Quantum dots laser desorption/ionization MS: multifunctional CdSe quantum dots as the matrix,concentrating probes and acceleration for microwave enzymatic digestion for peptide analysis and high resolution detection of proteins in a linear MALDI‐TOF MS

We report the first use of functionalized cadmium selinide quantum dots (CdSe QDs) with 11‐mercaptoundecanoic acid (MUA) as the matrix for the selective ionization of proteins with high resolution and rapid analysis of amino acids and peptides by using quantum dots laser desorption/ionization mass spectrometry (QDLDI‐MS). The mercaptocarboxylic groups of CdSe QDs have been known to be an effective affinity probe to interact with the biomolecules at low abundance level. Using these QDs as the matrix, sensitivity of the method was greatly enhanced and the LOQ of peptides was found to be 100 pM with RSD <10%. The QDLDI‐MS is capable for the selective ionization of insulin, lysozyme and myoglobin with high resolution, which is not observed with sinapic acid (SA) as the matrix. The QDLDI‐MS technique offers many advantages for the analysis of amino acids, peptides and proteins with regard to simplicity, rapidity, sensitivity and the mass spectra were generated in the presence of signal suppressors such as urea and Trition X‐100. In addition, the CdSe QDs have been successfully applied as preconcentrating probes for the analysis of digested peptides in lysozyme from chicken egg white by microwave‐assisted enzymatic digestion. This indicates that the QDs are able to absorb radiation from microwave and their ability to trap peptides from microwave‐digested lysozyme. These results demonstrate that the CdSe QDs are promising candidates for the selective ionization of the analytes with an accurate platform to the rapid screening of biomolecules.     

铜绿假单胞菌的MALDI-TOF-MS检测方法的建立

目的 建立利用基质辅助激光解吸电离飞行时间质谱仪( MALDI-TOF-MS)对铜绿假单胞菌的快速检测方法.方法 通过MALDI-TOF-MS法对铜绿假单胞菌进行检测分析,并与生化鉴定方法相比较.结果 MALDI-TOF-MS对铜绿假单胞菌的检测后得到肽指纹图片及相关质谱数据,建立MALDI-TOF-MS对铜绿假单胞菌的快速检测方法.结论 MALDI-TOF-MS方法检测铜绿假单胞菌准确快速、操作简单等特点,可发展成为食品检验铜绿假单胞菌的重要(辅助)工具.     

MALDI-TOF-MS方法快速鉴定食品中空肠弯曲菌的技术研究

运用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)技术快速鉴定食品中空肠弯曲菌.通过对该方法的样品前处理的选择、稳定性、特异性等方面进行研究,确定了方法...     

运用蛋白质组学技术研究与氨甲蝶呤耐药性相关的蛋白质

氨甲蝶呤（MTX）是一种重要的常用化疗药物,然而由于肿瘤细胞对其耐药性的增强而经常导致其疗效大大降低。为了寻找并鉴定与MTX耐药性相关的蛋白质从而为进一步闸明MTX的耐药机制提供线索,培养来源于小鼠NIH3T3的小鼠胚胎成纤维细胞系3T3R500与其耐300μmol／L MTX的细胞系MTX300,提取上述两种细胞系的总蛋白质,双向凝胶电泳分离蛋白质组成分,扫描并通过软件分析考马斯亮蓝染色的2-DE凝胶,选取表达差异最显著的点,胶内酶切后MALDI-TOF-MS进行肽指纹图谱（PMF）鉴定。图像分析显示,实验组和对照组的蛋白质组图谱之间,一些蛋白质点的表达有明显的变化。通过MALDI-TOF-MS和数据库查询,成功鉴定了耐药后表达变化最显著的蛋白质点为二氢叶酸还原酶（DHFR）,并通过Western blot验证了该结果,提示DHFR在MTX耐药机制中发挥重要作用。     

Proteomic Analysis for the Identification of Proteins Related to Methotrexate Resistance

Methotrexate(MTX) is one of the most important and frequently used drugs in cancer therapy, but the efficacy of this drug is often compromised by the development of resistance in cancer cells. To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance, proteins from cell line MTX300 (resistant to 300 μmol/L MTX) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis (2-DE). The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis, which revealed several spots with high levels of differential expression between MTX300 and 3T3R500. The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS analysis and database searches revealed it to be dihydrofolate reductase (DHFR), which was subsequently confirmed by Western blot. The result suggested that DHFR might play an important role in the MTX resistance.     

Identification of synaptic plasma membrane proteins co-precipitated with fibrillar beta-amyloid peptide

The beta-amyloid peptide that is overproduced in Alzheimer's disease rapidly forms fibrils, which are able to interact with various molecular partners. This study aimed to identify abundant synaptosomal proteins binding to the fibrillar beta-amyloid (fAbeta) 1-42. Triton X-100-soluble proteins were extracted from the rat synaptic plasma membrane fraction. Interacting proteins were isolated by co-precipitation with fAbeta, or with fibrillar crystallin as a negative control. Protein identification was accomplished (1) by separating the tryptically digested peptides of the protein pellet by one-dimensional reversed-phase HPLC and analysing them using an ion-trap mass spectrometer with electrospray ionization; and (2) by subjecting the precipitated proteins to gel electrophoretic fractionation, in-gel tryptic digestion and to matrix-assisted laser desorption/ionization time-of-flight mass measurements and post-source decay analysis. Six different synaptosomal proteins co-precipitated with fAbeta were identified by both methods: vacuolar proton-pump ATP synthase, glyceraldehyde-3-phosphate dehydrogenase, synapsins I and II, beta-tubulin and 2',3'-cyclic nucleotide 3'-phosphodiesterase. Most of these proteins have already been associated with Alzheimer's disease, and the biological and pathophysiological significance of their interaction with fAbeta is discussed.     

Analysis of lysozyme in cheese by immunocapture mass spectrometry

The enzyme lysozyme is used as a preservative to prevent late blowing of ripened cheese, caused by Clostridium tyrobutyricum. Since the enzyme is extracted from hen egg white, lysozyme has to be declared on food product labels as a potential allergen. Here, a method is reported that combines immunocapture purification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis for the detection of lysozyme in cheese samples. Cheese extracts were treated with magnetic particles coated with a monoclonal antibody directed against lysozyme. After immunocapture purification, lysozyme was detected by MALDI-TOF-MS. The limit of detection of the assay was about 5 mg/kg lysozyme in cheese. The method reliably distinguished between cheese samples which had been produced with and without lysozyme. Thus, the novel assay allows the reliable, sensitive, and specific detection of lysozyme in a food matrix. The assay could be easily adapted to other target peptides and proteins in complex food matrices and, therefore, has a broad application potential, e.g. for the analysis of allergens.     

A new application of microwave technology to proteomics

Two-dimensional electrophoresis (2-DE) combined with mass spectrometry has significantly improved the possibilities of large-scale identification of proteins. However, 2-DE is limited by its inability to speed up the in-gel digestion process. We have developed a new approach to speed up the protein identification process utilizing microwave technology. Proteins excised from gels are subjected to in-gel digestion with endoprotease trypsin by microwave irradiation, which rapidly produces peptide fragments. The peptide fragments were further analyzed by matrix-assisted laser desorption/ionization technique for protein identification. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several proteins, including lysozyme, albumin, conalbumin, and ribonuclease A. The method reduced the required time for in-gel digestion of proteins from 16 hours to as little as five minutes. This new application of microwave technology to protein identification will be an important advancement in biotechnology and proteome research.     

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.     

大鼠海马的表达蛋白质组学实验研究

目的：用蛋白质组学方法初步分析大鼠海马蛋白质的表达。方法：提取大鼠海马蛋白质样品后,用双向凝胶电泳对其分离,经考马斯亮蓝染色后,产生大鼠海马蛋白质双向凝胶电泳图谱。从凝胶上切割分离的蛋白质,经胰蛋白酶胶内酶解,通过基质辅助激光解吸／电离飞行时间质谱（MALDI-TOF-MS）对酶解后的肽段进行分析。根据肽段质谱数据,经数据库（NCBI）检索,对蛋白质进行鉴定。结果：鉴定了37种具有明确功能的蛋白质,它们分别属于代谢酶、细胞骨架蛋白、热休克蛋白、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、神经元特异蛋白及神经胶质蛋白。另外,鉴定了3种未知功能蛋白。结论：为建立大鼠海马蛋白质组数据库提供必要的资料,为在大鼠模型上研究神经疾病发病机理奠定基础。     

Metal imaging in non-denaturating 2D electrophoresis gels by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for the detection of metalloproteins

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was developed as a powerful analytical technique for metal imaging of 2D gels for the detection of metalloproteins in rat kidney after electrophoretic separation. Protein complexes, extracted with water, were separated in their native state in the first and second dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, manganese and lead, were monitored by LA-ICP-MS after gel ablation by a focused laser beam in a way that the total surface of a selected fragment of the gel was totally ablated. The metal distribution of this part of the gel was then constructed by plotting the metal (isotope) signal intensity as a function of the x,y (isoelectric point, molecular mass) coordinates of the gel. The proteins at locations rich in metals were cut out, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Online microwave D-cleavage LC-ESI-MS/MS of intact proteins: site-specific cleavages at aspartic acid residues and disulfide bonds

An online nonenzymatic digestion method utilizing a microwave-heated flow cell and mild acid hydrolysis at aspartic acid (D) for rapid protein identification is described. This methodology, here termed microwave D-cleavage, was tested with proteins ranging in size from 5 kDa (insulin) to 67 kDa (bovine serum albumin) and a bacterial cell lysate ( Escherichia coli). A microwave flow cell consisting of a 5 microL total volume reaction loop connected to a sealed reaction vessel was introduced into a research grade microwave oven. With this dynamic arrangement, the injected sample was subjected to microwave radiation as it flowed through the reaction loop and was digested in less than 5 min. Different digestion times can be achieved by varying the sample flow rate and/or length of the loop inside the microwave flow cell. The microwave flow cell can be operated individually with the output being collected for matrix assisted laser ionization/desorption (MALDI) mass spectrometry (MS) or connected online for liquid chromatography (LC) electrospray ionization (ESI)-MS. In the latter configuration, the microwave flow cell eluates containing digestion products were transferred online to a reversed phase liquid chromatography column for direct ESI-MS and ESI-MS/MS analyses (specifically, Collision Induced Dissociation, CID). Concurrently with the microwave D-cleavage step, disulfide bond reduction/cleavage was achieved by the coinjection of dithiothreitol (DTT) with the sample prior to online microwave heating and online LC-MS analysis and so eliminating the need for alkylation of the reduced protein. All protein standards, protein mixtures, and proteins in a bacterial cell lysate analyzed by this new online methodology were successfully identified via a SEQUEST database search of fragment ion mass spectra. Overall, online protein digestion and identification was achieved in less than 40 min total analysis time, including the chromatographic step.     

Mass spectrometric analysis of affinity-captured proteins on a dendrimer-based immunosensing surface: investigation of on-chip proteolytic digestion

The monolayer of fourth-generation poly(amidoamine) dendrimers was adopted to construct the immunoaffinity surface of an antibody layer. The antibody layer as a bait on the dendrimer monolayer was found to result in high binding capacity of antigenic proteins and a reliable detection. The affinity-captured protein at the immunosensing surface was subjected to direct on-chip tryptic digestion, and the resulting proteolytic peptides were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The performance of the on-chip digestion procedure was investigated with respect to the ratio of trypsin to protein, digestion time, composition of a reaction buffer, and the amount of affinity-captured protein on a surface. Addition of a water-miscible organic solvent to a reaction buffer had no significant effect on the digestion efficiency under the optimized digestion conditions. The on-chip digestion method identified the affinity-captured bovine serum albumin (BSA), lysozyme, and ferritin at the level of around 100 fmol. Interestingly, the detected number of peptide hits through the on-chip digestion was almost similar regardless of the amount of captured protein ranging from low- to high-femtomole levels, whereas the efficiency of in-solution digestion decreased significantly as the amount of protein decreased to low-femtomole levels. The structural alignment of the peptide fragments from on-chip-digested BSA revealed that the limited exterior of the captured protein is subjected to attack by trypsin. The established detection procedures enabled the identification of BSA in the biological mixtures at the level of 0.1 ng/mL. The use of antibodies against the proteins involved in the metabolic pathway of L-threonine in Escherichia coli also led to discrimination of the respective target proteins from cell lysates.     

Laser desorption/ionization—Mass spectrometry using mesoporous silicate as matrix for the analysis of various molecules

In this study, mesoporous silicate was applied as a matrix for the analysis of various molecules from small molecules to medium sized peptides in laser desorption/ionization mass spectrometry. In contrast with conventional matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), the proposed approach desorption/ionization on mesoporous silicate mass spectrometry (DIOM-MS), significantly reduces the problem of matrix interference in low mass region and can be applied to the analysis of versatile chemicals including amino acids, synthetic drugs, peptides and others. In addition, distinctive advantage of DIOM-MS showed higher salt tolerance and could be applied to identify the proteins from the analysis of tryptically digested peptides. DIOM-MS has several availabilities such as easy sample preparation, rapid analysis of small molecules without noise, peptide analysis without organic matrix, high salt tolerance, versatile coupling with other separation techniques, and high throughput manner.     

Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry: a new approach to protein digestion in minutes

Accelerated proteolytic cleavage of proteins under controlled microwave irradiation has been achieved. Selective peptide fragmentation by endoproteases trypsin or lysine C led to smaller peptides that were analyzed by matrix-assisted laser desorption ionization (MALDI) or liquid chromatography-electrospray ionization (LC-ESI) techniques. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several biologically active proteins, including cytochrome c, ubiquitin, lysozyme, myoglobin, and interferon alpha-2b. Most important, using this novel approach digestion of proteins occurs in minutes, in contrast to the hours required by conventional methods.     

Can electromagnetic fields influence the structure and enzymatic digest of proteins? A critical evaluation of microwave-assisted proteomics protocols

This study reevaluates the putative advantages of microwave-assisted tryptic digests compared to conventionally heated protocols performed at the same temperature. An initial investigation of enzyme stability in a temperature range of 37-80°C demonstrated that trypsin activity declines sharply at temperatures above 60°C, regardless if microwave dielectric heating or conventional heating is employed. Tryptic digests of three proteins of different size (bovine serum albumin, cytochrome c and β-casein) were thus performed at 37°C and 50°C using both microwave and conventional heating applying accurate internal fiber-optic probe reaction temperature measurements. The impact of the heating method on protein degradation and peptide fragment generation was analyzed by SDS-PAGE and MALDI-TOF-MS. Time-dependent tryptic digestion of the three proteins and subsequent analysis of the corresponding cleavage products by MALDI-TOF provided virtually identical results for both microwave and conventional heating. In addition, the impact of electromagnetic field strength on the tertiary structure of trypsin and BSA was evaluated by molecular mechanics calculations. These simulations revealed that the applied field in a typical laboratory microwave reactor is 3-4 orders of magnitude too low to induce conformational changes in proteins or enzymes.     

Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The attachment protein or G protein of the A2 strain of human respiratory syncytial virus (RSV) was digested with trypsin and the resultant peptides separated by reverse-phase high-performance liquid chromatography (HPLC). One tryptic peptide produced a mass by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) corresponding to residues 152-187 with the four Cys residues of the ectodomain (residues 173, 176, 182, and 186) in disulfide linkage and absence of glycosylation. Sub-digestion of this tryptic peptide with pepsin and thermolysin produced peptides consistent with disulfide bonds between Cys173 and Cys186 and between Cys176 and Cys182. Analysis of ions produced by post-source decay of a peptic peptide during MALDI-TOF-MS revealed fragmentation of peptide bonds with minimal fission of an inter-chain disulfide bond. Ions produced by this unprecedented MALDI-induced post-source fragmentation corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products. These findings indicate that the ectodomain of the G protein has a non-glycosylated subdomain containing a "cystine noose."     

生物质谱技术在糖蛋白结构分析中的应用

生物质谱包括基质辅助激光解吸附飞行时间质谱及电喷雾质谱被广泛应用于生物样品如多肽、蛋白质及核酸的分析,由于这种具有软电离方式的质谱具有极高的灵敏度及准确度,目前也被成功地用于糖蛋白的结构分析,与普通的化学方法相比,质谱法快速、简单,结合网上数据库检索、凝集素亲和提取、二维凝胶电泳以及靶上直接酶切等新方法,可以提供糖蛋白的一级结构乃至高级结构的信息。     

Proteomics Approaches for Identification of Tumor Relevant Protein Targets in Pulmonary Squamous Cell Carcinoma by 2D-DIGE-MS

Potential markers for progression of pulmonary squamous cell carcinoma (SCC) were identified by examining samples of lung SCC and adjacent normal tissues using a combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF). The PANTHER System was used for gel image based quantification and statistical analysis. An analysis of proteomic data revealed that 323 protein spots showed significantly different levels of expression (P≤0.05) in lung SCC tissue compared to expression in normal lung tissue. A further analysis of these protein spots by MALDI-TOF-MS identified 81 different proteins. A systems biology approach was used to map these proteins to major pathways involved in numerous cellular processes, including localization, transport, cellular component organization, apoptosis, and reproduction. Additionally, the expression of several proteins in lung SCC and normal tissues was examined using immunohistochemistry and western blot. The functions of individual proteins are being further investigated and validated, and the results might provide new insights into the mechanism of lung SCC progression, potentially leading to the design of novel diagnostic and therapeutic strategies.     

MALDI-TOF质谱技术分析与鉴定病原细菌研究

本文通过基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术分析病原细菌的方法进行研究, 阐明影响分析结果的重要因素, 并建立了MALDI-TOF-MS 分析病原细菌的标准方法。对不同属、种和亚种的12株植物病原细菌进行全细胞分析结果表明：MALDI-TOF-MS能快速而准确的区分和鉴定病原细菌, 分析过程简单、灵敏度高。此法在细菌属、种、亚种和菌株水平上, 可快速、准确地区分和鉴定。