Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis

We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 immobilized pH gradients (IPGs) over 54 cm (Poland et al., Electrophoresis 2003, 24, 1271). Here we show that similar results can be conveniently achieved using commercially available IPGs of appropriate pH ranges positioned end-on-end in series during electrophoresis, which we term "daisy chain IEF". Proteins efficiently electrophorese from one IPG to another during IEF by traversing buffer-filled porous bridges between the serial IPGs. A variety of materials can function as bridges, including paper, polyacrylamide gels or even IPGs. The quality of two-dimensional (2-D) protein patterns is not apparently worse than that generated by conventional IEF using the same individual IPGs. A major advantage of this method is that sample is consumed efficiently, without the requirement for preliminary steps, such as chamber IEF. This advantage is pronounced when working with extremely limited sources of samples, such as with clinical biopsies or cellular subfractions. The present study was limited by the commercial availability of suitable pH gradients. Proteomics analyses could be further improved if commercial vendors would manufacture IPGs with suitable pH ranges to achieve high resolution (approximately 100 cm) IEF separation of proteins in one electrophoretic step over the pH range 2-12.     

Evaluation of peptide fractionation strategies used in proteome analysis

Peptide fractionation is extremely important for the comprehensive analysis of complex protein mixtures. Although a few comparisons of the relative separation efficiencies of 2‐D methodologies using complex biological samples have appeared, a systematic evaluation was conducted in this study. Four different fractionation methods, namely strong‐cation exchange, hydrophilic interaction chromatography, alkaline‐RP and solution isoelectric focusing, which can be used prior to LC‐MS/MS analysis, were compared. Strong‐cation exchange × RPLC was used after desalting the sample; significantly more proteins were identified, compared with the nondesalted sample (1990 and 1375). We also found that the use of a combination of analytical methods resulted in a dramatic increase in the number of unique peptides that could be identified, compared with only a small increase in protein levels. The increased number of distinct peptides that can be identified is especially beneficial, not only for unequivocally identifying proteins but also for proteomic studies involving posttranslational modifications and peptide‐based quantification approaches using stable isotope labeling. The identification and quantification of more peptides per protein provide valuable information that improves both the quantification of, and confidence of protein identification.     

Liquid chromatography/mass spectrometry-based metabolic profiling to elucidate chemical differences of tobacco leaves between Zimbabwe and China

An approach was developed for extracting and analyzing the chemical components of tobacco leaves based on solvent extraction and rapid & resolution liquid chromatography/quadrupole time-of-flight mass spectrometry analysis. Two solvents with different polarities were used to extract hydrophilic components and hydrophobic components, respectively, the combined analytical data can provide a "global" view of metabolites. Based on the evaluation of parallel samples, it was found that this approach provided good repeatability, accurate and reliable profiling data, and is suitable for the metabolomics study of tobacco leaves. In order to find the chemical component differences of tobacco leaves, 56 samples from Zimbabwe and China were analyzed using the developed method. The metabolite data were processed by multivariate statistic technique; an obvious group classification between Zimbabwe and China was observed, 14 significantly changed compounds were found, and 9 of them were identified.     

Development of a simple ampholyte-free isoelectric focusing slab electrophoresis for protein fractionation

Sample preparation is often necessary to separate and concentrate various compounds prior to analysis of complex samples. In this regard, isoelectric focusing (IEF) is one of the best sample preparation methods. With this approach, however, carrier ampholytes have to be introduced into the samples, which may result in matrix interferences. In this paper, a simple ampholyte-free IEF free-flow electrophoresis design was developed for the separation of proteins. beta-Lactoglobulin, hemoglobin, myoglobin and cytochrome c were selected as model analytes. The experimental design took advantage of the electrolysis-driven production of H(+) and OH(-) ions that migrated from the anode and cathode, respectively, establishing a pH gradient spanning from 2.3 to 8.9. The separation chamber was filled with silanized glass beads as a support medium. Dialysis membranes were mounted at the two sides of the separation chamber (made of glass slides) and sealed with 2% agarose gel. The separated proteins drained from the outlets of the separation chamber and could be successfully collected into small glass tubes. The focusing process was visually observed and the separation was confirmed by capillary isoelectric focusing (cIEF) with pI markers.     

Low-molecular weight protein profiling of genetically modified maize using fast liquid chromatography electrospray ionization and time-of-flight mass spectrometry

In this work, the use of liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC-TOFMS) has been evaluated for the profiling of relatively low-molecular weight protein species in both genetically modified (GM) and non-GM maize. The proposed approach consisted of a straightforward sample fractionation with different water and ethanol-based buffer solutions followed by separation and detection of the protein species using liquid chromatography with a small particle size (1.8 μm) C(18) column and electrospray-time-of-flight mass spectrometry detection in the positive ionization mode. The fractionation of maize reference material containing different content of transgenic material (from 0 to 5% GM) led to five different fractions (albumins, globulins, zeins, zein-like glutelins, and glutelins), all of them containing different protein species (from 2 to 52 different species in each fraction). Some relevant differences in the quantity and types of protein species were observed in the different fractions of the reference material (with different GM contents) tested, thus revealing the potential use of the proposed approach for fast protein profiling and to detect tentative GMO markers in maize.     

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.     

A generic approach to the impurity profiling of drugs using standardised and independent capillary zone electrophoresis methods coupled to electrospray ionisation mass spectrometry

Three standardised, capillary zone electrophoresis-electrospray ionisation mass spectrometry (CZE-ESI-MS) methods were developed for the analysis of six drug candidates and their respective process-related impurities comprising a total of 22 analytes with a range of functional groups and lipophilicities. The selected background electrolyte conditions were found to be: 60/40 v/v 10 mM ammonium formate pH 3.5/organic, 60/40 v/v 10 mM ammonium acetate pH 7.0/organic and 10 mM piperidine, pH 10.5, where the organic solvent is 50/50 v/v methanol/acetonitrile. The coaxial sheath flow consisted of either 0.1% v/v formic acid in 50/50 v/v methanol/water, or 10 mM ammonium acetate in 50/50 v/v methanol/water, depending on the mixture being analysed. Factor analysis and informational theory were used to quantify the orthogonality of the methods and predict their complementarities. The three selected CZE-ESI-MS methods allowed the identification of 21 out of 22 of all the drug candidates and their process-related impurities and provided orthogonality with four established high-performance liquid chromatography-mass spectrometry (HPLC-MS) methods. These methodologies therefore form the basis of a generic approach to impurity profiling of pharmaceutical drug candidates and can be applied with little or no analytical method development, thereby offering significant resource and time savings.     

Glossary of terms for separations coupled to mass spectrometry

This document is a glossary of terms for separations coupled to mass spectrometry. It covers gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and supercritical fluid chromatography/mass spectrometry and the sample introduction, ionization, and data analysis methods used with these combined techniques.     

Systematic approach to optimize a pretreatment method for ultrasensitive liquid chromatography with tandem mass spectrometry analysis of multiple target compounds in biological samples

In current approaches for new drug development, highly sensitive and robust analytical methods for the determination of test compounds in biological samples are essential. These analytical methods should be optimized for every target compound. However, for biological samples that contain multiple compounds as new drug candidates obtained by cassette dosing tests, it would be preferable to develop a single method that allows the determination of all compounds at once. This study aims to establish a systematic approach that enables a selection of the most appropriate pretreatment method for multiple target compounds without the use of their chemical information. We investigated the retention times of 27 known compounds under different mobile phase conditions and determined the required pretreatment of human plasma samples using several solid‐phase and liquid–liquid extractions. From the relationship between retention time and recovery in a principal component analysis, appropriate pretreatments were categorized into several types. Based on the category, we have optimized a pretreatment method for the identification of three calcium channel blockers in human plasma. Plasma concentrations of these drugs in a cassette‐dose clinical study at microdose level were successfully determined with a lower limit of quantitation of 0.2 pg/mL for diltiazem, 1 pg/mL for nicardipine, and 2 pg/mL for nifedipine.     

Recent trends in analytical methods and separation techniques for drugs of abuse in hair

Hair analysis of drugs of abuse has been a subject of growing interest from a clinical, social and forensic perspective for years because of the broad time detection window after intake in comparison to urine and blood analysis. Over the last few years, hair analysis has gained increasing attention and recognition for the retrospective investigation of drug abuse in a wide variety of contexts, shown by the large number of applications developed. This review aims to provide an overview of the state of the art and the latest trends used in the literature from 2005 to the present in the analysis of drugs of abuse in hair, with a special focus on separation analytical techniques and their hyphenation with mass spectrometry detection. The most recently introduced sample preparation techniques are also addressed in this paper. The main strengths and weaknesses of all of these approaches are critically discussed by means of relevant applications.     

Liquid chromatography-electrospray ionization tandem mass spectrometry for on-line characterization, monitoring and isotopic profiling of the main selenium-metabolite in human urine after consumption of Se-rich and Se-enriched food

The metabolism of selenium (Se) in the human body has yet not completely been unravelled and hence, an efficient method for characterization and on-line monitoring of the main Se-compound in human urine after consumption of Se-rich food was developed. Total Se-concentration in human urine after consumption of several Se-rich products was measured with inductively coupled plasma mass spectrometry (ICP-MS). The highest Se concentration in urine was observed after 4-10 h. The urine samples were brought onto a reversed phase column and the Se was detected by ICP-MS. Parameters for liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) measurements were optimized by using commercially available sugars, because it is known that some of the urinary metabolites contain a sugar moiety. In order to characterize the predominant Se-metabolite, it was necessary to extensively clean-up the sample and preconcentrate the species. The main metabolite was measured on its precursor ion on three different m/z according to three isotopes of Se. Relative peak surfaces matched the relative abundances of the isotopes. The product ions could be measured in a human urine sample in accordance to the product ions of the commercially available sugars. Moreover, the evidence of a selenosugar was demonstrated by the use of the Se-isotopes when measuring the product ions. LC-ESI-MS-MS was proven to be very efficient for the characterization of the main urinary Se-metabolite and can be used for on-line monitoring of the compound in urine samples. The method can be extended for clinical screening after consumption of Se-(en)rich(ed) food by use of the Se-isotopic profile and/or of the typical product ions of (methyl)-N-acetyl-hexosamines.     

Mass spectrometry-based survey of age-associated protein carbonylation in rat brain mitochondria

There is a body of evidence lending credence to the idea that oxidative stress may be responsible for age-related deleterious changes in brain function, and that protein carbonylation is a potential marker for such changes. An investigation of oxidative damage to mitochondrial proteins from aged rat brains was done using gel electrophoresis coupled with carbonylation-specific immunostaining. Six proteins that appeared to be susceptible to oxidative modification were identified by in-gel trypsin digestion followed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry. Two subunits of the H(+)-transporting ATP synthase, adenine nucleotide translocator, voltage-dependent anion channel, glutamate oxaloacetate transaminase, and aconitase were identified as likely targets of age-associated carbonylation.     

Rapid confirmatory method for the determination of 11 nitroimidazoles in egg using liquid chromatography tandem mass spectrometry

A rapid confirmatory method has been developed and validated for the simultaneous identification, confirmation and quantitation of 11 nitroimidazoles in eggs by liquid chromatography tandem mass spectrometry (LC–MS/MS). The method is validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), ipronidazole (IPZ) and their hydroxy metabolites MNZ-OH, HMMNI (hydroxymethyl, methyl nitroimidazole), IPZ-OH. The method is also capable of analysing carnidazole (CRZ), ornidazole (ORZ), tinidazole (TNZ) and ternidazole (TRZ). MNZ, DMZ and RNZ have been assigned a recommended level (RL) of 3 μg kg⁻¹ by the Community Reference Laboratory (CRL) in Berlin. The developed method described in this study is easily able to detect all the nitroimidazole compounds investigated at this level and below. Egg samples are extracted with acetonitrile, and NaCl is added to help remove matrix contaminants. The acetonitrile extract undergoes a liquid–liquid wash step with hexane; it is then evaporated and reconstituted in mobile phase. The reconstituted samples are analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS). The decision limits (CCα) range from 0.33 to 1.26 μg kg⁻¹ and the detection capabilities (CCβ), range from 0.56 to 2.15 μg kg⁻¹. The results of the inter-assay study, which was performed by fortifying hen egg samples (n = 18) on three separate days, show the accuracy calculated for the various analytes to range between 87.2 and 106.2%. The precision of the method, expressed as %CV values for the inter-assay variation of each analyte at the three levels of fortification (3, 4.5 and 6.0 μg kg⁻¹), ranged between 3.7 and 11.3%. A Day 4 analysis was carried out to examine species variances in eggs from different birds such as duck and quail and investigating differences in various battery and free range hen eggs.     

Liquid chromatography tandem mass spectrometry for the simultaneous determination of mequindox and its metabolites in porcine tissues

A rapid liquid chromatography tandem mass spectrometric method was developed for the simultaneous determination of mequindox and its five metabolites (2-isoethanol mequindox, 2-isoethanol 1-desoxymequindox, 1-desoxymequindox, 1,4-bisdesoxymequindox, and 2-isoethanol bisdesoxymequindox) in porcine muscle, liver, and kidney, fulfilling confirmation criteria with two transitions for each compound with acceptable relative ion intensities. The method involved acid hydrolysis, purification by solid-phase extraction, and subsequent analysis with liquid chromatography tandem mass spectrometry using electrospray ionization operated in positive polarity with a total run time of 15 min. The decision limit values of five analytes in porcine tissues ranged from 0.6 to 2.9 μg/kg, and the detection capability values ranged from 1.2 to 5.7 μg/kg. The results of the inter-day study, which was performed by fortifying porcine muscle (2, 4, and 8 μg/kg), liver, and kidney (10, 20, and 40 μg/kg) samples on three separate days, showed that the accuracy of the method for the various analytes ranged between 75.3 and 107.2% with relative standard deviation less than 12% for each analyte.     

Interfaces for coupled liquid-phase separation/mass spectrometry techniques. An update on recent developments

An update is presented covering the latest developments in the interfacing of liquid-phase separation systems and mass spectrometers. The interfacing devices presented are those developed for continuous-flow matrix-assisted laser desorption/ionization, micro- and nano-liquid chromatography/masspectrometry (MS), capillary electrophoresis/MS and on-chip separation technologies/MS. From the information that can be found in the most recent literature on the topic, it is evident that the trend towards the miniaturization of separation and interface devices is gaining ground. This can be rationalized by the substantial gains in sensitivity for the detection and study of extremely low levels of analytes and especially of high molecular mass biopolymers.     

Recent advances in electrophoretic techniques for the characterization of protein biomolecules: a poker of aces

The four classical modes of electrophoresis of protein molecules (sodium dodecyl sulphate electrophoresis, SDS-PAGE, isoelectric focusing, IEF, and immobilized pH gradients, IPGs, two-dimensional maps, 2D, and capillary electrophoresis, CE) are here reviewed, with special emphasis on recent innovations. Thus, in the case of SDS-PAGE, a novel method, consisting in focusing SDS-protein micelles against a gradient of cationic charges grafted onto a polyacrylamide gel is presented. In the case of IEF, the recent decoding of the structure, polydispersity, molecular mass distribution and buffering properties of the soluble carrier ampholyte buffers are here discussed. In regard to two dimensional mapping, recent instrumentation for performing 2D maps in horizontal, large gel slabs (up to 30 cm × 40 cm) and in a radial format for the SDS dimension is here evaluated. Finally, in the case of CE, three major applications are presented: a thorough study of capillary IEF and of all experimental variables, a method of importance in screening of rDNA products; the possibility of running proteins and peptide separations in very acidic, amphoteric, isoelectric buffers in absence of any capillary coating; finally, the possibility of producing a facile, user friendly, covalent coating of the wall silanols via bonding of quaternarized piperazines endowed with an iodinated tail. In acidic, volatile buffers, such protein/peptide runs can be directly interfaced with mass spectrometry instrumentation.     

Recent advances in computational analysis of mass spectrometry for proteomic profiling

The proteome, defined as an organism's proteins and their actions, is a highly complex end-effector of molecular and cellular events. Differing amounts of proteins in a sample can be indicators of an individual's health status; thus, it is valuable to identify key proteins that serve as 'biomarkers' for diseases. Since the proteome cannot be simply inferred from the genome due to pre- and posttranslational modifications, a direct approach toward mapping the proteome must be taken. The difficulty in evaluating a large number of individual proteins has been eased with the development of high-throughput methods based on mass spectrometry (MS) of peptide or protein mixtures, bypassing the time-consuming, laborious process of protein purification. However, proteomic profiling by MS requires extensive computational analysis. This article describes key issues and recent advances in computational analysis of mass spectra for biomarker identification.     

Analysis of amicarbazone and its two metabolites in grains and soybeans by liquid chromatography with tandem mass spectrometry

A sensitive, simple and reliable analytical method based on a modified quick, easy, cheap, effective, rugged, safe sample preparation and liquid chromatography with tandem mass spectrometry detection was developed for the simultaneous determination of amicarbazone and its two major metabolites desamino amicarbazone and isopropyl‐2‐hydroxy‐desamino amicarbazone residues in grains (rice, wheat, corn, buckwheat) and soybean. Several parameters, including liquid chromatography and tandem mass spectrometry conditions, extraction approaches and the adsorbents for clean‐up, which might influence the accuracy of the method, were extensively investigated. The established method was further validated by determining the linearity (R² > 0.99), fortified recovery (79–118%), precision (1–12%) and sensitivity (limit of quantification, 5 μg/kg for amicarbazone and desamino amicarbazone, and 10 μg/kg for isopropyl‐2‐hydroxy‐desamino amicarbazone). Finally, the established method was successfully applied to determine the residues of amicarbazone and its metabolites in 49 real samples of grain and soybean.     

Metabolomic profiling of human urine in hepatocellular carcinoma patients using gas chromatography/mass spectrometry

With the technique of metabolomics, gas chromatography/mass spectrometry (GC/MS), urine or serum metabolites can be assayed to explore disease biomarkers. In this work, we present a metabolomic method to investigate the urinary metabolic difference between hepatocellular carcinoma (HCC, n = 20) male patients and normal male subjects (n = 20). The urinary endogenous metabolome was assayed using chemical derivatization followed by GC/MS. After GC/MS analysis, 103 metabolites were detected, of which 66 were annotated as known compounds. By a two sample t-test statistics with p < 0.05, 18 metabolites were shown to be significantly different between the HCC and control groups. A diagnostic model was constructed with a combination of 18 marker metabolites or together with alphafetoprotein, using principal component analysis and receiver-operator characteristic curves. The multivariate statistics of the diagnostic model yielded a separation between the two groups with an area under the curve value of 0.9275. This non-invasive technique of identifying HCC biomarkers from urine may have clinical utility.