidated oligoGalA to be used as substrates and/or inhibitors for the characterization of pectolytic enzymes acting on the homogalacturonan backbone. The reactions were performed with varying concentrations of ammonia or methylamine and monitored in time using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that allows sensitive monitoring of the reactions. MALDI-TOF MS reveals the degree of amidation (DAm) and extent of hydrolysis of methyl-esters. Using this technique the conditions for each of the reactions was optimized. Amidation was performed best under anhydrous conditions at a concentration of 4 M ammonia or methylamine at ambient temperature. Amidation using methylamine reached almost completeness (DAm 95) without hardly any hydrolysis of methyl-esters while amidation with ammonia reached a DAm of 70 on average. After an initial fast amidation, precipitation of the partly amidated oligoGalA reduced the reaction rate enormously. The use of ammonia in aqueous solutions instead off anhydrous ammonia resulted in 6–10% lower DAm values due to the hydrolysis of methyl-esters. Therefore, anhydrous conditions are preferred during amidation. Furthermore, methylamine is a better reagent for amidation of oligoGalA and pectins then ammonia, but also results in totally different products with other properties.     

枯草芽孢杆菌JA产生的脂肽类抗生素－iturin A的纯化 及电喷雾质谱鉴定

枯草芽孢杆菌JA产生的抗生素对植物病原真菌具有广谱抗性,明确抗生素的种类是进一步研究的基础.用6mol/L盐酸沉淀JA菌株的去菌体培养基,再用甲醇抽提获得抗生素的粗提物.利用反相HPLC系统,将粗提物过Diamonsil C18柱,收集有抗小麦赤霉病等病原真菌活性的化合物1、2.运用电喷雾质谱法(ESI/MS)测得其分子量分别为1042.4D和1056.5D.再利用碰撞诱导解离(CID)技术获得化合物的典型结构特征离子碎片,结果表明分子量为1042.4D的化合物一级结构为Pro-Asn-Tyr-βAA-Asn-Tyr-Asn-Gln(βAA为14个碳原子的氨基脂肪酸),属于脂iturin A.化合物1、2为相差一个亚甲基(-CH2)的iturin A同系物.研究结果提供了一种从枯草芽孢杆菌发酵液中快速分离纯化和鉴定脂肽类抗生素iturin A的新方法.     

基质辅助激光解吸电离飞行时间质谱在微生物鉴定中的研究进展

与传统的微生物鉴定技术相比,基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS)是一种准确、可靠和快速的鉴定和分型的技术。本文通过检索近年来国内外相关研究论文,总结最新的研究进展,发现MALDI-TOF MS在临床病原微生物、食源性微生物以及环境微生物等鉴定中有较大的优势,加快了微生物鉴定的进程,同时探索该技术在新领域的最新进展和面临的挑战,以期为我国基质辅助激光解吸电离飞行时间质谱技术的发展提供参考。     

基质辅助激光解析电离飞行时间质谱在医学真菌领域的应用进展

基质辅助激光解析电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS）是近年来新兴的微生物检测技术,通过核糖体蛋白分析实现对真菌快速、准确鉴定。本文针对MALDI-TOF MS用于致病真菌鉴定、分类、体外抗真菌药物敏感性检测以及临床微生物样本直接检测等方面作一综述。     

A study on in vitro glycation processes by matrix-assisted laser desorption ionization mass spectrometry

The number of glucose molecules condensed on glycated bovine serum albumin have been easily determined by means of matrix-assisted laser desorption/ionization mass spectrometry. Measurements were carried out on samples from incubation of the proteins with glucose at different concentrations (0.02 M, 0.2 M, 2 M and 5 M). A clear increase in molecular mass of BSA with respect to incubation time is detected. In contrast to what is observed with fluorescence, the plots of molecular mass increase vs. incubation time show tha occurrence of a steady state, corresponding to the complete saturation of all the protein sites against glucose. Comparison of fluorescence and molecular mass data reveals that some further reactions, different from condensation, must take place, which could be in principle either intramolecular or originated by reactivity of modified condensed gluocse moieties vs. free glucose.     

Combination of Micropreparative Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Peptide Analysis

Signal suppression is a problem in matrix-assisted laser desorption/ionization mass spectrometry of peptides prepared by capillary electrophoresis. Many common electrolytes that are efficient for separation, such as sodium phosphate, also are strongly suppressive during laser desorption/ionization. We have tested individual electrolytes for highest performance in each step of separation and collection, respectively. Suppression is not observed if citrate, trifluoroacetic acid, or hydrochloric acid is used for collection, while phosphate still can be employed in the capillary providing excellent resolution. Low concentrations of hydrochloric acid added to the sample/matrix mixture generate mass spectra with better ion intensities than if trifluoroacetic acid or citrate is used.     

Synthesis and matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry study of phosphopeptide

Summary The symmetry phosphoramidite reagents were synthesized via a two-step route and used for the ‘one pot’ synthesis of the phosphoserine building block Fmoc-Ser(PO(OBzl)OH)-OH. The procedure is simple and rapid, and product is obtained in high yield. When using this building block, the phosphopeptide (RKGS(PO₃H₂)SSNEPSSDSLSSPTLLAL) related to the C-terminal of c-Fos protein was synthesized manually by Fmoc/t-Bu procedure and characterized by matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. In the fragment ion of phosphopeptide in the MALDI-TOF mass spectrometry with a curved field reflection, the acquired information can be used to identify the sequences and the phosphorylation sites of the peptide.     

Imaging mass spectrometry: principle and application

Imaging mass spectrometry (IMS) is two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules, which does not need either separation or purification of target molecules, and enables us to monitor not only the identification of unknown molecules but also the localization of numerous molecules simultaneously. Among the ionization techniques, matrix assisted laser desorption/ionization (MALDI) is one of the most generally used for IMS, which allows the analysis of numerous biomolecules ranging over wide molecular weights. Proper selection and preparation of matrix is essential for successful imaging using IMS. Tandem mass spectrometry, which is referred to MSⁿ, enables the structural analysis of a molecule detected by the first step of IMS. Applications of IMS were initially developed for studying proteins or peptides. At present, however, targets of IMS research have expanded to the imaging of small endogenous metabolites such as lipids, exogenous drug pharmacokinetics, exploring new disease markers, and other new scientific fields. We hope that this new technology will open a new era for biophysics.     

基质辅助激光解吸电离飞行时间质谱技术用于常见益生菌鉴定的初步研究

目的建立基质辅助激光解吸电离飞行时间质谱(MADLI-TOF MS)技术鉴定常见益生菌的实验方法并对MADLI-TOF MS技术的适用性进行初步评价。方法对MADLI-TOF MS技术鉴定常见益生菌过程中各影响因素进行考察,筛选出最佳的实验条件。利用19株供试菌株所得的蛋白指纹图谱对MADLI-TOF MS技术的适用性进行研究。结果建立了MADLI-TOF MS技术鉴定常见益生菌的最佳实验方法。初步证明MADLI-TOF MS技术具备在属、种、亚种以及菌株水平上鉴定常见益生菌的能力。结论建立的实验方法稳定性高、重复性好,可以作为MADLI-TOF MS技术鉴定常见益生菌的参考方法。MADLI-TOF MS技术可以作为常见益生菌鉴定的方法之一。     

Locally Addressable Electrochemical Patterning Technique (LAEPT) applied to poly(L-lysine)-graft-poly(ethylene glycol) adlayers on titanium and silicon oxide surfaces

The protein-resistant polycationic graft polymer, poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), was uniformly adsorbed onto a homogenous titanium surface and subsequently subjected to a direct current (dc) voltage. Under the influence of an ascending cathodic and anodic potential, there was a steady and gradual loss of PLL-g-PEG from the conductive titanium surface while no desorption was observed on the insulating silicon oxide substrates. We have implemented this difference in the electrochemical response of PLL-g-PEG on conductive titanium and insulating silicon oxide regions as a biosensing platform for the controlled surface functionalization of the titanium areas while maintaining a protein-resistant background on the silicon oxide regions. A silicon-based substrate was micropatterned into alternating stripes of conductive titanium and insulating silicon oxide with subsequent PLL-g-PEG adsorption onto its surfaces. The surface modified substrate was then subjected to +1800 mV (referenced to the silver electrode). It was observed that the potentiostatic action removed the PLL-g-PEG from the titanium stripes without inducing any polyelectrolyte loss from the silicon oxide regions. Time-of-flight secondary ions mass spectroscopy and fluorescence microscopy qualitatively confirmed the PLL-g-PEG retention on the silicon oxide stripes and its absence on the titanium region. This method, known as "Locally Addressable Electrochemical Patterning Technique" (LAEPT), offers great prospects for biomedical and biosensing applications. In an attempt to elucidate the desorption mechanism of PLL-g-PEG in the presence of an electric field on titanium surface, we have conducted electrochemical impedance spectroscopy experiments on bare titanium substrates. The results showed that electrochemical transformations occurred within the titanium oxide layer; its impedance and polarization resistance were found to decrease steadily upon both cathodic and anodic polarization resulting in the polyelectrolyte desorption from the titanium surface.     

Paper spray ionization mass spectrometry: recent advances and clinical applications

ABSTRACT

Introduction: Paper spray mass spectrometry has provided a rapid, quantitative ambient ionization method for xenobiotic and biomolecule analysis. As an alternative to traditional sample preparation and chromatography, paper spray demonstrates the sampling ionization of a wide range of molecules and significant sensitivity from complex biofluids. The amenability of paper spray with dried blood spots and other sampling types shows strong potential for rapid, point-of-care (POC) analysis without time-consuming separation procedures.

Areas covered: This special report summarizes the current state and advances in paper spray mass spectrometry that relate to its applicability for clinical analysis. It also provides our perspectives on the future development of paper spray mass spectrometry and its potential roles in clinical settings.

Expert commentary: Paper spray has provided the fundamental aspects of ambient ionization needed for implementation at the POC. With further clinical management and standardization, paper spray has the potential to replace traditional complex analysis procedure for rapid quantitative detection of illicit drugs, therapeutic drugs and metabolites. Surface and substrate modifications also offer significant improvement in desorption and ionization efficiencies, resulting in enhanced sensitivity. Comprehensive analysis of metabolites and lipids will further extend the implementation of paper spray ionization mass spectrometry into clinical applications.     

Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.     

Synthesis and matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry study of phosphopeptide

The symmetry phosphoramidite reagents were synthesized via atwo-step route and used for the `one pot' synthesis of thephosphoserine building block Fmoc-Ser(PO(OBzl)OH)-OH. Theprocedure is simple and rapid, and product is obtained in highyield. When using this building block, the phosphopeptide(RKGS(PO₃H₂)SSNEPSSDSLSSPTLLAL) related to theC-terminal of c-Fos protein was synthesized manually byFmoc/t-Bu procedure and characterized by matrix assisted laserdesorption/ionization time of flight (MALDI-TOF) massspectrometry. In the fragment ion of phosphopeptide in theMALDI-TOF mass spectrometry with a curved field reflection, theacquired information can be used to identify the sequences andthe phosphorylation sites of the peptide.     

Localization of water-soluble carbohydrates in wheat stems using imaging matrix-assisted laser desorption ionization mass spectrometry

The pool of endogenous water-soluble oligosaccharides found in the stems of wheat (Triticum aestivum) is being investigated as a potential indicator of grain yield. Techniques such as liquid chromatography with mass spectrometry (LC-MS) can profile these analytes but provide no spatial information regarding their distribution in the wheat stem. The imaging matrix-assisted laser desorption ionization (MALDI) mass spectrometry technique has not been utilized for the analysis of oligosaccharides in plant systems previously. Imaging MALDI mass spectrometry was used to analyse cross and longitudinal sections from the stems of Triticum aestivum. A range of oligosaccharides up to Hex(11) were observed. Water-soluble oligosaccharides were ionized as potassiated molecules, and found to be located in the stem pith that is retained predominantly around the inner stem wall. Imaging MALDI analyses provided spatial information on endogenous oligosaccharides present in wheat stems. The technique was found to offer comparable sensitivities for oligosaccharide detection to those of our established LC-MS method, and has potential for broad application in studying the in situ localization of other compound types in plant material.     

Ammonia desorption chemical ionization mass spectrometry of phospholipids

Ammonia desorption chemical ionization mass spectra (NH₃-DCIMS) of phosphatidyl-sulfocholines, a mixture of a homologous series of phosphatidylsulfocholines (PSC) and a mixture of a PSC phosphatidylcholine (PC), were measured by a flash heating method involving introduction of 1 μg of the samples on a tungsten wire, quickly heated, into the ion source of a Finnigan 4000/INCOS quadrupole instrument. It was possible to observe mass spectra during several seconds.The first spectra recorded of the PSCs gave essentially only the quasi-molecular [M + 18]⁺ peaks and the ‘A’ (see text) peaks. Spectra of a mixture of three homologous PSCs clearly showed three pairs of the above peaks. In contrast spectra of the PCs gave principally the [M + 1]⁺ and the ‘A’ peaks after 2s so that one can distinguish the diagnostic peaks in a mixture of a PC and a PSC.These results show that ammonia desorption chemical ionization by fast heating is a suitable technique for the charaterization of some head groups of phospholipids as well as a promising method for the analysis of phospholipid mixtures.