A highly efficient and automated method of purifying and desalting PCR products for analysis by electrospray ionization mass spectrometry

In this work we present a rapid and fully automated method to purify and desalt PCR products prior to analysis by electrospray ionization mass spectrometry. The protocol employs a commercial pipette tip packed with an anion-exchange resin and comprises four primary steps: tip pretreatment, sample loading, rinsing, and sample elution. This tip-based purification/desalting protocol has two distinct advantages over previously published methods. First, the protocol can be performed either manually (1-12 samples at a time), using a standard p10 manual pipette, or in a fully automated microtiter plate format (96 samples at a time) employing standard laboratory robotics. Additionally, the entire protocol from crude PCR product to an "electrosprayable" analyte solution requires only 10 microl of crude product and takes less than 20 min. Using capillary gel electrophoresis, we demonstrate an overall recovery efficiency of approximately 80% and demonstrate the exquisite desalting efficiency with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Using an internal mass standard we demonstrate sub-ppm mass measurement error which provides an unambiguous base composition for a 120-mer PCR product.     

Analysis of tetracyclines in raw urine by column-switching high-performance liquid chromatography and tandem mass spectrometry

The aim of the project was to develop a fast and reliable method for the quantification of the three tetracyclines: tetracycline, oxytetracycline and chlortetracycline in urine. The method is based on column-switching high-performance liquid chromatography with detection by MS–MS. Buffer is added to the sample before it is injected into the chromatographic system, and the first column which is an internal surface reversed-phase column separates the tetracyclines from the bulk of other compounds in urine. The tetracyclines are collected and concentrated on the analytical column before they are separated and eluted into the mass spectrometer in which the tetracycline are detected. The mass spectrometer is a triple quadrupole instrument and is equipped with an electrospray ion source. The MH⁺ ions are selected in the first quadrupole and collisionally activated in the collision cell. Upon collision, activation all three tetracyclines form fragment ions which could be assigned as: [M+H–H₂O–NH₃]⁺ which are selected in the sond mass filter. The detection limits for all three tetracyclines are about 10 ppb, and the calibration curves are linear from 10 to 1000 ppb.     

Removal of urea from urea-rich protein samples using metal ions in a microfluidic device

Urea is commonly used to lyse cultured cells and solubilize proteins from a biological source. In this study, after extracting biomolecules using a lysis buffer that included urea for an effective cleaning of protein from a urea-rich protein sample, a five-flow microfluidic desalting system was applied using the metal ions of Mn²⁺, Zn²⁺ and Fe³⁺, which have urea affinity-capturing properties. This device effectively removed urea from the sample phase of the microfluidic channel via the diffusion, with a difference of the concentration from the sample flow to both sides of the buffer flow, and an affinity of metal ions into the urea between the buffer phase and the affinity phase. The removal efficiency for the urea was 67, 64, and 63%, with concentrations of 50 mM Mn²⁺, 10 mM Zn²⁺, and 5 mM Fe³⁺ metal ions in the affinity phase, respectively. In addition, protein after desalting with the microfluidic device was improved to more than 10% of the relative activity, with a significant improvement of the signal of mass spectrum shown by MALDI-MS.     

Automatic Sampling and Analysis of Organics and Biomolecules by Capillary Action-Supported Contactless Atmospheric Pressure Ionization Mass Spectrometry

Contactless atmospheric pressure ionization (C-API) method has been recently developed for mass spectrometric analysis. A tapered capillary is used as both the sampling tube and spray emitter in C-API. No electric contact is required on the capillary tip during C-API mass spectrometric analysis. The simple design of the ionization method enables the automation of the C-API sampling system. In this study, we propose an automatic C-API sampling system consisting of a capillary (∼1 cm), an aluminium sample holder, and a movable XY stage for the mass spectrometric analysis of organics and biomolecules. The aluminium sample holder is controlled by the movable XY stage. The outlet of the C-API capillary is placed in front of the orifice of a mass spectrometer, whereas the sample well on the sample holder is moved underneath the capillary inlet. The sample droplet on the well can be readily infused into the C-API capillary through capillary action. When the sample solution reaches the capillary outlet, the sample spray is readily formed in the proximity of the mass spectrometer applied with a high electric field. The gas phase ions generated from the spray can be readily monitored by the mass spectrometer. We demonstrate that six samples can be analyzed in sequence within 3.5 min using this automatic C-API MS setup. Furthermore, the well containing the rinsing solvent is alternately arranged between the sample wells. Therefore, the C-API capillary could be readily flushed between runs. No carryover problems are observed during the analyses. The sample volume required for the C-API MS analysis is minimal, with less than 1 nL of the sample solution being sufficient for analysis. The feasibility of using this setup for quantitative analysis is also demonstrated.     

The Synthesis of Gibberethiones,Gibberellin-related Diterpenoids

The factors affecting desalting by gel chromatography were investigated both experimentally and theoretically. In most cases, the desalting of bovine serum albumin dissolved in an ammonium sulfate solution on a Sephadex G-25 Coarse column was chosen as a model experiment, and the effects of sample volume and concentration on the desalting were examined. The elution curves observed were in fairly good agreement with those calculated on the basis of a mass balance model, when the concentration of ammonium sulfate was lower than 0.4 m. Otherwise, the concentrations of solutes in the eluate were considerably diluted in comparison with the calculated results. Finally, useful charts were illustrated, from which appropriate operational conditions for desalting could be predicted.     

Simple and sensitive liquid chromatography-tandem mass spectrometry methods for quantification of paraquat in plasma and urine: application to experimental and clinical toxicological studies

Simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed and validated for quantification of paraquat (PQ) in plasma and urine. Plasma and urine sample preparation were carried out by one-step protein precipitation using cold acetonitrile (-20 to -10 °C). After centrifugation, an aliquot of 10 μL of supernatant was injected into a Kinetex? hydrophilic interaction chromatography (HILIC) column with a KrudKatcher? Ultra in-line filter. The chromatographic separation was achieved using the mobile phase mixture of 250 mM ammonium formate (with 0.8% aqueous formic acid) in water and acetonitrile at a flow rate of 0.3 mL/min. Detection was performed using an API2000 triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear over the concentration range of 10-5000 ng/mL, with an LLOQ of 10 ng/mL. The inter- and intra-day precision (% R.S.D.) were <8.5% and 6.4% for plasma and urine, respectively with the accuracies (%) within the range of 95.1-102.8%. PQ in plasma and urine samples was stable when stored at -70 °C for three freeze-thaw cycles. The methods were successfully applied to determine PQ concentration in rat and human samples.     

Quantitative analysis of an N-oxide metabolite by fast atom bombardment tandem mass spectrometry

An assay for the N-oxide metabolite of a benzazepine drug by fast atom bombardment ionization with tandem mass spectrometric analysis on a triple quadrupole mass spectrometer has been developed and validated for urine and plasma samples. This methodology allows analysis of this metabolite directly in crude sample extracts, without the need for extensive chromatography or sample derivatization. Quantification was accomplished with the use of a stable isotope analog of the analyte as an internal standard, using the selected reaction monitoring mode of operation.     

Gas chromatographic-negative-ion chemical ionization mass spectrometric determination of a new dihydropyridine calcium antagonist (MPC-1304) and its metabolites in human plasma and urine

A gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed for the determination of a new calcium antagonist, (±)-methyl 2-oxopropyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicar☐ylate, and its metabolites in plasma and urine. The sample was extracted with n-hexane-diethyl ether. The dried organic layer was subjected to acetylation: the aqueous layer was acidified and extracted with ethyl acetate, and after the ethyl acetate extract was dried the resulting residue was subjected to methylation. Aliquots of each reactant solution were injected into the gas chromatograph-mass spectrometer, equipped with a chemical ionization source and negative-ion monitoring mode, and analysed by the elected-ion monitoring method using deuterium-labelled internal standards. Detection was limited to 0.02–0.05 ng/ml of plasma and urine for each metabolite. A precise and sensitive assay for the determination of a new dihydropyridine calcium antagonist and its metabolites in plasma and urine was thus established.     

Evaluation of a novel, integrated approach using functionalized magnetic beads, bench-top MALDI-TOF-MS with prestructured sample supports, and pattern recognition software for profiling potential biomarkers in human plasma.

The advantage of using proteins and peptides as biomarkers is that they can be found readily in blood, urine, and other biological fluids. Such sample types are easily obtained and represent a potentially rich palette of biologically informative molecules. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) represents a key tool for rapidly interrogating such sample types. The goal of clinical proteomics is to harness the power of this tool for identifying novel, condition-specific protein fingerprints that may, in turn, lead to the elucidation and use of diseasespecific biomarkers that may be used to diagnose disease as well as to evaluate disease severity, disease progression, and intervention efficacy. Here we have evaluated a simple, affordable bench-top MALDI-TOF mass spectrometer to generate protein profiles from human plasma samples of asthma patients and healthy individuals. We achieve this profiling by using C8-functionalized magnetic beads that enrich a specific subset of plasma proteins based on their absorption by this resin. This step is followed by elution, transfer onto a prestructured sample support (AnchorChip technology), and analysis in a bench-top MALDI-TOF mass spectrometer (OmniFLEX) with AutoXecute acquisition control which enables automated operation with reproducible results. Resulting spectra are compiled and analyzed through the pattern recognition component of ClinProTools software. This approach in combination with ClinProTools software permits the investigator to rapidly scan for potential biomarker peptides/proteins in human plasma. The reproducibility of plasma profiles within and between days has been evaluated. The results show that the novel and facile approach with manual magnetic-bead sample preparation and a low-cost bench-top MALDI-TOF mass spectrometer is suitable for preliminary biomarker discovery studies.     

Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis.

We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer. The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis.     

High-performance liquid chromatographic separation and on-line mass spectrometric detection of saturated and unsaturated oligogalacturonic acids

A method for the simultaneous determination of saturated and unsaturated oligogalacturonic acids up to degree of polymerization (dp) of 7 by high-performance liquid chromatography (HPLC) is presented. For this purpose, a Cyclobond I 2000 column and a volatile mobile phase consisting of ammonium formate and methanol were used, allowing direct coupling of HPLC to a mass spectrometer via an electrospray interface (ESI-MS) without additional desalting. The analytical system was used for the characterization of digests obtained by incubation of polygalacturonic acid with commercial enzyme preparations.     

Characterization and quantification of Bcl-2 antisense G3139 and metabolites in plasma and urine by ion-pair reversed phase HPLC coupled with electrospray ion-trap mass spectrometry

A novel ion-pair reversed phase electrospray ionization (IP-RP-ESI) liquid chromatography-mass spectrometry (LC-MS) method has been developed for identification and quantification of Bcl-2 antisense phosphorothioate oligonucleotides G3139 and metabolites in plasma. This method utilized solid phase extraction for desalting and matrix removal and detection by an ion trap mass spectrometer. Resolution was accomplished on a micro C18 column eluted with a mobile phase consisting of hexafluoro-2-propanol and triethylamine in methanol at 50 degrees C. Five G3139 metabolites were identified in plasma and urine from treated patients and rats. A cassette HPLC-MS/MS quantification method for G3139 and three metabolites was developed and validated with a limit of quantification (LOQ) of 17.6 nM in human and rat plasma with acceptable precision and accuracy. Plasma pharmacokinetics of G3139 and metabolites in these species were described.     

Proteome analysis of intact proteins in complex mixtures

Analysis of intact protein mixtures by electrospray ionization mass spectrometry requires the resolution of a complex, overlapping set of multiply charged envelopes. To ascertain the ability of a moderate resolution mass spectrometer to resolve such mixtures, we have analyzed the soluble proteins of adult chick skeletal muscle. This is a highly specialized tissue showing a marked bias in expression of glycolytic enzymes in the soluble fraction. SDS-PAGE-resolved proteins were first identified by a combination of matrix-assisted laser desorption ionization time-of-flight (TOF) and electrospray ionization tandem mass spectrometry. Then the mixture of intact proteins was introduced into the electrospray source of a Q-TOF mass spectrometer either by direct infusion or via a C4 desalting trap. In both instances, the complex pattern of peaks could be resolved into true masses, and these masses could in many instances be reconciled with the masses predicted from the known protein sequences when qualified by expected co- and post-translational modifications. These included loss of the N-terminal initiator methionine residue and N-terminal acetylation. The ability to resolve such a complex mixture of proteins with a routine instrument is of considerable value in analyses of protein expression and in the confirmation of post-translational changes in mature proteins.     

蛋白质的肽质谱指纹图分析方法的优化

肽质谱指纹图分析是一种常用的蛋白质的鉴定方法.为了提高这种方法鉴定蛋白质时序列覆盖率和准确度,以6个标准蛋白质为分析样品,对几种不同的酶解肽段的浓缩、脱盐和点样方法进行了检验和优化.结果发现,将酶解肽段的浓缩体积控制在5μl以下和采用10mmolL柠檬酸铵缓冲液板上脱盐能提高蛋白质鉴定的准确度;在点样的时候,采用先点样品再点基质的方法能明显提高匹配肽段的个数和信噪比.这些优化的样品制备方法明显地提高了MALDITOF质谱肽质谱指纹图分析方法鉴定蛋白质的可靠性.     

Automating MALDI sample plate loading

We describe the design and implementation of a generic robotic solution to automate the loading of MALDI sample plates into a mass spectrometer. The soft- and hardware aspects are described together with the various safety issues that need to be addressed. The automation increases throughput by a factor of between 5- and 80-fold.     

Simple urinary sample preparation for proteomic analysis

Since the completion of the human genome sequence, attention has now focused on establishing reference maps of body fluids such as plasma and urine for detecting diagnostic markers of disease. Although some progress has been made, challenges still remain in the development of an optimal sample preparation method for proteomic analysis of urine. We have developed a simple and efficient urine preparation method for two-dimensional (2-D) gel electrophoresis which involves precipitation of proteins with simultaneous desalting. Acetonitrile precipitation produced 2-D gel separations with the highest resolution and the greatest number of protein spots compared to precipitation by other organic solvents. The method was applied to observe changes in the urinary proteome over a 6 week period and to establish a reference map of a healthy subject. A total of 339 proteins from 159 genes was identified from healthy male urine by peptide mass fingerprinting. The profiles of the urinary proteome at three times in 1 day and on four different days were compared and were found to vary in number and spatial location of the proteins on the map. The method was also shown to be applicable to the higher concentrations of protein found in the urine of an ovarian cancer subject. We have developed a facile and robust method for preparing urine for 2-D gels that will encourage further use of urine.     

Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g., dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.     

Preconcentration of organic solutes in urine by bubble bursting

Introduction

The chemical sensitivity of urine metabolomics analysis is greatly compromised due to the large amounts of inorganic salts in urine (NaCl, KCl), which are detrimental to analytical instrumentation, e.g. chromatographic columns or mass spectrometers. Traditional desalting approaches applied to urine pretreatment suffer from the chemical losses, which reduce the information depth of analysis.

Objectives

We aimed to test a simple approach for the simultaneous preconcentration and desalting of organic solutes in urine based on the collection of induced bursting bubble aerosols above the surface of urine samples.

Method

Bursting bubbles were generated at ambient conditions by feeding gas through an air diffuser at the bottom of diluted (200 times in ultrapure water) urine solution (50–500 mL). Collected aerosols were analyzed by the direct-infusion electrospray ionization mass spectrometry (ESI–MS).

Results

The simultaneous preconcentration (ca. 6–12 fold) and desalting (ca. six–tenfold) of organic solutes in urine was achieved by the bursting bubble sample pretreatment, which allowed ca. three-times higher number of identified urine metabolites by high-resolution MS analysis. No chemical losses due to bubbling were observed. The increased degree of MS data clustering was demonstrated on the principal component analysis of data sets from the urine of healthy people and from the urine people with renal insufficiency. At least ten times higher sensitivity of trace drug detection in urine was demonstrated for clenbuterol and salbutamol.

Conclusion

Our results indicate the high versatility of bubble bursting as a simple pretreatment approach to enhance the chemical depth and sensitivity of urine analysis. The approach could be attractive for personalized medicine as well as for the diagnostics of renal disorders of different etiology (diabetic nephropathy, chronic renal failure, transplant-associated complications, oncological disorders).

Graphical Abstract

Urine desalting and preconcentration in bursting bubbles.

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Thermospray liquid chromatography-mass spectrometry of nucleosides and of enzymatic hydrolysates of nucleic acids.

Nucleosides dissolved in aqueous buffered solutions undergo ionization during direct introduction of the solution into a mass spectrometer using a thermospray interface. The principal ions formed represent the protonated molecule, the corresponding protonated free base, and sugar. In addition to potential utility for characterization of new nucleosides, the technique can be used to monitor nucleosides separated from enzymatic hydrolysates by liquid chromatography. The selectivity of chromatographic detection is significantly greater than with UV absorbance alone so that independent detection of components of unresolved chromatographic peaks is usually possible. Detection limits, with signal/noise greater than 10 for most nucleosides, are approximately 0.1-1 ng per component for selected ion monitoring and 10-50 ng for full-scan mass spectra. Examples are given from the detection of modified nucleosides in enzymatic hydrolysates of 0.05 A260 units (2.5 micrograms) of rabbit liver tRNAVal and of unfractionated H. volcanii tRNA.     

Sensitive headspace gas chromatography-mass spectrometry determination of trihalomethanes in urine

A sensitive and straightforward method for the determination of trihalomethanes (THMs) in urine by using headspace extraction technique has been developed. Chemical and instrumental variables were studied in order to optimize the method for sensitivity: an excess of KCl (4 g per 12 ml of urine), an oven temperature of 85 degrees C and an equilibration time of 30 min were selected. The use of the mass spectrometer in selected ion monitoring mode allows achieving linear ranges between 10 and 5000 ng/l and detection limits from 3 to 10 ng/l, for 12 ml of urine. The stability of the urine sample during storage at 4 and -20 degrees C was also evaluated: THMs remained stable for up to 2 days and 2 months, respectively. Finally, the method was successfully applied to study the THM uptake from swimmers of an indoor swimming pool, as well as non-swimmers. This study revealed that the concentrations of THMs in urine increased approximately three times for chloroform and bromodichloromethane after swimming activity. In addition, THMs in unchanged form were mainly excreted within 2-3h after the end of exposure.