Continuous Flow Atmospheric Pressure Laser Desorption/Ionization Using a 6–7-μm-Band Mid-Infrared Tunable Laser for Biomolecular Mass Spectrometry

A continuous flow atmospheric pressure laser desorption/ionization technique using a porous stainless steel probe and a 6–7-µm-band mid-infrared tunable laser was developed. This ion source is capable of direct ionization from a continuous flow with a high temporal stability. The 6–7-µm wavelength region corresponds to the characteristic absorption bands of various molecular vibration modes, including O–H, C=O, CH₃ and C–N bonds. Consequently, many organic compounds and solvents, including water, have characteristic absorption peaks in this region. This ion source requires no additional matrix, and utilizes water or acetonitrile as the solvent matrix at several absorption peak wavelengths (6.05 and 7.27 µm, respectively). The distribution of multiply-charged peptide ions is extremely sensitive to the temperature of the heated capillary, which is the inlet of the mass spectrometer. This ionization technique has potential for the interface of liquid chromatography/mass spectrometry (LC/MS).     

Recent Advances in Bacteria Identification by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using Nanomaterials as Affinity Probes

Identifying trace amounts of bacteria rapidly, accurately, selectively, and with high sensitivity is important to ensuring the safety of food and diagnosing infectious bacterial diseases. Microbial diseases constitute the major cause of death in many developing and developed countries of the world. The early detection of pathogenic bacteria is crucial in preventing, treating, and containing the spread of infections, and there is an urgent requirement for sensitive, specific, and accurate diagnostic tests. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an extremely selective and sensitive analytical tool that can be used to characterize different species of pathogenic bacteria. Various functionalized or unmodified nanomaterials can be used as affinity probes to capture and concentrate microorganisms. Recent developments in bacterial detection using nanomaterials-assisted MALDI-MS approaches are highlighted in this article. A comprehensive table listing MALDI-MS approaches for identifying pathogenic bacteria, categorized by the nanomaterials used, is provided.     

Access of Hydrogen-Radicals to the Peptide-Backbone as a Measure for Estimating the Flexibility of Proteins Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

A factor for estimating the flexibility of proteins is described that uses a cleavage method of “in-source decay (ISD)” coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The MALDI-ISD spectra of bovine serum albumin (BSA), myoglobin and thioredoxin show discontinuous intense ion peaks originating from one-side preferential cleavage at the N-Cα bond of Xxx-Asp, Xxx-Asn, Xxx-Cys and Gly-Xxx residues. Consistent with these observations, Asp, Asn and Gly residues are also identified by other flexibility measures such as B-factor, turn preference, protection and fluorescence decay factors, while Asp, Asn, Cys and Gly residues are identified by turn preference factor based on X-ray crystallography. The results suggest that protein molecules embedded in/on MALDI matrix crystals partly maintain α-helix and that the reason some of the residues are more susceptible to ISD (Asp, Asn, Cys and Gly) and others less so (Ile and Val) is because of accessibility of the peptide backbone to hydrogen-radicals from matrix molecules. The hydrogen-radical accessibility in MALDI-ISD could therefore be adopted as a factor for measuring protein flexibility.     

Protein Profiling of Blood Samples from Patients with Hereditary Leiomyomatosis and Renal Cell Cancer by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an extremely rare syndrome with autosomal dominant inheritance. HLRCC is characterized by a predisposition to leiomyomas of the skin and the uterus as well as renal cell carcinoma. The disease-related gene has been identified as fumarate hydratase (fumarase, FH), which encodes an enzyme involved in the mitochondrial tricarboxylic acid cycle. Protein profiling may give some insight into the molecular pathways of HLRCC. Therefore, we performed protein profiling of blood samples from HLRCC patients, their family members, and healthy volunteers, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) coupled with IMAC-Cu chips. For hierarchical clustering analysis, we used the 45 peaks that revealed significant differences in single-marker analysis over the range from 1500 to 15,000 m/z. Heat map analysis based on the results of clustering distinguished the HLRCC kindred from non-HLRCC subjects with a sensitivity of 94% and a specificity of 90%. SELDI-TOF MS profiling of blood samples can be applied to identify patients with HLRCC and to assess specific molecular mechanisms involved in this condition.     

Analysis of Insect Cuticular Hydrocarbons Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Insect cuticular hydrocarbons (CHCs) were probed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry with a lithium 2,5-dihydroxybenzoate matrix. CHC profiles were obtained for 12 species of diverse insect taxa (termites, ants, a cockroach, and a flesh fly). MALDI spectra revealed the presence of high molecular weight CHCs on the insect cuticle. Hydrocarbons with more than 70 carbon atoms, both saturated and unsaturated, were detected. When compared with gas chromatography/mass spectrometry (GC/MS), MALDI-TOF covered a wider range of CHCs and enabled CHCs of considerably higher molecular weight to be detected. Good congruity between GC/MS and MALDI-TOF was observed in the overlapping region of molecular weights. Moreover, a number of previously undiscovered hydrocarbons were detected in the high mass range beyond the analytical capabilities of current GC/MS instruments. MALDI was shown to hold potential to become an alternative analytical method for insect CHC analyses. The ability of MALDI to discriminate among species varying in the degree of their relatedness was found to be similar to GC/MS. However, neither MALDI-MS nor GC/MS data were able to describe the phylogenetic relationships.     

Current Status and Future Perspectives of Mass Spectrometry Imaging

Mass spectrometry imaging is employed for mapping proteins, lipids and metabolites in biological tissues in a morphological context. Although initially developed as a tool for biomarker discovery by imaging the distribution of protein/peptide in tissue sections, the high sensitivity and molecular specificity of this technique have enabled its application to biomolecules, other than proteins, even in cells, latent finger prints and whole organisms. Relatively simple, with no requirement for labelling, homogenization, extraction or reconstitution, the technique has found a variety of applications in molecular biology, pathology, pharmacology and toxicology. By discriminating the spatial distribution of biomolecules in serial sections of tissues, biomarkers of lesions and the biological responses to stressors or diseases can be better understood in the context of structure and function. In this review, we have discussed the advances in the different aspects of mass spectrometry imaging processes, application towards different disciplines and relevance to the field of toxicology.     

聚环氧丙烷的电喷雾质谱分析

采用电喷雾质谱（ESI—MS）分析得到了聚环氧丙烷（PPO）的一级质谱和二级质谱。通过正负2种离子化模式计算一级质谱中各分子离子峰m／z的差值为58,证实了单体为环氧丙烷（PO）。并在二级质谱中通过对一些化合物碎片峰的解析,得到了PPO的裂解方式。同时利用碎片分析,推断出此PPO起始剂为丙三醇。     

氨苄西林质谱解析及其离子化方式研究

通过对于氨苄西林正离子模式质谱谱图的解析,探讨了氨苄西林的离子化方式和裂解规律。研究表明：氨苄西林存在3种质子化方式,不同的质子化方式所形成分子离子具有不同的裂解方式;质子化的质子具有明显的活性,是质谱进一步裂解的主要原因;离子化方式对于质谱峰的丰度会产生重要影响。本研究为氨苄西林质谱分析和构效关系研究提供了重要的实验基础与理论依据。     

基质辅助激光解析电离-飞行时间质谱在高分子聚合物研究应用中的样品制备方法

在基质辅助激光解析电离．飞行时间质谱（Matxa ssisted laser desorption／ionization time—of-flight massspectrum,MALDI—TOF—MS）研究高分子聚合物的过程中,样品的制备方法对获得准确的质谱图非常关键,样品制备必须选择合适的溶剂、基质和阳离子化试剂。文章就近年来MALDI-TOF—MS在高分子聚合物研究中的有关样品制备方法的发展进行综述。     

Optimization and Evaluation of Magnetic Bead Separation Combined with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectroscopy (MALDI-TOF MS) for Proteins Profiling of Peritoneal Dialysis Effluent

Peritoneal dialysis effluent (PDE) potentially carries an archive of peptides relevant to pathological processes in abdominal and surrounding tissues. Magnetic beads and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is one such approach that offers a unique tool for profiling of peptides, but this approach has not been used in the PDE analysis. In this study, we developed a strategy for screening PDE proteins <15 kDa and applied this technique to identify potential biomarkers for peritonitis. We examined four kinds of magnetic beads, including a carbon series (C3, C8), weak cation exchange (WCX) and immobilized metal-affinity chromatography (IMAC-Cu) beads. Samples processed with IMAC-Cu magnetic beads consistently showed more MS signals across all beads within the measured mass range. Moreover, there was no difference in the number and morphology of MS signals between concentrated and unconcentrated samples. The PDE peptidome pattern, based on a panel of 15 peaks, accurately recognized peritonitis PD patients from peritonitis-free patients with sensitivity of 90.5% and specificity of 94.7% respectively. Therefore, IMAC-Cu magnetic beads and unconcentrated samples can be used as a fast and cost-effective approach for sample preparation prior to more in-depth discovery of predictive biomarkers of disease in patients on dialysis.     

MALDI Imaging Mass Spectrometry (MALDI-IMS)-Application of Spatial Proteomics for Ovarian Cancer Classification and Diagnosis

MALDI imaging mass spectrometry (MALDI-IMS) allows acquisition of mass data for metabolites, lipids, peptides and proteins directly from tissue sections. IMS is typically performed either as a multiple spot profiling experiment to generate tissue specific mass profiles, or a high resolution imaging experiment where relative spatial abundance for potentially hundreds of analytes across virtually any tissue section can be measured. Crucially, imaging can be achieved without prior knowledge of tissue composition and without the use of antibodies. In effect MALDI-IMS allows generation of molecular data which complement and expand upon the information provided by histology including immuno-histochemistry, making its application valuable to both cancer biomarker research and diagnostics. The current state of MALDI-IMS, key biological applications to ovarian cancer research and practical considerations for analysis of peptides and proteins on ovarian tissue are presented in this review.     

Towards Lipidomics of Low-Abundant Species for Exploring Tumor Heterogeneity Guided by High-Resolution Mass Spectrometry Imaging

Many studies have evidenced the main role of lipids in physiological and also pathological processes such as cancer, diabetes or neurodegenerative diseases. The identification and the in situ localization of specific low-abundant lipid species involved in cancer biology are still challenging for both fundamental studies and lipid marker discovery. In this paper, we report the identification and the localization of specific isobaric minor phospholipids in human breast cancer xenografts by FTICR MALDI imaging supported by histochemistry. These potential candidates can be further confirmed by liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS) after extraction from the region of interest defined by MALDI imaging. Finally, this study highlights the importance of characterizing the heterogeneous distribution of low-abundant lipid species, relevant in complex histological samples for biological purposes.     

Evaluation of Proteomic and Lipidomic Changes in Aeromonas-Infected Trout Kidney Tissue with the Use of FT-IR Spectroscopy and MALDI Mass Spectrometry Imaging

Aeromonas species are opportunistic bacteria causing a vast spectrum of human diseases, including skin and soft tissue infections, meningitis, endocarditis, peritonitis, gastroenteritis, and finally hemorrhagic septicemia. The aim of our research was to indicate the molecular alterations in proteins and lipids profiles resulting from Aeromonas sobria and A. salmonicida subsp. salmonicida infection in trout kidney tissue samples. We successfully applied FT-IR (Fourier transform infrared) spectroscopy and MALDI-MSI (matrix-assisted laser desorption/ionization mass spectrometry imaging) to monitor changes in the structure and compositions of lipids, secondary conformation of proteins, and provide useful information concerning disease progression. Our findings indicate that the following spectral bands’ absorbance ratios (spectral biomarkers) can be used to discriminate healthy tissue from pathologically altered tissue, for example, lipids (CH₂/CH₃), amide I/amide II, amide I/CH₂ and amide I/CH₃. Spectral data obtained from 10 single measurements of each specimen indicate numerous abnormalities concerning proteins, lipids, and phospholipids induced by Aeromonas infection, suggesting significant disruption of the cell membranes. Moreover, the increase in the content of lysolipids such as lysophosphosphatidylcholine was observed. The results of this study suggest the application of both methods MALDI-MSI and FT-IR as accurate methods for profiling biomolecules and identifying biochemical changes in kidney tissue during the progression of Aeromonas infection.     

大气压离子化质谱技术研究N-聚氧乙烯醚衍生物

利用大气压离子化质谱技术 ,采用直接进样方式研究了N 聚氧乙烯醚衍生物的氧乙烯链聚合度 (EO数 )分布 ,并对O 聚氧乙烯醚衍生物及壬基酚聚氧乙烯醚进行了测定。当化合物的分子质量小于 1 0 0 0u时 ,采用大气压化学离子源和大气压电喷雾离子源两种离子化质谱技术均可得到正态的EO数分布曲线 ,最大n值与根据环氧乙烷加入量计算出的平均加成数基本吻合。采用直接进样质谱技术可同步分析含氧乙烯链的不同结构化合物的EO数分布曲线。选择大气压化学离子源检测时 ,样品与溶剂间的极性差异越小越好 ;而采用大气压电喷雾离子源时 ,溶剂极性对EO数的正态分布影响较小。对于EO数较大的化合物 ,宜采用大气压电喷雾源离子化 ,并可观察到多电荷体系 ;调节质谱参数将影响多电荷体系的分布。直接进样质谱技术为含氧乙烯链化合物的EO数分布提供了一条快速、简捷的分析途径。     

Zinc Deficiency Leads to Lipid Changes in Drosophila Brain Similar to Cognitive-Impairing Drugs: An Imaging Mass Spectrometry Study

Several diseases and disorders have been suggested to be associated with zinc deficiency, especially learning and memory impairment. To have better understanding about the connection between lipid changes and cognitive impairments, we investigated the effects of a zinc-chelated diet on certain brain lipids of Drosophila melanogaster by using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The data revealed that there are increases in the levels of phosphatidylcholine and phosphatidylinositol in the central brains of the zinc-deficient flies compared to the control flies. In contrast, the abundance of phosphatidylethanolamine in the brains of the zinc-deficient flies is lower. These data are consistent with that of cognitive-diminishing drugs, thus providing insight into the biological and molecular effects of zinc deficiency on the major brain lipids and opening a new treatment target for cognitive deficit in zinc deficiency.     

Rapid Quantification of Low-Viscosity Acetyl-Triacylglycerols Using Electrospray Ionization Mass Spectrometry

Acetyl‐triacylglycerols (acetyl‐TAG) possess an sn‐3 acetate group, which confers useful chemical and physical properties to these unusual triacylglycerols (TAG). Current methods for quantification of acetyl‐TAG are time consuming and do not provide any information on the molecular species profile. Electrospray ionization mass spectrometry (ESI–MS)‐based methods can overcome these drawbacks. However, the ESI–MS signal intensity for TAG depends on the aliphatic chain length and unsaturation index of the molecule. Therefore response factors for different molecular species need to be determined before any quantification. The effects of the chain length and the number of double‐bonds of the sn‐1/2 acyl groups on the signal intensity for the neutral loss of short chain length sn‐3 groups were quantified using a series of synthesized sn‐3 specific structured TAG. The signal intensity for the neutral loss of the sn‐3 acyl group was found to negatively correlated with the aliphatic chain length and unsaturation index of the sn‐1/2 acyl groups. The signal intensity of the neutral loss of the sn‐3 acyl group was also negatively correlated with the size of that chain. Further, the position of the group undergoing neutral loss was also important, with the signal from an sn‐2 acyl group much lower than that from one located at sn‐3. Response factors obtained from these analyses were used to develop a method for the absolute quantification of acetyl‐TAG. The increased sensitivity of this ESI–MS‐based approach allowed successful quantification of acetyl‐TAG in various biological settings, including the products of in vitro enzyme activity assays.     

Supramolecular Analysis of Monosaccharide Derivatives Using Cucurbit[7]uril and Electrospray Ionization Tandem Mass Spectrometry

Identification of monosaccharide derivatives using an analytical technique is difficult due to their isomeric structures and similar physical properties. Although mass spectrometry (MS) has emerged as a prominent technique for this purpose, a comprehensive MS-based method for analyzing diverse monosaccharide species is yet to be developed. Herein, we employ host-guest chemistry using cucurbit[7]uril (CB[7]) with MS to identify four different monosaccharide derivative species. Tandem MS analysis distinguishes the monosaccharide derivatives based on the unique fragmentation pattern produced when forming gas-phase complexes with CB[7]. The ion mobility studies combined with computational calculations reveal that subtle differences in isomers cause distinct orientations inside CB[7] cavity, resulting in different fragmentation patterns of the isomers. This unique study demonstrates that host-guest chemistry combined with MS can be used for developing effective isomer analysis techniques.     

Lipid and Phospholipid Profiling of Biological Samples Using MALDI Fourier Transform Mass Spectrometry

Here we describe a study of the feasibility of lipid and phospholipid (PL) profiling using matrix assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS) for two different applications. In this work PL profiles of different mammalian tissues as well as those of whole cell organisms were examined. In particular, comparative analysis of lipid and PL profiles of tissues from mice fed different diets was done and, in another application, MALDI FTMS was used to analyze PL profiles of genetically modified Saccharomyces cerevisiae. Computational sorting of the observed ions was done in order to group the lipid and PL ions from complex MALDI spectra. The PL profiles of liver tissues from mice fed different diets showed a cross correlation coefficient of 0.2580, indicating significant dissimilarity, and revealed more than 30 significantly different peaks at the 99.9% confidence level. Histogram plots derived from the spectra of wild type and genetically modified yeast resulted in a cross correlation coefficient 0.8941 showing greater similarity, but still revealing a number of significantly different peaks. Based on these results, it appears possible to use MALDI FTMS to identify PLs as potential biomarkers for metabolic processes in whole cells and tissues.     

超高效液相色谱–电喷雾串联质谱测定蔬菜水果中27种常见农药残留

建立了蔬菜水果中27种常见农药残留超高效液相色谱–电喷雾串联质谱(UPLC–ESI–MS/MS)测定方法。样品用乙腈均质提取,加入无水硫酸镁和氯化钠盐析分层,经CARBON/NH2固相萃取柱净化,乙腈–甲苯(3∶1,v/v)洗脱,旋转蒸发浓缩,用乙腈–0.1%甲酸(1∶1,v/v)溶解,以电喷雾串联质谱在多反应监测模式(MRM)下进行测定。分析方法结果表明,多数组分在5～100μg/L范围内线性良好(r2=0.991～0.999),在10～50μg/kg范围内,27种农药的回收率为65.81%～124.7%,相对标准偏差为3.6%～19.2%。方法的检出限和定量限范围分别为0.05～5μg/L和0.2～18μg/L。该方法操作简便、快捷、灵敏,灵敏度、准确度和精密度均能满足农药多残留检测的技术要求,适用于蔬菜水果中常见农药残留的检测。     

Analysis of Molecular Species Profiles of Ceramide-1-phosphate and Sphingomyelin Using MALDI-TOF Mass Spectrometry

Ceramide‐1‐phosphate (C1P) is a potential signaling molecule that modulates various cellular functions in animals. It has been known that C1P with different N‐acyl lengths induce biological responses differently. However, molecular species profiles of the C1P in animal tissues have not been extensively examined yet. Here, we developed a method for determination of the molecular species of a C1P using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry with Phos‐tag, a phosphate capture molecule. The amounts of total C1P in skin, brain, liver, kidney and small intestine of mice were determined to be 344, 151, 198, 96 and 90 pmol/g wet weight, respectively. We found a C1P species having an α‐hydroxypalmitoyl residue (h‐C1P, 44 pmol/g wet weight) in mouse skin. The h‐C1P was detected only in the skin, and not other tissues of mice. The same analysis was applied to sphingomyelin after conversion of sphingomyelin to C1P by Streptomyces chromofuscus phospholipase D. We found that molecular species profiles of sphingomyelin in skin, kidney and small intestine of mice were similar to those of C1P in corresponding tissues. In contrast, molecular species profiles of sphingomyelin in liver and brain were quite different from those of C1P in these tissues, indicating selective synthesis or degradation of C1P in these tissues. The method described here will be useful for detection of changes in molecular species profiles of C1P and sphingomyelin.