一种基于肽质量指纹谱技术鉴定蓖麻毒素的新方法

应用基质辅助激光解吸电离飞行时间质谱(MALD I-TOF/MS)法实现了对蓖麻毒素(R ic in)的鉴定。测定蓖麻毒素的分子量为62925Da,实现了蓖麻粗毒的凝胶电泳分离,并通过胶内酶切获得了蓖麻毒素的肽质量指纹谱(PMF);经过数据库检索,在输入检索的22条肽段中有17条获得了匹配。检索结果显示,利用生物质谱技术是鉴定蓖麻毒素的最有效的新方法之一。     

Rapid and sensitive identification of ricin in environmental samples based on lactamyl agarose beads using LC‐MS/MS (MRM)

Ricin, a plant‐derived toxin extracted from the seeds of Ricinus communis (castor bean plant), is one of the most toxic proteins known. Ricin's high toxicity, widespread availability, and ease of its extraction make it a potential agent for bioterrorist attacks. Most ricin detection methods are based on immunoassays. These methods may suffer from low efficiency in matrices containing interfering substances, or from false positive results due to antibody cross reactivity, with highly homologous proteins. In this study, we have developed a simple, rapid, sensitive, and selective mass spectrometry assay, for the identification of ricin in complex environmental samples. This assay involves three main stages: (a) Ricin affinity capture by commercial lactamyl‐agarose (LA) beads. (b) Tryptic digestion. (c) LC‐MS/MS (MRM) analysis of tryptic fragments. The assay was validated using 60 diverse environmental samples such as soil, asphalt, and vegetation, taken from various geographic regions. The assay's selectivity was established in the presence of high concentrations of competing lectin interferences. Based on our findings, we have defined strict criteria for unambiguous identification of ricin. Our novel method, which combines affinity capture beads followed by MRM‐based analysis, enabled the identification of 1 ppb ricin spiked into complex environmental matrices. This methodology has the potential to be extended for the identification of ricin in body fluids from individuals exposed (deliberately or accidentally) to the toxin, contaminated food or for the detection of the entire family of RIP‐II toxins, by applying multiplex format.     

Determination of ricin by nano liquid chromatography/mass spectrometry after extraction using lactose-immobilized monolithic silica spin column

Ricin is a glycosylated proteinous toxin that is registered as toxic substance by Chemical Weapons convention. Current detection methods can result in false negatives and/or positives, and their criteria are not based on the identification of the protein amino acid sequences. In this study, lactose-immobilized monolithic silica extraction followed by tryptic digestion and liquid chromatography/mass spectrometry (LC/MS) was developed as a method for rapid and accurate determination of ricin. Lactose, which was immobilized on monolithic silica, was used as a capture ligand for ricin extraction from the sample solution, and the silica was supported in a disk-packed spin column. Recovery of ricin was more than 40%. After extraction, the extract was digested with trypsin and analyzed by LC/MS. The accurate masses of molecular ions and MS/MS spectra of the separated peptide peaks were measured by Fourier transform-MS and linear iontrap-MS, respectively. Six peptides, which were derived from the ricin A-(m/z 537.8, 448.8 and 586.8) and B-chains (m/z 701.3, 647.8 and 616.8), were chosen as marker peptides for the identification of ricin. Among these marker peptides, two peptides were ricin-specific. This method was applied to the determination of ricin from crude samples. The monolithic silica extraction removed most contaminant peaks from the total ion chromatogram of the sample, and the six marker peptides were clearly detected by LC/MS. It takes about 5 h for detection and identification of more than 8 ng/ml of ricin through the whole handling, and this procedure will be able to deal with the terrorism using chemical weapon.     

Identification and quantification of ricin in biomedical samples by magnetic immunocapture enrichment and liquid chromatography electrospray ionization tandem mass spectrometry

Ricin is a toxic protein derived from castor beans and composed of a cytotoxic A chain and a galactose-binding B chain linked by a disulfide bond, which can inhibit protein synthesis and cause cell death. Owing to its high toxicity, ease of preparation, and lack of medical countermeasures, ricin has been listed as both chemical and biological warfare agents. For homeland security or public safety, the unambiguous, sensitive, and rapid methods for identification and quantification of ricin in complicated matrices are of urgent need. Mass spectrometric analysis, which provides specific and sensitive characterization of protein, can be applied to confirm and quantify ricin. Here, we report a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method in which ricin was extracted and enriched from serum by immunocapture using anti-ricin monoclonal antibody 3D74 linked to magnetic beads, then digested by trypsin, and analyzed by LC-ESI-MS/MS. Among 19 distinct peptides observed in LC-quadrupole/time of flight-MS (LC-QTOF-MS), two specific and sensitive peptides, T_7A (⁴⁹VGLPINQR⁵⁶) and T_14B (¹⁸⁸DNCLTSDSNIR¹⁹⁸), were chosen, and a highly sensitive determination of ricin was established in LC-triple quadrupole-MS (LC-QqQ-MS) operating in multiple reaction monitoring mode. These specific peptides can definitely distinguish ricin from the homologous protein Ricinus communis agglutinin (RCA120), even though the amino acid sequence homology of the A-chain of ricin and RCA120 is up to ca. 93 % and that of B-chain is ca. 85 %. Furthermore, peptide T_7A was preferred in the quantification of ricin because its sensitivity was at least one order of magnitude higher than that of the peptide T_14B. Combined with immunocapture enrichment, this method provided a limit of detection of ca. 2.5 ng/mL and the limit of quantification was ca. 5 ng/mL of ricin in serum, respectively. Both precision and accuracy of this method were determined and the RSD was less than 15 %. This established method was then applied to measure ricin in serum samples collected from rats exposed to ricin at the dosage of 50 μg/kg in an intravenous injection manner. The results showed that ca. 10 ng/mL of the residual ricin in poisoned rats serum could be detected even at 12 h after exposure.     

Coupling of CE‐MS for protein and peptide analysis

The review is focused on the latest developments in the analysis of proteins and peptides by capillary electrophoresis techniques coupled to mass spectrometry. First, the methodology and instrumentation are overviewed. In this section, recent progress in capillary electrophoresis with mass spectrometry interfaces and capillary electrophoresis with matrix‐assisted laser desorption/ionization is mentioned, as well as separation tasks. The second part is devoted to applications—mainly bottom‐up and top‐down proteomics. It is obvious that capillary electrophoresis with mass spectrometry methods are well suited for peptide and protein analysis (proteomic research) and it is described how these techniques are complementary and not competitive with the often used liquid chromatography with mass spectrometry methods.     

Rapid screening and determination of designer drugs in saliva by a nib‐assisted paper spray‐mass spectrometry and separation technique

A method for the rapid screening and determination of amphetamine‐type designer drugs in saliva by a novel nib‐assisted paper spray‐mass spectrometry procedure is described. Under optimized conditions, the limit of detections for amphetamine derivatives (model samples: o‐, m‐, p‐chloroamphetamine and o‐, m‐, p‐fluoroamphetamine, respectively) were determined to 0.1 μg/mL by the nib‐assisted paper spray‐mass spectrometry method. This method is easier and has a higher sensitivity than similar methodologies, including atmospheric pressure/matrix‐assisted laser desorption ionization mass spectrometry and electrospray‐assisted laser desorption ionization/mass spectrometry. Data obtained using more classical separation methods, including liquid chromatography and capillary electrophoresis, are also reported.     

剧毒性Ⅱ型核糖体失活蛋白蓖麻毒素和相思子毒素的检测鉴定方法研究进展

Ⅱ型核糖体失活蛋白(RIPs)是一类重要的蛋白毒素,该类毒素大都具有一对二硫键连接的A-B链结构特征,B链具有半乳糖结合特性,能够与真核细胞膜表面受体特异性结合,将具有N-糖苷酶活性的A链导入细胞,与核糖体特定位点发生脱嘌呤作用使核糖体失活,最终通过抑制蛋白质合成而展现出细胞毒性。Ⅱ型RIPs毒素毒性极强,来源于植物的蓖麻毒素(ricin)和相思子毒素(abrin)的毒性分别是神经性毒剂维埃克斯(Vx)的385倍和2885倍。同时,该类毒素来源广泛、易于制备、稳定性好,成为一类潜在化生恐怖战剂,受到国内外广泛关注,其中蓖麻毒素作为唯一的蛋白毒素被收录于禁止化学武器公约禁控清单。近年来发生的多次蓖麻毒素邮件恐怖事件,进一步促进了有关Ⅱ型RIPs毒素的准确、灵敏、快速的检测鉴定技术的发展。剧毒性Ⅱ型RIPs毒素的检测鉴定方法主要涉及免疫分析法为代表的特异性识别和生物质谱分析为主的定性定量检测方法,以及基于脱嘌呤反应活性和细胞毒性的毒素活性检测方法。基于抗原-抗体作用的免疫检测法及基于寡核苷酸适配体的特异性识别检测法具有速度快、灵敏度高的优势,但对于复杂样品中高度同源蛋白的检测,易产生假阳性结果。随着生物质谱技术的快速发展,电喷雾离化(ESI)或基质辅助激光解吸离化(MALDI)等技术广泛应用于蛋白质的准确鉴定,不仅能够提供蛋白毒素的准确分子量和结构序列信息,而且能够实现准确定量。酶解质谱法是应用最为广泛的检测鉴定方法,通过酶解肽指纹谱分析,实现蛋白毒素的准确鉴定;对于复杂样品中蛋白毒素的分析,通过多种蛋白酶酶解策略获得丰富的特异性肽段标志物,然后进行肽段标志物的靶向质谱分析从而获得准确的定性及定量信息,方法有效提升了Ⅱ型RIPs毒素鉴定的准确度和灵敏度。免疫分析法和生物质谱法能够准确鉴定Ⅱ型RIPs毒素,但无法识别毒素是否还保持毒性。对于Ⅱ型RIPs毒素的活性分析,主要包括基于N-糖苷酶活性的脱嘌呤反应测定法和细胞毒性测定法,两种方法均可实现毒素毒性的简便、快速、灵敏的分析检测,是Ⅱ型RIPs毒素检测方法的有效补充。由于该类毒素的高度敏感性,国际禁止化学武器组织(OPCW)对相关样品中Ⅱ型RIPs毒素的分析提出了唯一性鉴定的技术要求。该文引用了Ⅱ型RIPs毒素及其检测方法相关的70篇文献,综述了以上Ⅱ型RIPs毒素的结构性质、中毒机理及典型剧毒性Ⅱ型RIPs毒素检测方法的研究进展,对不同检测方法的特点和应用潜力进行了总结,并结合OPCW对Ⅱ型RIPs毒素唯一性鉴定的技术需求,展望了未来Ⅱ型RIPs毒素检测技术研究的发展趋势。     

Development and validation of an ELISA kit for the detection of ricin toxins from biological specimens and environmental samples

Ricin is a toxin that can be easily extracted from seeds of Ricinus communis plants. Ricin is considered to be a major bio-threat as it can be freely and easily acquired in large quantities. A deliberate release of such toxin in civilian populations would very likely overwhelm existing public health systems, resulting in public fear and social unrest. There is currently no commercially available or FDA-approved prophylaxis such as vaccines, or therapeutic antitoxins or antidotes, available for ricin intoxication. Patient treatment is typically supportive care based on symptoms, often designed to reinforce the body’s natural response. This paper describes the development and validation of a robust ELISA test kit, which can be used to screen for ricin in biological specimens such as whole blood and faeces. Faecal specimens are shown in this study to have better diagnostic sensitivity and a wider diagnostic window compared to whole blood. From these results, it is concluded that faeces is the most suitable clinical specimen for diagnosis of ricin poisoning via the oral route. The ELISA test kit can also detect ricin in environmental samples. An advantage of this ELISA kit over other commercial off-the-shelf (COTS) detection kits currently on the market that are developed to screen environmental samples only is its ability to diagnose ricin poisoning from clinical specimens as well as detect ricin from environmental samples.     

Mechanistic Insights into the Synthesis of Fully Condensed Polyhedral Octaphenylsilsesquioxane

A comprehensive study on the efficient one‐pot synthesis of polyhedral octaphenylsilsesquioxane (OPS) is reported via the hydrolytic condensation of phenyltrimethoxysilane (PTMS) in the presence of basic catalyst to investigate the specific synthesis mechanism. The synthetic reactions are monitored with real time infrared (RTIR) spectroscopy. Then RTIR coupled with ²⁹Si nuclear magnetic resonance spectroscopy (NMR) and matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF‐MS) are used to monitor the reactions and identify the intermediary species during the reaction. The rapid hydrolysis of PTMS is detected by RTIR. Contrary to previous reports, the ladder‐like structured species are identified as intermediates during the reaction process. It is suggested that formation of caged T₈ OPS is realized through the chain break and rearrangement of the ladder‐like phenyltrimethoxysilanes. Accordingly, a scheme from hydrolysis of the PTMS to formation of the OPS is provided.     

Study of the forced degradation behavior of prasugrel hydrochloride by liquid chromatography with mass spectrometry and liquid chromatography with NMR detection and prediction of the toxicity of the characterized degradation products

Prasugrel was subjected to forced degradation studies under conditions of hydrolysis (acid, base, and neutral), photolysis, oxidation, and thermal stress. The drug showed liability in hydrolytic as well as oxidative conditions, resulting in a total of four degradation products. In order to characterize the latter, initially mass fragmentation pathway of the drug was established with the help of mass spectrometry/time‐of‐flight, multiple stage mass spectrometry and hydrogen/deuterium exchange data. The degradation products were then separated on a C₁₈ column using a stability‐indicating volatile buffer method, which was later extended to liquid chromatography‐mass spectrometry studies. The latter highlighted that three degradation products had the same molecular mass, while one was different. To characterize all, their mass fragmentation pathways were established in the same manner as the drug. Subsequently, liquid chromatography‐nuclear magnetic resonance (NMR) spectroscopy data were collected. Proton and correlation liquid chromatography with NMR spectroscopy studies highlighted existence of diastereomeric behavior in one pair of degradation products. Lastly, toxicity prediction by computer‐assisted technology (TOPKAT) and deductive estimation of risk from existing knowledge (DEREK) software were employed to assess in silico toxicity of the characterized degradation products.     

Improved in‐solution trypsin digestion method for methanol–chloroform precipitated cellular proteomics sample

Methanol–chloroform based protein precipitation is an essential step in many liquid chromatography–tandem mass spectrometry‐based cellular proteomics applications. However, re‐solubilization of the total protein precipitate is difficult using regular in‐solution digestion protocol. Sodium deoxycholate is reported as an efficient surfactant for re‐solubilization of membrane fractions. In this study, we demonstrated an application combining methanol–chloroform based protein precipitations and deoxycholic acid assisted re‐solubilization of pellets to evaluate the improvement of protein identifications in mass spectrometry‐based bottom‐up proteomics. We evaluated the modified method using an equal amount of Raw 264.7 mouse macrophage cell lysate. Detailed in‐solution trypsin digestion studies were presented on methanol–chloroform precipitated samples with or without deoxycholic acid treatments and compared with popular sample digestion methods. A mass spectrometric analysis confirmed an 82% increase in protein identification in deoxycholic acid‐treated samples compared to other established methods. Furthermore, liquid chromatography–tandem mass spectrometry analysis of an equal amount of proteins from methanol–chloroform precipitated, and methanol–chloroform/deoxycholic acid‐treated macrophage cell lysate showed a 14% increase and 27% unique protein identifications. We believe this improved digestion method could be a complementary or alternative method for mammalian cell sample preparations where sodium dodecyl sulfate based lysis buffer is frequently used.     

Carrier ampholyte‐free isoelectric focusing on a paper‐based analytical device for the fractionation of proteins

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte‐free isoelectric focusing on paper‐based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometry. This paper‐based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost‐effective protein sample clean‐up method for target protein analysis with mass spectrometry.     

The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry

Qualitative and quantitative analysis of post‐translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well‐characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined. Our results clearly confirm that the ion yields are generally lower and the signal intensities are reduced with phosphopeptides than with their nonphosphorylated analogs and that this has to be taken into account in MALDI and ESI mass spectrometry. However, the average reduction of ion yield caused by phosphorylation is more pronounced with MALDI than with ESI. The unpredictable impact of phosphorylation does not depend on the hydrophobicity and net charge of the peptide, indicating that reliable quantification of phosphorylation by mass spectrometry requires the use of internal standards. In contrast to phosphorylation, mono‐ and dimethylated peptides frequently exhibit increased signal intensities in MALDI mass spectrometry (MALDI‐MS). Despite minor matrix‐dependent variability, MALDI methods are well suited for the sensitive detection of dimethylated arginine and lysine peptides. Mono‐ and dimethylation of the arginine guanidino group did not significantly influence the ionization efficiency of peptides in ESI‐MS. Copyright © 2009 John Wiley & Sons, Ltd.     

MALDI‐TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varieties

A procedure for identification of malting barley varieties using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of ethanol‐soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI‐TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry‐based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd.     

Verification of cheeses authenticity by mass spectrometry

Cheeses are a group of fermented dairy products that are produced all over the world in various forms and flavours. Milk, especially sheep or goat milk, is still regarded as an expensive raw material in the world, which makes milk and milk products highly attractive as a fraud target. Most often, such fraud includes partial or complete substitution with cheaper sorts of milk (e.g. bovine milk). The aim of this work was to verify the authenticity of 27 cheeses commonly emerging on the Czech food market. The cheeses were distinguished on the basis of milk animal species origin. For this purpose, two mass spectrometry techniques were used: matrix‐assisted laser desorption/ionization with time of flight mass spectrometry together with principal component analysis method and liquid chromatography coupled with electrospray ionization and quadrupole time‐of‐flight mass spectrometry. The results were a partial success, because the cheeses could only be partially distinguished with the first mass spectrometry technique probably because of the influence of some protein additive materials in cheeses. The second technique allowed for collecting higher quality results and thus appears to be highly suitable for the research task.     

Decellularization of intact tissue enables MALDI imaging mass spectrometry analysis of the extracellular matrix

Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful molecular mapping technology that offers unbiased visualization of the spatial arrangement of biomolecules in tissue. Although there has been a significant increase in the number of applications employing this technology, the extracellular matrix (ECM) has received little attention, likely because ECM proteins are mostly large, insoluble and heavily cross‐linked. We have developed a new sample preparation approach to enable MALDI IMS analysis of ECM proteins in tissue. Prior to freezing and sectioning, intact tissues are decellularized by incubation in sodium dodecyl sulfate. Decellularization removes the highly abundant, soluble species that dominate a MALDI IMS spectrum while preserving the structural integrity of the ECM. In situ tryptic hydrolysis and imaging of tryptic peptides are then carried out to accommodate the large sizes of ECM proteins. This new approach allows the use of MALDI IMS for identification of spatially specific changes in ECM protein expression and modification in tissue. Copyright © 2015 John Wiley & Sons, Ltd.     

Acid hydrolysis followed by matrix‐assisted laser desorption/ionization mass spectrometry for the rapid diagnosis of serum protein biomarkers in patients with major depression

We have developed a technique combining acid hydrolysis with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) for the rapid study of the changes in the levels of positive and negative acute phase protein biomarkers in the sera of patients suffering from major depression. The serum proteins were first precipitated using an organic solvent; after separation, they were subjected to hydrochloric acid hydrolysis (6 M HCl) for 10 min. The resulting peptides were characterized using MALDI‐TOF MS. Short‐term treatment of the serum proteins with HCl efficiently removed interference from the abundant protein – albumin – and produced abundant peptide ion signals in the range of m/z 4000–10 000. This approach allowed us to rapidly detect the peptide ions originating from transferrin (a common negative acute phase protein) and fibrinogen (a common positive acute phase protein). The average ratios and (standard deviations) of the ion signals derived from transferrin/fibrinogen were 3.58 (±1.93) for the healthy control subjects and 1.02 (±0.52) for the patients suffering from major depression. The differences in transferrin/fibrinogen ratios between healthy controls and patients suggest that major depression will induce internal inflammation and cause either an increase in the level of fibrinogen or a decrease in the level of transferrin. Copyright © 2009 John Wiley & Sons, Ltd.     

Detection of ricin in food using electrochemiluminescence-based technology

Ricin is a toxic ribosome inactivating protein (RIP-II) present in beans of the castor plant, Ricinus communis. Its potential as a biodefense threat has made the rapid, sensitive detection of ricin in food important to the U.S. Food and Drug Administration. Samples of juice, dairy products, soda, vegetables, bakery products, chocolate, and condiments were spiked with varying concentrations of ricin and analyzed using a 96-well format, electrochemiluminescence (ECL) immunoassay. Assay configurations included the use of a monoclonal capture antibody coupled with either a polyclonal or monoclonal detector antibody. The samples and detector antibodies were either added sequentially or in combination during the capture step. Using the polyclonal antibody, 0.04 ng/mL ricin was detected in analytical samples prepared from several beverages. By simultaneously incubating the sample with detector antibody, it was possible to decrease the assay time to a single 20 min incubation step with a limit of detection <10 ng/mL. Assays run according to this single incubation step exhibited a hook effect (decrease in signal at high concentrations of ricin), but because of the large signal-to-noise ratio associated with the ECL assay, the response remained above background and detectable. Thus, the ECL assay was uniquely suited for the screening of samples for ricin.     

Characterization of novel hydrolysis products of carbapenems by electrospray ionization mass spectrometry

Carbapenems, including meropenem and imipenem, exhibit low stability against acid or base reagents. The fragmentation behavior of meropenem and its acid hydrolysis products was investigated by Fourier transform ion cyclotron resonance electrospray ionization tandem mass spectrometry and ion trap tandem multi‐stage mass spectrometry in both positive and negative ion mode. Only one neutral loss of CO₂ was observed from the precursor ion to the MS⁴ product ions for the acid hydrolysis product and this behavior did not correspond to that expected for the previously accepted 1‐pyrroline or 2‐pyrroline structure with two carbonyl acid units. The unknown product was then proposed to be 2‐(4‐(5‐(dimethylcarbamoyl)pyrrolidin‐3‐ylthio)‐5‐imino‐3‐methyl‐6‐oxotetrahydro‐2H‐pyran‐2‐yl)‐3‐hydroxybutanoic acid on the basis of the multi‐stage mass spectrometric and accurate mass data. A similar acid hydrolysis product of imipenem was also identified by mass spectrometry, confirming that these carbapenems had the same acid hydrolysis behavior. The proposed structures were further confirmed by NMR experiments. Copyright © 2009 John Wiley & Sons, Ltd.     

Sequential electroelution and mass spectroscopic identification of intact sodium dodecyl sulfate-proteins labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester

A gel electrophoresis apparatus capable of scanning the migration path fluorometrically and of computer-directed electroelution of bands was applied to the mass spectrometric identification of sequentially electroeluted 5(6)-carboxyfluorescein-N-hydrosuccinimide ester (FLUOS)-labeled sodium dodedyl sulfate (SDS)-proteins. The masses of four electroeluted SDS-proteins under study determined by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry are changed by 1% due to their reaction with FLUOS in a 1:5 molar ratio of protein:label, allowing for the identification of the labeled intact proteins on the basis of mass. More importantly, the partial (10 or 50%) derivatization of proteins with FLUOS does not preclude their tryptic hydrolysis, and identification of the protein on the basis of the mass spectrometric analysis of its tryptic peptides. Potentially, the procedure allows for the automated mass spectrometric identification of SDS-proteins globally labeled with FLUOS and electrophoretically separated, without need for any gel sectioning.