UPLC–MS-Based Non-targeted Analysis of Endogenous Metabolite Changes in the Leaves of Scabiosa tschiliensis Grüning Induced by 6-Benzylaminopurine and Kinetin

In vitro propagation technology with plant growth regulators (PGRs) is generally applied in the cultivation of Scabiosa tschiliensis, which can solve collection difficulties and limited resources of S. tschiliensis. Nevertheless, comprehensive metabolomic evaluation on S. tschiliensis with PGR effects is still lacking. In this work, a non-targeted metabolomics approach, coupled with statistical and pathway enrichment analysis, was used to assess the regulatory influences of 6-benzylaminopurine (6-BA) and kinetin (KT) applied in S. tschiliensis. The results showed that the PGRs affect metabolism differentially, and the addition of 6-BA and KT can increase different secondary metabolites. In the two PGR groups, some primary metabolites such as L-phenylalanine, L-tyrosine, L-arginine, L-asparagine, and D-proline were significantly reduced. We suspect that under the action of PGRs, these decreased amino acids are derived into secondary metabolites such as umbelliferone, chlorogenic acid, and glutathione. Additionally, some of those secondary metabolites have a biological activity and can also promote the plant growth. Our results provide a basis for the targeted cultivation and utilization of S. tschiliensis, especially the expression of metabolites related to PGR application.

MALDI-MSI Analysis of Endogenous Lipid Changes in Zebrafish Exposures to Water Eutrophication

斑马鱼作为一种常见的模式物种,体内易富集水中污染物,常用于农药、有机物等污染水质监测与评估,但其响应富营养化水质污染的分子机制尚不明确。基质辅助激光解吸电离质谱成像(matrix assisted laser desorption ionization mass spectrometry imaging, MALDI-MSI)能有效检测和反映分子空间分布及原位含量信息,已被广泛应用于动植物学、基础医学、药学、微生物等诸多研究领域。采用MALDI-MSI技术,针对富营养化污染水质环境下斑马鱼内源性脂质代谢分子开展原位分析表征;利用多元统计学方法和相关性分析整合数据;进一步挖掘有效关键信息,旨在从脂质分子层面上初步揭示斑马鱼响应富营养化污染水质的相关分子变化规律。研究结果显示:在m/z 500～1 000的质量检测范围内,富营养化水质处理组和未处理组斑马鱼有超过50%原位检测的脂质分子存在明显表达与空间分布差异。主成分分析(principal component analysis, PCA)、偏最小二乘法判别分析(partial least squares discriminant analysis, PLS-DA)及皮尔逊相关聚类热图分析(pearson correlation cluster heatmap analysis)进一步证实了上述差异结果,且统计发现磷脂分子的表达受富营养化污染水质的干扰尤为突出,主要包括PC(32∶0)、PC(34∶1)、PC(34∶2)、PC(36∶0)、PC(38∶6)、PC(40∶6)、PE(36∶8)、PE(36∶0)、PE(38∶0)、PE(40∶9)共10种脂质分子。研究结果为进一步深度探究斑马鱼响应富营养化污染水质体内脂质代谢分子机制奠定了一定的实验基础,也证实了MALDI-MSI作为一种新型分子影像技术应用于生态/环境毒理学监测、评估研究的潜在价值。

Copper adhesive tape attached to the reverse side of a non-conductive glass slide to achieve protein MALDI-imaging in FFPE-tissue sections

Herein, copper adhesive tape attached to the reverse side of a glass slide was developed as a new method to achieve protein in situ detection and imaging in a formalin fixed paraffin-embedded (FFPE) tissue section on a non-conductive glass slide by MALDI-MSI. The use of this new method led to 223 protein ions being imaged from a rat brain FFPE-tissue section on a non-conductive glass slide by MALDI-MS, compared to only 145 and 163 protein ions detected on an ITO glass slide and an AnchorChip target plate, respectively. This new method has great potential to become standard practice for protein MALDI-imaging in FFPE-tissue sections on non-conductive glass slides.

Absolute quantification of 2‐hydroxyglutarate on tissue by matrix‐assisted laser desorption/ionization mass spectrometry imaging for rapid and precise identification of isocitrate dehydrogenase mutations in human glioma

Isocitrate dehydrogenase (IDH) gene mutations are important predictive molecular markers to guide surgical strategy in brain cancer therapy. Herein, we presented a method using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) for absolute quantification of 2-hydroxyglutarate (2-HG) on tissues to identify IDH mutations and evaluate tumor residue. This analytical method was tested among 34 glioma patients and validated with gold standard clinical technologies. The cut-off value of 2-HG was set as 0.81 pmol/mug to identify IDH mutant (IDHmt) gliomas with 100% specificity and sensitivity. In addition, 2-HG levels and tumor cell density (TCD) showed positive correlation in IDHmt gliomas by this spatial method. This MALDI MSI-based absolute quantification method has great potentiality for incorporating into surgical workflow in the future.

Metabolite changes associated with earthworms (Eisenia fetida) graphene exposure revealed by matrix-assisted laser desorption/ionization mass spectrometry imaging

The increased production and environmental release of graphene nanoparticles has raised concerns about its environmental impact, but the effects of graphene on living organisms at the metabolic level remain unknown. In this study, we used matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)-based untargeted metabolomics to investigate the metabolic response of juvenile earthworms (Eisenia fetida) to graphene exposure in soil tests for the first time. Our results reveal that graphene-exposure significantly disturbs earthworm metabolome, and graphene toxicity on earthworm shows non-concentration-dependent effect. Alanine, phenylalanine, proline, glutamate, arginine, histidine, maltose, glucose, malate, succinate, myo-inositol, and spermidine were successfully screened as significantly change compounds in earthworms for the exposure of graphene. The heterogeneous distributions of these metabolites in earthworm were also clearly imaged by MALDI-MSI. Our MSI results fully showed that the metabolite expression levels in juvenile earthworms significantly changed (up-/down-regulation) after exposure to graphene nanoparticles. This work improves our understanding of graphene nanoparticle toxicity to juvenile earthworms and also enables the continued progression of MALDI-MSI-based metabolomics as an emerging, reliable, and rapid ecotoxicological tool for assessing contaminant toxicity.

Simultaneous colorimetric sensing of malachite & leucomalachite green in aquatic products based on novel ionic associate self-visualization HPTLC strips

The development of simultaneous and rapid screening of the residues of veterinary drugs and their metabolites is up to now still a challenge. In this study, a novel strategy of read-out analysis is proposed based on high-performance thin-layer chromatography (HPTLC) separation coupled with self-visualization detection of trace of leucomalachite Green (LMG) and malachite green (MG) in aquatic products. The novel self-visualization nanomaterial was successfully synthesized and subsequently fully characterized. The ionic associate MG-I-3 formed on the visualization sensing interface was effectively attested by MALDI-TOF/TOF-MS for the first time. A serials of visualization reactions were conducted on the target zone surface of HPTLC after separation. Under the optimum conditions, the calibration curves of LMG and MG were obtained with good linearity in the range of 0.25-8.0 mu g mL(-1) and 0.125-4.0 mu g mL(-1), respectively. The limits of detection and limit of quantification were calculated as 1.71 and 5.19 mu g kg(-1) for LMG and 0.9 and 2.74 mu g kg(-1) for MG, respectively. Real sample analysis and validation provided satisfactory results. The proposed method has the potential to detect simultaneously, rapidly, and accurately LMG and MG in aquatic products during storage, handling, and sales.

Complement Factor H Displays Opposite Expression Patterns Under Two Situations of Methamphetamine Administration: Acute Exposure and Chronic Dependence

Dear Editor,Methamphetamine(METH)is a highly addictive central nervous system stimulant that has severe physical and psychological side-effects,including loss of appetite, hyperactivity, dysphoria, and depression[1].Due to its illegal production, distribution, sale, and possession it has become a worldwide burden.METH is directly toxic to dopaminergic and serotoninergic neurons, resulting in excitotoxicity,oxidative stress,and other processes[2].Research on biomolecules associated with these processes will be useful for identifying potential markers, exploring the mechanism of METH dependence, and even developing prevention and treatment strategies.

Proteomics: recent advances in the analysis of diabetic kidney disease

糖尿病肾病(diabetic kidney disease, DKD)是糖尿病的主要并发症之一,严重威胁人类健康与生命.截至目前, DKD的致病机制尚未阐释清楚,且临床常用诊断方法的灵敏性和准确性并不十分理想,从而导致DKD确诊后治疗方案的确定比一般性肾脏疾病更为棘手.蛋白质作为生命活动的主要承担者与体现者,直接参与和调控各种生命过程.从蛋白质组学水平开展DKD研究,能够从整体、动态、互作网络等视角探究该疾病相关分子机制.针对不同生理病理条件下的DKD临床样本开展蛋白质组学研究,可全面探查与DKD显著相关的关键蛋白质;通过对这些蛋白质进行深入分析和验证,能够更直观地理解DKD发生发展的分子机制,并获得DKD进程相关候选标志物和后续疾病的潜在治疗靶点,为DKD的早期诊断和治疗新方法的探究奠定基础.近年来,随着蛋白质组学技术的不断发展,在蛋白质分离、质谱鉴定、生物信息学分析等蛋白质组学核心技术基础上衍生出了许多新兴技术,进一步推动了蛋白质组学在疾病生物标志物筛选、致病分子机制揭示、药物作用蛋白质靶点等研究中的应用.本文基于蛋白质组学研究技术,主要从DKD致病机制研究、早期诊断潜在生物标志物筛选、治疗靶点及效果评估三个方面对蛋白质组学在DKD研究中的应用进展进行了系统性综述.尽管蛋白质组学在DKD研究中取得了长足的进步,但仍具有较大的发展空间,特别是现已识别的大量潜在DKD分子标志物的相关性分析、药物蛋白质作用靶点临床验证与应用将是DKD未来研究的重点.

Pd/Cu-Catalyzed Cascade C(sp3)–H Arylation and Intramolecular C–N Coupling: A One-Pot Synthesis of 3,4-2H-Quinolinone Skeletons

In this letter, we successfully explored a cascade Pd/Cu-catalyzed intermolecular C(sp(3))-H arylation of amides and intramolecular C-N coupling reaction. This method provides a one-pot strategy to synthesize 3,4-2 H-quinolinone with good regioselectivity of C-H arylation and C-N coupling from C-I and C-X bonds from readily available starting materials.

3,4-Dimethoxycinnamic Acid as a Novel Matrix for Enhanced In Situ Detection and Imaging of Low-Molecular-Weight Compounds in Biological Tissues by MALDI-MSI

Enhanced in situ detection and imaging of lipids in biological tissues by using 2,3-dicyanohydroquinone as a novel matrix for positive-ion MALDI-MS imaging

A fluorescent dye, 2,3-dicyanohydroquinone (DCH), was optimized for use as a new matrix for positive-ion MALDI-MS imaging, and it provided enhanced lipid detection and imaging in biological tissues. The properties of DCH, include a strong ultraviolet absorption, low volatility in a high-vacuum (similar to 10(-7) mbar) source, super chemical stability, mu m-sized matrix crystals, uniform matrix deposition, and high ionization efficiency for the detection of lipids.

Recent Advances in Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) for in situ analysis of endogenous molecules in plants

Mass spectrometry imaging(MSI), an emerging technology for molecular imaging in biological tissue, enables simultaneous examination and characterization of the spatial distributions and relative abundances of endogenous and exogenous compounds directly from the surface of a thinly cut tissue slice, such as proteins, peptides, lipids, metabolites,and drugs. In the past decade, MSI has been widely applied in a broad range of fields, including basic medical science,pharmaceutical science, microbiology, and zoology. MSI has been applied to investigate plant biology because it can be used for in situ label-free molecular imaging. This review provides an overview of MSI technology, describes the standard operating procedures of different types of MSI, and outlines the applications of MSI for in situ analysis of endogenous molecules in plant tissue, particularly in medicinal plant materials. Finally, the technical limitations and challenges of MSI are summarized, which must be addressed to allow for effective and widespread application of MSI in plants. Further improvements in MS technology will enhance the overall performance of MSI in plant studies

A novel method for nondenaturing protein extraction from Populus euphratica leave

为探索适用于胡杨叶片非变性蛋白质的提取方法,以采自内蒙古额济纳旗天然胡杨林中胡杨成熟叶片为材料,采用咪唑法、Bis-Tris法、Tris-HCl法和新构建的Tris-SSAD法4种非变性蛋白质提取策略,分别用于提取胡杨叶片蛋白质样品;随后进行第一维非变性聚丙烯酰胺凝胶电泳(1~(st)-DE:Native-PAGE)。相比之下,只有Tris-SSAD法获得了清晰、条带数目较多的Native-PAGE图谱,且结合第二维变性凝胶电泳(2~(nd)-DE:SDS-PAGE)可成功分离出较多蛋白质复合体亚基或相互作用蛋白质分子,证明了Tris-SSAD法非变性温和提取特性。研究建立并优化了适用于胡杨叶片的非变性蛋白质提取方法,为后续进一步分析蛋白质与蛋白质相互作用及蛋白质复合体功能提供了实验基础。

Metabolomic insights into the effects of thyroid hormone on Rana [Lithobates] catesbeiana metamorphosis using whole-body Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI)

Anuran metamorphosis involves the transformation of an aquatic tadpole into a juvenile frog. This process is completely dependent upon thyroid hormones (THs). Although much research has been focused on changes in gene expression programs during this postembryonic developmental period, transitions in the metabolic profiles are relatively poorly understood. Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) is a technique that generates highly multiplexed mass spectra while retaining spatial location information on a thin tissue section. Reconstructed ion heat maps are correlated with morphology of the tissue section for biological interpretation. The present study is the first to use whole-body MALDI-MSI on tadpoles to gain insights into anuran metamorphosis. Approximately 1000 features were detected in each of five tissues examined (brain, eye, liver, notochord, and tail muscle) from premetamorphic North American bullfrog (Rana [Lithobates] catesbeiana) tadpoles. Of these detected metabolites, 1700 were unique and 136 were significantly affected by exposure to 50nM thyroxine for 48h. Of the significantly-affected metabolites, 64 features were tentatively identified using the MassTRIX annotation tool. All tissues revealed changes in lipophilic compounds including phosphatidylcholines, phosphatidylinositols, phosphatidylglycerols, phosphatidylethanolamines, and phosphatidylserines. These lipophilic compounds made up the largest portion of significantly-affected metabolites indicating that lipid signaling is a major target of TH action in frog tadpoles.

Mass spectrometry imaging and its application to in situ analysis of endogenous molecules in plants

质谱成像作为一种新型分子影像技术,可在组织或细胞水平上同时获得样本表面多种分子组成、丰度及原位空间分布信息.近年来,随着该技术的不断发展与完善,质谱成像正逐渐成为质谱分析领域的研究前沿与热点.目前,该技术已被广泛应用于诸多领域:如基础医学、药学、微生物学、动物学等.值得注意的是,在植物学研究领域,质谱成像技术也正逐渐受到重视并被广泛应用.本文基于多种质谱成像方法及相关技术流程,综述了该技术在植物内源性分子原位表征中的应用进展,尤其是在药用植物有效成分成像表征中的应用,并对质谱成像技术现有缺点与不足、潜在价值与应用前景进行了总结与展望.

Metabolomic profiling of prostate cancer by matrix assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry imaging using Matrix Coating Assisted by an Electric Field (MCAEF)

In this work, we combined the use of two MALDI matrices (quercetin and 9-aminoacridine), a recently developed new matrix coating technique - matrix coating assisted by an electric field (MCAEF), and matrix-assisted laser desorption/ionization - Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) to detect and image endogenous compounds in the cancerous and non-cancerous regions of three human prostate cancer (stage II) tissue specimens. After three rounds of imaging data acquisitions (i.e., quercetin for positive and negative ion detection and 9-aminoacridine for negative ion detection), and metabolite identification, a total of 1091 metabolites including 1032 lipids and 59 other metabolites were routinely detected and successfully localized. Of these compounds, 250 and 217 were only detected in either the cancerous or the non-cancerous regions respectively, although we cannot rule out the presence of these metabolites at concentrations below the detection limit. In addition, 152 of the other 624 metabolites showed differential distributions (p

Poplar catkin: A natural biomaterial for highly specific and efficient enrichment of sialoglycopeptides

Sialic acids as terminal entities of larger glycans linked to proteins and lipids are involved in multiple different pathological and physiological processes. Structural characterisation of sialoglycoconjugates is required to understand their biological function. However, a comprehensive sialylation analysis of sialoglycoconjugates has remained challenges. In this study, we employ a natural biomaterial, poplar catkin derived from white poplar tree (Populus tomentosa Cam), to develop a novel capturing microtip for selective and efficient enrichment of sialoglycopeptides, without losses of sialic acid residues and water molecules from sialoglycopeptides. Scanning electron microscopy and Fourier-transform infrared spectroscopy analysis, along with Maule and Wiesner staining assays, indicated that the main components on the outer layer of the poplar catkin are syringyl and guaiacyl lignins which play a key role in enriching sialoglycopeptides from complex peptide mixture.(C) 2017 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.

Matrix coating assisted by an electric field (MCAEF) for enhanced tissue imaging by MALDI-MS

A novel technique, termed matrix coating assisted by an electric field (MCAEF), for enhancing tissue imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was developed in this study. In this technique a static and uniform electric field is applied to sliced tissue sections during matrix spray-coating, resulting in the enrichment of positively or negatively chargeable analytes in the MALDI matrix layer. Experimental results show that MCAEF not only increased the sensitivity of lipid and protein detection across the board in the subsequent MALDI-MS analyses, but also resulted in successful imaging of a larger number of analytes. MALDI imaging enhancement with MCAEF was observed for various tissues (rat liver, rat brain, and porcine adrenal gland) and with different MALDI matrices (e.g., quercetin, 2-mercaptobenzothiazole, dithranol, 9-aminoacridine, and sinapinic acid) and the sensitivity increases were independent of the solvent compositions and pH values of the matrix solutions. Taking rat brain as an example, MCAEF led to the on-tissue detection and imaging of 648 identified lipids by combining positive and negative ion detection by MALDI-Fourier transform ion cyclotron resonance MS and with quercetin as the matrix, as compared to only 344 lipids without MCAEF. For protein imaging, up to 232 protein signals were successfully detected in rat brain tissue sections by MALDI-time-of-flight MS within a mass range of 3500 to 37 000 Da, as compared to 119 without MCAEF. MCAEF also enabled the detection of higher molecular-weight proteins. These results demonstrate the advantages of MCAEF for overall performance improvements in MALDI imaging and we believe that this technique has the potential to become a standard practice for MALDI tissue imaging.

The use of matrix coating assisted by an electric field (MCAEF) to enhance mass spectrometric imaging of human prostate cancer biomarkers

In this work, we combined a newly developed matrix coating technique - matrix coating assisted by an electric field (MCAEF) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to enhance the imaging of peptides and proteins in tissue specimens of human prostate cancer. MCAEF increased the signal-to-noise ratios of the detected proteins by a factor of 2 to 5, and 232 signals were detected within the m/z 3500-37500 mass range on a time-of-flight mass spectrometer and with the sinapinic acid MALDI matrix. Among these species, three proteins (S100-A9, S100-A10, and S100-A12) were only observed in the cancerous cell region and 14 proteins, including a fragment of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 2, a fragment of cAMP-regulated phosphoprotein 19, 3 apolipoproteins (C-I, A-I, and A-II), 2 S100 proteins (A6 and A8), beta-microseminoprotein, tumor protein D52, alpha-1-acid glycoprotein 1, heat shock protein beta-1, prostate-specific antigen, and 2 unidentified large peptides at m/z 5002.2 and 6704.2, showed significantly differential distributions at the p < 0.05 (t-test) level between the cancerous and the noncancerous regions of the tissue. Among these 17 species, the distributions of apolipoprotein C-I, S100-A6, and S100-A8 were verified by immunohistological staining. In summary, this study resulted in the imaging of the largest group of proteins in prostate cancer tissues by MALDI-MS reported thus far, and is the first to show a correlation between S100 proteins and prostate cancer in a MS imaging study. The successful imaging of the three proteins only found in the cancerous tissues, as well as those showing differential expressions demonstrated the potential of MCAEF-MALDI/MS for the in situ detection of potential cancer biomarkers.

Change in IgG1FcN-linked glycosylation in human lung cancer: Age- and sex-related diagnostic potential

The interactions of IgG Fc region with Fc receptors are optimized by the tailoring of a single-conserved N-linked glycosylation site at Asn-297. Our previous study has demonstrated that the age-related Fc-glycosylation change is featured by sex specificity and that the Fc-glycosylation has the potential for disease discrimination. Here, we conducted a Fourier transform ion cyclotron resonance MS-based profiling study involving 410 control individuals and 259 lung cancer (LC) patients. As compared to healthy controls, the marked increase in IgG1 Fc-agalactosylation and decrease in galactosylation were observed in LC patients. The binary logistic regression in combination with the receiver operating characteristic curve was used to determine the diagnostic ability of IgG1 Fc-glycosylation. It was found that this diagnostic ability was both sex and age dependent. Additionally, the change in Fc-glycosylation upon many different physiological and pathological conditions was retrospectively discussed. The data furthered the understanding of the immune-associated change in human LC, and also might be useful in the future attempts for Fc-glycosylation-associated diagnostic evaluations and clinical assays.

Dephosphorylation of intact glycoprotein to greatly improve digestion efficiency coupled with matrix-assisted laser desorption/ionization–Fourier transform ion cyclotron resonance mass spectrometric analysis

Sialylation is essential for a variety of cellular functions. Herein, we used bovine fetuin with three potential N-linked glycosylation sites containing complex-type glycan structures, four potential O-linked glycosylation sites and six potential phosphorylation sites as a model compound to develop a highly-efficient digestion strategy for sialylated glycoproteins and efficient enrichment strategy for sialylated glycopeptides using titanium dioxide. The former according to the process of alkaline phosphatase digestion followed by tryptic digestion and then proteinase K digestion could greatly improve the enzymatic efficiency on fetuin, and the latter could obviously enhance the enrichment efficiency for multisialylated glycopeptides using phosphoric acid solution as elution buffer. The mass spectra of the enriched glycopeptides derived from fetuin reveal that several series of the ion clusters with mass difference of 291 Da correspond to the presence of multisialylated glycopeptides. In addition, the approach was applied to characterize the sialylated status of alpha2-macroglobulin and transferrin, respectively, from the sera of healthy subjects and sex- and age-matched patients with thyroid cancer, and their spectra indicate that the change in the amount of the glycoforms containing different number of sialic acid (SA) residues from one glycosylation site may be used to differentiate between healthy subjects and cancer cases.

Hydroxyflavones as a New Family of Matrices for MALDI Tissue Imaging

The discovery of new matrices that are suitable for in situ analysis of low molecular-weight compounds by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an important technological aspect of tissue imaging. In this work, ten natural flavonoid compounds, including flavone and nine of its mono- or polyhydroxyl-substituted analogues (3-hydroxyflavone, 5-hydroxyflavone, 3,7-dihydroxyflavone, chrysin, 7,3',4'-trihydroxyflavone, fisetin, luteolin, quercetin, and morin) were evaluated as potential MALDI matrices for the profiling and imaging of endogenous lipids in mouse liver, using a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer with a 355-nm Nd:YAG UV laser, in the positive ion mode. When an electronic sprayer was used for matrix coating and with a high-pH (0.1-0.5% ammonia hydroxide) matrix solvent, eight of the ten compounds, all of which had at least one OH group at the C3 or C5 position of the flavone structure, enabled the successful detection of 77 to 161 phospholipids and other lipids. The best results were observed with two penta-OH flavones (i.e., quercertin and morin). Taking quercetin as an example, this matrix showed characteristics superior to those of commonly used MALDI matrices, such as DHB (2,5-dihydroxybenzoic acid), CHCA (alpha-cyano-4-hydroxycinnamic acid), and 2-mercaptobenzothiazole (2-MBT). These characteristics were: mum-sized matrix crystals, uniform matrix coating, low volatility in the high vacuum (~10(-7) mbar) source, good chemical stability, low yield of matrix-related ions, low matrix consumption, low power threshold for laser desorption/ionization, and improved safety of handling. The use of quercetin led to improved lipid imaging, with 212 lipids being successfully imaged from rat brain in a single experiment and with asymmetric distributions of some lipids in left and right brain hippocampus being observed for the first time.

Elevated Serum Levels of Circulating Immunoinflammation-Related Protein Complexes Are Associated with Cancer

Disease-specific immune response-related protein complexes in the bloodstream are associated with disease status. We used proteomic technologies to screen novel circulating immunoinflammation-related protein complexes (IIRPCs) and to evaluate their diagnostic accuracy. The discovery study included 96 gastric cancer patients and 83 healthy controls and was designed to isolate and identify the IIRPCs. Then an independent validation study including 1366 patients with lung, colorectal, pancreatic, gastric, or thyroid cancer, 141 patients with other types of cancer, 376 patients with benign lung, colorectal, pancreatic, gastric, or thyroid diseases, and 3707 healthy controls was performed. We observed seven major patterns of the IIRPCs and confirmed the IIRPCs as personalized biomarkers of cancers. The levels of the IIRPCs were significantly increased in cancer patients compared with controls and benign patients (p < 0.0001). Each of the IIRPCs (a2 to a4, a6, a7, and b3 to b5) shows excellent discriminating power for lung, colorectal, pancreatic, and gastric cancer, with the areas under the receiver operating characteristic curves (AUCs) from 0.95 to 0.99 (95% CIs 0.91-1.00), and for thyroid cancer, with the AUCs from 0.87 to 0.96 (95% CIs 0.80-0.98). The IIRPCs can be used as a novel type of broad-spectrum and supramolecular biomarker for personalized cancer diagnosis.

Comprehensive Imaging of Porcine Adrenal Gland Lipids by MALDI-FTMS Using Quercetin as a Matrix

Adrenal glands synthesize and release functional zone-specific steroid and catecholamine hormones to regulate mammalian stress responses. Lipids such as sphingolipids have been shown to control the steroid hormone biosynthesis in adrenal glands, indicating their important roles in endocrine organs. Molecular imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a well-established analytical technique for determining both the spatial location and the relative abundances of various lipids on tissue. To better understand the overall roles of different lipid classes that play in the mammalian adrenal glands, it is necessary to comprehensively determine the spatial distributions of various lipids in the different functional zones of adrenal glands. However, the potential of this technique has not been fully reached, considering there are thousands of lipid species in a cell or tissue. To achieve this, we used quercetin as a MALDI matrix for negative ion detection of endogenous lipids on tissue sections of porcine adrenal glands by MALDI-Fourier-transform ion cyclotron resonance (FTICR) MS. As a result of these experiments, 409 endogenous compounds were detected in the negative ion mode. Combining both the positive and negative ion detection led to successful determination of the spatial distribution patterns of 555 unique endogenous compounds that were identified as 544 lipid entities and 11 nonlipid metabolites. Many classes of these lipids showed distinct distribution patterns in different functional zones of the adrenal gland. To the best of our knowledge, this work presents the largest group of lipid entities that have been analyzed in a single MS imaging study so far, and comprehensive profiles of the spatial distributions of lipids in porcine adrenal glands are shown here for the first time.

Lipid profiling for early diagnosis and progression of colorectal cancer using direct-infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

RATIONALE: Colorectal cancer (CRC) has attracted increasing attention due to its common occurrence and worldwide distribution. METHODS: Direct-infusion positive and negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (DI-ESI(+/-)-FTICR MS) was applied to analyze the serum metabolites from 52 CRC patients and 52 healthy controls. Metabolites whose inter-group intensities were determined to be statistically significant by univariate and multivariate statistical analyses were further identified by a combination of the Human Metabolome Database, accurate mass measurement, isotopic abundance distribution simulation, and tandem mass spectrometry. Orthogonal partial least square discriminant analysis (OPLS-DA), based on the data from DI-ESI(+/-)-FTICR MS, revealed a remarkable discrimination among early stage patients, late stage patients, and healthy controls. RESULTS: A total of 15 differentially expressed metabolites were identified and categorized into four lipid classes. Each lipid class demonstrated specific changing trends in CRC progression. Biomarker panel 1 containing palmitic amide, oleamide, hexadecanedioic acid, octadecanoic acid, eicosatrienoic acid, LPC(18:2), LPC(20:4), LPC(22:6), myristic acid and LPC(16:0) achieved excellent diagnostic accuracy with area under the ROC curve (AUC) of 0.991, a sensitivity of 0.981 and a specificity of 1.000 for differentiating early stage patients from healthy controls, which was better than the carcinoembryonic antigen biomarker. CONCLUSIONS: Our study revealed that the consideration of CRC stages would be necessary in diagnostic biomarker discovery, as well as that attention should be paid to the facile loss of methyl chloride from the [M + Cl](-) form of LPC(16:0) in its tandem mass spectrum.

Human IgG Fc-glycosylation profiling reveals associations with age, sex, female sex hormones and thyroid cancer

Broad-spectrum Four-dimensional Orthogonal Electrophoresis: A Novel Comprehensively Feasible System for Protein Complexomics Investigation

The major challenge of "protein complexomics" is to separate intact protein complexes or interactional proteins without dissociation or denaturation from complex biological samples and to characterize structural subunits of protein complexes. To address these issues, we developed a novel approach termed "broad-spectrum four-dimensional orthogonal electrophoresis (BS4-DE) system," which is composed of a nondenaturing part I and denaturing part II. Here we developed a mild acidic-native-PAGE to constitute part I, together with native-thin-layer-IEF and basic-native-PAGE, widening the range of BS4-DE system application for extremely basic proteins with the range of pI from about 8 to 11 (there are obviously 1000 kinds of proteins in this interval), and also speculated on the mechanism of separating. We first proposed ammonium hydroxide-ultrasonic protein extractive strategy as a seamless connection between part I and part II, and also speculated on the extractive mechanism. More than 4000 protein complexes could be theoretically solved by this system. Using this approach, we focus on blood rich in protein complexes which make it challenging to sera/plasma proteome study. Our results indicated that the BS4-DE system could be applied to blood protein complexomics investigation, providing a comprehensively feasible approach for disease proteomics.

Probing gender-specific lipid metabolites and diagnostic biomarkers for lung cancer using Fourier transform ion cyclotron resonance mass spectrometry

BACKGROUND: There are no effective clinical biomarkers for early and specific detection of lung cancer (LC). The changes in the levels of some serum metabolites of LC patients are associated with patient gender and LC stages. METHODS: Serum metabolites of the LC patients (n=58) and healthy controls (n=495) were performed using direct-infusion positive ion electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry in combination with univariate and partial least squares discriminant analyses (PLS-DA). RESULTS: Univariate analysis indicated that 141 of the 212 serum metabolites were significantly changed in the LC patients compared with healthy controls. PLS-DA model, based on the 141 metabolites, demonstrated the differential metabolite distribution in single-sex LC patients compared with the corresponding healthy controls, and also in LC females compared with LC males. Several lipids comprising fatty acid derivations, sphingomyelin (SM) and lysophosphatidylcholine (LPC) were associated with the LC progression. Classification using oleamide, long chain acyl carnitines, LPC(18:1), LPC(20:4), LPC(20:3), LPC(22:6), and SM(16:0/1) as a biomarker panel resulted in remarkable separation between the LC patients and healthy controls with sensitivity and specificity of 100% and 91%, respectively. CONCLUSION: The serum metabolites found in this study may play essential roles in gender-specific LC detection and early diagnosis of cancer.

Separation and Identification of HSP-Associated Protein Complexes from Pancreatic Cancer Cell Lines Using 2D CN/SDS-PAGE Coupled with Mass Spectrometry

Protein complexes are a cornerstone of many biological processes and together they form various types of molecular machinery. A broad understanding of these protein complexes is crucial for revealing and building models of protein function and regulation. Pancreatic cancer is a highly lethal disease which is difficult to diagnose at early stage and even more difficult to cure. In this study, we applied a gradient clear native gel system combined with subsequent second-dimensional SDS-PAGE to separate protein complexes from cell lysates of SW1990 and PANC-1 pancreatic cancer cell lines with different degrees of differentiation. Ten heat-shock-protein- (HSP-) associated protein complexes were separated and identified, and the differentially expressed proteins related to cancers were also found, such as HSP60, protein disulfide-isomerase A4 (ERp72), and transitional endoplasmic reticulum ATPase (TER ATPase).

Proteomics-based Characterization of Protein Complexes from Human Pancreatic Cancer Cell Line

To develop an economical and feasible approach to probe protein complexes, differential centrifugation and three-dimensional polyacrylamide gel electrophoresis (PAGE) were performed to separate protein complexes from the cell lysate of human pancreatic cancer cell line, SW1990, followed by mass spectrometric identification. Four macromolecular protein complexes were separated and identified unambiguously.

In vitro methylation by methanol: Proteomic screening and prevalence investigation

It is assumed that much more functional importance for protein activity than expected may be granted by methylation that occurs at the side-chain of aspartate or glutamate residue. In vitro methylation mainly comes from the use of methanol in sample preparation prior to MS analysis. In this study, we first performed the methylation site-directed proteomic screening of bovine serum albumin, ovalbumin and 20S proteasome for gel staining using a meaningfully indicative MS-pattern of peak tag (termed as 4P tag) and manual inspection for mass spectral data. As a result, there were 17 proteolytic peptides with 20 modified sites confirmed to be in vitro methylated. Subsequently, the prevalence investigation was performed, focusing on the reaction kinetic behavior of in vitro methylation. This study provided a simple and robust approach for confirmation of in vitro methylation by methanol, as well as the precautious guide for the use of methanol in proteomic study.

Four-Dimensional Orthogonal Electrophoresis System for Screening Protein Complexes and Protein−Protein Interactions Combined with Mass Spectrometry

Most current approaches for purification and identification of protein complexes adopt affinity purifications combined with mass spectrometry, such as co-immunoprecipitation and tandem affinity purification. Herein, we propose a new approach, termed as the four-dimensional orthogonal electrophoresis (4-DE) system, to find and analyze the cytoplasmic protein complexes. 4-DE system is composed of two parts: nondenaturing part (Part I) and denaturing part (Part II). Through Part I and decision procedure separations, six protein complex candidates 20S core particle of proteasome (CP), hemoglobin (Hb, alpha2beta2), Hb (alpha2delta2), peroxiredoxin-2 (PRDX2), carbonic anhydrase-1 (CAH1), and heat shock protein 60 (HSP60) were separated. CP, Hb (alpha2beta2), PRDX2, and HSP60 with different MWs and pI's were chosen for Part II proteomic analysis. The results indicate that 4-DE is not only suitable for studying protein complexes and protein-protein interactions as well as structural proteomics from complex biological samples, but can also be easy to separate and concentrate intact protein complexes from dilute complex samples.

Chemical cross-linking and mass spectrometry for investigation of protein complex structure and protein-protein interaction

近年来化学交联法结合质谱分析法被广泛用于蛋白质复合体结构及蛋白质相互作用的研究。研究表明这两种方法的有机结合为研究蛋白质复合体结构及蛋白质相互作用提供了一条新的途径。文章对不同类型的化学交联剂、质谱分析中的Bottom-up与Top-down两种研究策略,以及化学交联法结合质谱分析法在蛋白质复合体结构、蛋白质相互作用研究中的应用进行综述。这两种方法的不断发展与完善,将会极大促进生物大分子复合体结构及蛋白质相互作用的研究。