Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins from Shewanella putrefaciens separated by two-dimensional polyacrylamide gel electrophoresis

The implementation of nanoflow liquid chromatography offers unique opportunities for automation of proteomics research. We demonstrate that automated nanoflow LC/MS/MS allowed the unambiguous identification of proteins from the omnipotent bacterium Shewanella putrefaciens, based on similarity searches against the completely determined genome of related microorganisms and against non-redundant databases. Total protein extracts were separated by 2-dimensional polyacrylamide electrophoresis. Only 1/20th of a tryptic digest mixture obtained from a single Coomassie Blue stained spot was loaded on the nanoflow LC column using a preconcentration/desalting step, and analyzed on-line on a hybrid quadrupole time-of-flight mass spectrometer with an automated MS-to-MS/MS switching protocol. This method allowed the de novo peptide sequence determination of several tryptic fragments and the identification of different proteins. CopyrightCopyright 2000 John Wiley & Sons, Ltd.     

Insoluble eggshell matrix proteins--their peptide mapping and partial characterization by capillary electrophoresis and high-performance liquid chromatography

Avian eggshell matrix proteins were studied by two analytical approaches. Peptide mapping was done by trypsin and pepsin followed by collagenase cleavage; analyses were carried out by capillary electrophoresis and reversed-phase high-performance liquid chromatography (HPLC). Comparison of peptide maps obtained by both methods revealed a complex mixture of peptides in the insoluble layers of the eggshell; it was concluded that there are at least three different insoluble protein/peptide layers in the avian eggshell (cuticle, palisade, and mammillary layer). Partial characterization of peptides in each layer was made by HPLC-mass spectrometry analysis. There is an evidence that the eggshell insoluble proteins contain species susceptible to collagenase cleavage, however, the sequences split by this enzyme probably are not those typical for the main triple-helical core of collagenous proteins. It is proposed that the action of collagenase upon eggshell proteins is caused by the side effect of collagenase described previously with synthetic peptides. Some of the proteins present are probably glycosylated. Fatty acid content in the insoluble eggshell layers (after decalcification) was in the range of 2-4% (which reflected both lipid and lipoproteins bound fatty acids). Porphyrin pigments are dominant in the cuticle layer.     

Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix

Summary A method is described for obtaining peptide fragments for sequence analysis from microquantities of proteins separated by 1- or 2-dimensional polyacrylamide gel electrophoresis. After separation by electrophoresis, the proteins were stained with Coomassie Blue and excised. Proteolytic digestion with trypsin was performed directly in the polyacrylamide matrix. The resulting peptide fragments were eluted, separated by reversed phase HPLC, collected and sequenced in a gas phase sequencer. Excellent peptide recoveries allowed generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequences that can be used to generate oligonucleotide probes for molecular cloning, to design synthetic peptides for inducing antibodies, and to search sequence databases for related proteins.     

Characterization of mixtures of biocidal oligoguanidines by capillary electrophoresis and high-performance liquid chromatography coupled to mass spectrometry

The polycondensation of guanidine hydrochloride and 2,2′-(ethylenedioxy)bis(ethylamine) leads to various types of oligomeric guanidines exhibiting a broad spectrum of biocidal activities. In the present work a reversed-phase high-performance liquid chromatographic method with a gradient consisting of aqueous 0.1% trifluoroacetic acid and acetonitrile as mobile phase has been developed to separate these oligoguanidines according to type and chain length. The combination with electrospray mass spectrometry allowed the identification of the various compounds. By this technique, some structures already suggested in the literature could be confirmed, and several additional oligoguanidines not yet reported could be identified. As a complementary technique, capillary zone electrophoresis was investigated. Best results were obtained with carrier electrolytes consisting of phosphoric acid in water/acetonitrile mixtures. Although the number of peaks that could be separated by the electrophoretic method was considerably lower than in case of the chromatographic method, capillary electrophoresis in combination with UV detection at 195 nm may still be a fast method suitable for quantitation of some of the major compounds and for monitoring the reaction rate during the polycondensation reaction.     

Proteomic analysis of microvesicles in human saliva by gel electrophoresis with liquid chromatography-mass spectrometry

Microvesicles (MVs) have been shown to affect the physiology of neighboring recipient cells in various ways. They play an important role in tumor progression/metastasis and angiogenesis in cancer and may be useful therapeutic tools, as well as a mechanism of cell-to-cell communication. They have been visioned as an important biomarker or biomarker source for the detection of different diseases. Human saliva is a biological fluid with enormous diagnostic potential, which harbors plenty of salivary MVs. The goal of this study is to investigate the proteomic profiling of MVs in human saliva through a simple preparation procedure by using filtration and centrifugation. Gel electrophoresis was combined with LC–MS/MS (liquid chromatography–mass spectrometry) for the proteomic analysis of MVs. After SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) protein separation, the whole lane was cut into 25 bands, and each band was subjected to in-gel trypsin digestion. The peptides extracted from each band were loaded to LC–MS/MS for protein identification. Through protein database search, 63 proteins were identified for human salivary MVs. Several members of different protein families were identified, including annexin, keratin, actin, immunoglobulin and S100. This study showed that although there was an overlap with the proteins from human saliva and salivary exosomes, salivary MVs contained their own unique proteins. These results will poise human salivary MVs as a non-invasive tool for the early detection of different diseases.     

Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis

Current analytical techniques in protein identification by mass spectrometry are based on the generation of peptide mass maps or sequence tags that are idiotypic for the protein sequence. This work reports on the development of the use of mass spectrometric methods for protein identification in research on metabolic pathways of a genetically modified strain of the baker's yeast Saccharomyces cerevisiae. This study describes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass mapping and liquid chromatography/quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) sequence tag analysis in identification of yeast proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The spots were selected for analysis in order to collect information for future studies, to cover the whole pI range from 3 to 10, and to evaluate information from spots of different intensities. Mass mapping as a rapid, high-throughput method was in most cases sensitive enough for identification. LC/MS/MS was found to be more sensitive and to provide more accurate data, and was very useful when analyzing small amounts of sample. Even one sequence tag acquired by this method could be enough for unambiguous identification, and, in the present case, successfully identified a point mutation.     

Analysis of major ovine milk proteins by reversed-phase high-performance liquid chromatography and flow injection analysis with electrospray ionization mass spectrometry

Ovine milk proteins were analyzed both by coupling HPLC and electrospray ionization mass spectrometry (ESI-MS) and by flow injection analysis and ESI-MS detection after separation and collection of fractions from gel permeation chromatography. These methods resolved the four ovine caseins and whey proteins and made it possible to study the complexity of these proteins associated with genetic polymorphism, post-translational changes (phosphorylation and glycosylation) and the presence of multiple forms of proteins. The experimental molecular masses of ewe milk proteins were: 19 373 for κ-casein 3P; 25 616 for _s2-casein 10P; 23 411 for _s1-casein C-8P; 23 750 for β-casein 5P; 18 170 and 18 148 for β-lactoglobulins A and B; 14 152 for -lactalbumin A and 66 322 for serum albumin.     

Accelerated tryptic digestion for the analysis of biopharmaceutical monoclonal antibodies in plasma by liquid chromatography with tandem mass spectrometric detection

Accelerated tryptic digestion of a therapeutic protein including microwave irradiation and thermal transfer by convection at 60 °C and 37 °C was investigated. An analytical setup was devised to follow the protein digestion rate using 1D gel electrophoresis and liquid chromatography coupled a triple quadrupole linear ion trap mass spectrometer. The formation kinetic of its tryptic peptides was monitored in the selected monitoring mode (LC-SRM/MS). Different digestion end points (e.g. 2, 5, 10, 15, 30 and 60 min) as well as an overnight digestion were tested using a therapeutic human monoclonal antibody (mAb) with the goal of its LC-SRM/MS quantification in human plasma. The peptides from the human mAb were generated at different rates and were classified into three categories: (1) the fast forming peptides, (2) the slow forming peptides and (3) the peptides degrading over time. For many monitored peptides, a heating temperature of 37 °C with a 750 rpm mixing applied for at least 30 min provided equivalent results to microwave-assisted digestion and generally allowed the achievement of an equivalent peptide concentration as an overnight digestion carried out at 37 °C. The disappearance of the protein of the heavy and light chains can be monitored by 1D gel electrophoresis but was found not to be representative of the final tryptic peptide concentrations. For quantitative purposes a stable isotope labeled version (¹³C₄, ¹⁵N₁) of the therapeutic protein was used. The labeled protein as internal standard was found to be very efficient to compensate for incomplete digestion or losses during sample preparation.     

Determination of bromazepam in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of bromazepam in human plasma, using lorazepam as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions: m/z 316 > 182 for bromazepam and m/z 321 > 275 for lorazepam. The method was linear over the studied range (1-100 ng ml(-1)), with r(2) > 0.98, and the run time was 2.5 min. The intra- and inter-assay precisions were 2.7-14.6 and 4.1-17.3%, respectively and the intra- and inter-assay accuracies were 87-111 and 75.8-109.5%, respectively. The mean recovery was 73.7%, ranging from 64.5 to 79.7%. The limit of quantification was 1 ng ml(-1). At this concentration the mean intra- and inter-assay precisions were 14.6 and 7.1%, respectively, and the mean intra- and inter-assay accuracies were 102.5 and 104%, respectively. Bromazepam stability was evaluated and the results showed that the drug is stable in standard solution and in plasma samples under typical storage and processing conditions. The method was applied to a bioequivalence study in which 27 healthy adult volunteers (14 men) received single oral doses (6 mg) of reference and test bromazepam formulations, in an open, two-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak plasma concentration), AUC(0-96) and AUC(0-inf) (area under the plasma concentration versus time curve from time zero to 96 h and to infinity, respectively) were within the range 80-125%, which supports the conclusion that the test formulation is bioequivalent to the reference formulation regarding the rate and extent of bromazepam absorption.     

Reversed-phase high-performance liquid chromatography prefractionation prior to two-dimensional difference gel electrophoresis and mass spectrometry identifies new differentially expressed proteins between striate cortex of kitten and adult cat

Two-dimensional difference gel electrophoresis (2-D DIGE), in combination with mass spectrometry, is a highly effective method for the rapid and reproducible detection of differentially expressed proteins. This approach, however, has the unfortunate drawback that it preferentially displays rather abundantly expressed proteins. Nevertheless, comparison of the protein expression levels of the striate cortex of adult cats and 30-day-old kittens, resulted in the identification of several proteins related to postnatal brain development and possibly age-dependent plasticity as well (Van den Bergh et al., J. Neurochem. 2003, in press). The goal of the present study was the selective enrichment and identification of less abundant proteins within the same paradigm. Hereto, we performed a reversed-phase chromatography prefractionation of our tissue lysate to separate the proteins in four fractions based on their hydrophobicity prior to 2-D DIGE analysis. This approach not only confirmed the differential expression levels of a number of proteins from the previous study, but also identified three additional proteins preferentially expressed in kitten visual cortex and five additional proteins with higher expression levels in adult cat visual cortex. These spots were not visible on the total tissue lysate protein maps, indicating that the high-performance liquid chromatography (HPLC) prefractionation enabled us to visualize additional, less abundant proteins.     

Influence of one- and two-dimensional gel electrophoresis procedure on metal-protein bindings examined by electrospray ionization mass spectrometry, inductively coupled plasma mass spectrometry, and ultrafiltration

Three independent methods, (i) electrospray ionization mass spectrometry (ESI-MS), (ii) carrying out the complete protein preparation procedure required for protein gel electrophoresis (GE) including extraction, precipitation, washing, and desalting with subsequent microwave digestion of the produced protein fractions for metal content quantification, and (iii) ultrafiltration for separating protein-bound and unbound metal fractions, were employed to elucidate the influences of protein sample preparation and GE running conditions on metal-protein bindings. A treatment of the protein solution with acetone instead of trichloroacetic acid or ammonium sulfate for precipitate formation led to a strongly enhanced metal binding capacity. The desalting step of the resolubilized protein sample caused a metal loss between 10 and 35%. The omission of some extraction buffer additives led to a diminished metal binding capacity of protein fractions obtained from the sample preparation procedure for GE, whereas a tenside addition to the protein solution inhibited metal-protein bindings. The binding stoichiometry of Cu and Zn-protein complexes determined by ESI-MS was influenced by the type of the metal salt which was applied to the protein solution. A higher pH value of the sample solution promoted the metal ion complexation by the proteins. Ultrafiltration experiments revealed a higher Cu- and Zn-binding capacity of the model protein lysozyme in both resolubilization buffers for 1D- and 2D-GE compared to the protein extraction buffer. Strongly diminished metal binding capacities of lysozyme were recorded in the running buffer of 1D-GE and in the gel staining solutions.     

Identification of unusual (modified and non-encoded) amino acid residues in peptides by combinations of high-performance liquid chromatography and high-performance capillary electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The techniques for micro-level analysis of some widespread unusual amino acids (phosphorylated and hydroxylated ones) as well as of some genetically non-encoded amino acids were developed for their subsequent identification in the peptide and protein amino acid sequence by narrow-bore column high-performance liquid chromatography (10 pmol of the sample), high-performance capillary electrophoresis (1–10 pmol), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (1–10 pmol), and automatic protein gas phase sequencing (1–50 pmol).     

Proteomics analysis of human breast milk by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry to assess breast cancer risk

Breast cancer (BC) is one of the most common cancers and one of the most common causes for cancer-related mortality. Discovery of protein biomarkers associated with cancer is considered important for early diagnosis and prediction of the cancer risk. Protein biomarkers could be investigated by large-scale protein investigation or proteomics, using mass spectrometry (MS)-based techniques. Our group applies MS-based proteomics to study the protein pattern in human breast milk from women with BC and controls and investigates the alterations and dysregulations of breast milk proteins in comparison pairs of BC versus control. These dysregulated proteins might be considered potential future biomarkers of BC. Identification of potential biomarkers in breast milk may benefit young women without BC, but who could collect the milk for future assessment of BC risk. Previously we identified several dysregulated proteins in different sets of human breast milk samples from BC patients and controls using gel-based protein separation coupled with MS. Here, we performed 2D-PAGE coupled with nano-liquid chromatography–tandem MS (nanoLC-MS/MS) in a small-scale study on a set of six human breast milk pairs (three BC samples vs. three controls) and we identified several dysregulated proteins that have potential roles in cancer progression and might be considered potential BC biomarkers in the future.     

Determination of apomorphine in canine plasma by liquid chromatography-electrospray ionization mass spectrometry and its application to a pharmacokinetic study

An LC-ESI-MS method was developed and validated for the assay of apomorphine in canine plasma using one-step liquid-liquid extraction. The analytes were separated on a Phenomenex Gemini C18 (150 mm x 2.0 mm id 3 microm) column and determined by MS in the positive ion mode. The linear range was 0.4-40 ng/mL with an LOD of 0.2 ng/mL for apomorphine in plasma. The intraday and interday precision and accuracy of quality control samples were < 5.9% RSD and < 7.5% bias for apomorphine. Extraction recoveries were > 80%. The validated method was successfully applied to analyze canine plasma samples in a pharmacokinetic study of apomorphine in dogs and detailed pharmacokinetic parameters were calculated.     

Determination of itopride in human plasma by liquid chromatography coupled to tandem mass spectrometric detection: application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of itopride in human plasma, using sulpiride as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 359.5 > 166.1 for itopride and m/z 342.3 > 111.6 for IS, respectively. Analytes were chromatographed on an YMC C₁₈ reverse-phase chromatographic column by isocratic elution with 1 mM ammonium acetate buffer-methanol (20: 80, v/v; pH 4.0 adjusted with acetic acid). Results were linear (r² = 0.9999) over the studied range (0.5-1000 ng mL⁻¹) with a total analysis time per run of 2 min for LC-MS/MS. The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.     

A novel approach for analysis of oligonucleotide-cisplatin interactions by continuous elution gel electrophoresis coupled to isotope dilution inductively coupled plasma mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry

In this work we present a novel approach for in vitro studies of cisplatin interactions with 8-mer oligonucleotides. The approach is based on the recently developed coupling of continuous elution gel electrophoresis (GE) to an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) with the aim of monitoring the interaction process between this cytostatic drug and the nucleotides. In contrast to existing methods, the electrophoretic separation conditions used here allow both the determination of the reaction kinetics in more detail as well as the observation of dominant intermediates. Two different nucleotides sequences have been investigated for comparison purposes, one containing two adjacent guanines (5'-TCCGGTCC-3') and one with a combination of thymine and guanine (5'-TCCTGTCC-3'), respectively. In order to gain further structural information, MALDI-TOF MS measurements have been performed after fraction collection. This allows for identification of the intermediates and the final products and confirms the stepwise coordination of cisplatin via monoadduct to bisadduct formation. Furthermore, the ICP-MS results were quantitatively evaluated in order to calculate the kinetics of the entire process.     

Determination of didanosine in human serum by on-line solid-phase extraction coupled to high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

A method based on solid-phase extraction coupled to liquid chromatography with positive ion electrospray ionization and tandem mass spectrometric detection was developed for the determination of didanosine in human serum, using lamivudine as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions m/z 237 --> 136.7 for didanosine and m/z 230 --> 111.7 for lamivudine. The method was linear over the range studied (10-1500 ng ml(-1)), with r(2) > 0.98, and the run time was 5 min. The intra- and inter-assay precisions were < or =10% and the intra- and inter-assay accuracies were >95%. The absolute recoveries were 99.8% (10 ng ml(-1)), 98.4% (30 ng ml(-1)), 91.5% (700 ng ml(-1)) and 94.7% (1200 ng ml(-1)). The limits of detection and quantitation were 5 and 10 ng ml(-1), respectively. The method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (200 mg) of reference and test didanosine formulations (buffered powder for oral solutions), in an open, two-way, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (area under the serum concentration versus time curve from time zero to infinity) were within the range 80-125%, which supports the conclusion that the two formulations are bioequivalent regarding the rate and extent of didanosine absorption.     

Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone

A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.