Cancer immunosensor based on apo and holo transferrin binding.

An electrochemical immunosensor was developed for the determination of apo-Tf (non-iron-bound) and holo-Tf (iron-bound) using polyclonal antibody transferrin (anti-Tf) immobilized at an electrode surface as a biorecognition platform. The monitoring was based on the anti-Tf binding with both Tf forms which allows the detection of cancer cells due to the constant iron cycle and the overexpression of anti-Tf on the cancer cell surface. The immunosensor characterization was performed using electrochemical impedance spectroscopy (EIS), which evaluated the impedimetric biorecognition of the antigens-antibody by the use of K4Fe(CN)6 redox group. The immunosensor was able to detect both forms of Tf in terms of charge transfer resistance (Rct). Analytical curves showed a limit of detection of 0.049 and 0.053 ng mL-1 for apo-Tf and holo-Tf, respectively. The immunosensor was applied to the detection of the two cancer cells A549 (lung carcinoma) and MCF-7 (breast carcinoma) and compared with BHK570, a healthy cell line. The impedimetric response of healthy cells differs significantly from that of the cancerous cells, as revealed by a Dunnett's test in 95% confidence level-ca. 102 cells mL-1-indicating the feasibility of the immunosensor to discriminate both types of cells. The indirect detection of anti-Tf based on apo-Tf and holo-Tf binding can be considered an advanced approach for cancer recognition. Graphical abstract.

Absolute Quantitation of Proteins by Coulometric Mass Spectrometry.

Accurate quantification is essential in the fields of proteomics, clinical assay, and biomarker discovery. Popular methods for absolute protein quantitation by mass spectrometry (MS) involve the digestion of target protein and employ isotope-labeled peptide internal standards to quantify chosen surrogate peptides. Although these methods have gained success, syntheses of isotope-labeled peptides are time-consuming and costly. To eliminate the need for using standards or calibration curves, herein we present a coulometric mass spectrometric (CMS) approach for absolute protein quantitation, based on the electrochemical oxidation of a surrogate peptide combined with mass spectrometric measurement of the oxidation yield. To demonstrate the utility of this method, several proteins were analyzed such as model proteins ß-casein, and apomyoglobin as well as circadian clock protein KaiB isolated from Escherichia coli. In our experiment, tyrosine-containing peptides were selected as surrogate peptides for quantitation, considering the oxidizable nature of tyrosine. Our data showed that the results for surrogate peptide quantity measured by our method and by traditional isotope dilution method are in excellent agreement, with the discrepancy of 0.3-3%, validating our CMS method for absolute quantitation. Furthermore, therapeutic monoclonal antibody (mAb) could be quantified by our method as well. Due to the high specificity and sensitivity of MS and no need to use isotope-labeled peptide standards, our CMS method would be of high value for the absolute proteomic quantification.

Top-Down ETD-MS Provides Unreliable Quantitation of Methionine Oxidation.

Methionine oxidation plays a critical role in many processes of biologic and biomedical importance, including cellular redox responses and stability of protein pharmaceuticals. Bottom-up methods for analysis of methionine oxidation can suffer from incomplete sequence coverage, as well as an inability to readily detect correlated oxidation between 2 or more methionines. However, the methodology for quantifying protein oxidation in top-down analyses is lacking. Previous work has shown that electron transfer dissociation (ETD)-based tandem mass spectrometry (MS/MS) fragmentation offers accurate and precise quantification of amino acid oxidation in peptides, even in complex samples. However, the ability of ETD-based MS/MS fragmentation to accurately quantify amino acid oxidation of proteins in a top-down manner has not been reported. Using apomyoglobin and calmodulin as model proteins, we partially converted methionines into methionine sulfoxide by incubation in H2O2. Using top-down ETD-based fragmentation, we quantified the amount of oxidation of various ETD product ions and compared the quantified values with those from traditional bottom-up analysis. We find that overall quantification of methionine oxidation by top-down MS/MS ranges from good agreement with traditional bottom-up methods to vast differences between the 2 techniques, including missing oxidized product ions and large differences in measured oxidation quantities. Care must be taken in transitioning ETD-based quantitation of oxidation from the peptide level to the intact protein level.

Visualized detection of apo-transferrin based on cyanine dye supramolecular assembly.

In this work, a new biological probe for human transferrin (hTf) detection based on a cyanine dye ETC supramolecular assembly was designed. In sub-micromolar level, apo-hTf could induced the ETC aggregations transferred from H-aggregations to J-band with color change from pink to blue, while holo-hTf hardly possessed the ability, indicating ETC could specifically identify apo-hTf. The present study allowed for apo-hTf detection in the range of 8-80 nM with a detection limit of 2.8 nM and the sensitivity of visualization was around 70 nM. To further examine the suitability, iron ions were added into apo-hTf to stimulate the transformation from open conformation to the closed one gradually. It has been confirmed through tryptophan internal fluorescence quenching and the decrease of ETC J-aggregation. The interaction between ETC and apo-hTf performed high affinity that Ka was reached to 106 M-1 with a high selectivity. The potential in practical applications of this method has been tested for detection apo-hTf in human serum.

High Speed Intact Protein Characterization Using 4X Frequency Multiplication, Ion Trap Harmonization, and 21 Tesla FTICR-MS.

Mass spectrometric characterization of large biomolecules, such as intact proteins, requires the specificity afforded by ultrahigh resolution mass measurements performed at both the intact mass and product ion levels. Although the performance of time-of-flight mass analyzers is steadily increasing, the choice of mass analyzer for large biomolecules (e.g., proteins >50 kDa) is generally limited to the Fourier transform family of mass analyzers such as Orbitrap and ion cyclotron resonance (FTICR-MS), with the latter providing unmatched mass resolving power and measurement accuracy. Yet, protein analyses using FTMS are largely hindered by the low acquisition rates of spectra with ultrahigh resolving power. Frequency multiple detection schemes enable FTICR-MS to overcome this fundamental barrier and achieve resolving powers and acquisition speeds 4× greater than the limits imposed by magnetic field strength. Here we expand upon earlier work on the implementation of this technique for biomolecular characterization. We report the coupling of 21T FTICR-MS, 4X frequency multiplication, ion trapping field harmonization technology, and spectral data processing methods to achieve unprecedented acquisition rates and resolving power in mass spectrometry of large intact proteins. Isotopically resolved spectra of multiply charged ubiquitin ions were acquired using detection periods as short as 12 ms. Large proteins such as apo-transferrin (MW = 78 kDa) and monoclonal antibody (MW = 150 kDa) were isotopically resolved with detection periods of 384 and 768 ms, respectively. These results illustrate the future capability of accurate characterization of large proteins on time scales compatible with online separations.

Surface-Induced Protein Unfolding in Submicron Electrospray Emitters.

The charging of protein ions formed by nanoelectrospray ionization (nanoESI) with tips that are between 1.5 µm and 250 nm in outer diameter is compared. More charging is obtained with the smaller tip sizes for proteins that have a net positive charge in solution, and additional high-charge-state distributions are often observed. A single charge-state distribution of holo-myoglobin ions is produced by nanoESI from a slightly acidified aqueous solution with the micron outer diameter tips, but some apo-myoglobin ions are produced with the submicron tips. In contrast, the charge-state distributions for proteins with a net negative charge in solution do not depend on tip size. Both the formation of high charge states and the appearance of higher-charge-state distributions, as well as the loss of the heme group from myoglobin, indicate that a fraction of the protein population is unfolding with the smaller tips. The increased charging with the smaller tip sizes for proteins with a net positive charge but not for proteins with a net negative charge indicates that the unfolding occurs prior to nanoelectrospray ionization as a result of Coulombic attraction between positively charged protein molecules in solution and the glass surfaces of the emitter tips that are negatively charged. These results demonstrate a novel method for producing highly charged protein ions that does not require exposing the proteins to additional chemicals either in solution or in the gas phase.

Differences in the Elemental Isotope Definition May Lead to Errors in Modern Mass-Spectrometry-Based Proteomics.

The elemental isotope definition used to calculate the theoretical masses and isotope distribution of (bio)molecules is considered to be a fixed, universal standard in mass-spectrometry-based proteomics. However, this is an incorrect assumption. In view of the ongoing advances in mass spectrometry technology, and in particular the ever-increasing mass precision, the elemental isotope definition and its variations should be taken into account. We illustrate the effect of the elemental isotope uncertainty on the theoretical and experimental masses with theoretical calculations and examples.

Aequorin-based luminescence imaging reveals differential calcium signalling responses to salt and reactive oxygen species in rice roots.

It is well established that both salt and reactive oxygen species (ROS) stresses are able to increase the concentration of cytosolic free Ca(2+) ([Ca(2+)]i), which is caused by the flux of calcium (Ca(2+)). However, the differences between these two processes are largely unknown. Here, we introduced recombinant aequorin into rice (Oryza sativa) and examined the change in [Ca(2+)]i in response to salt and ROS stresses. The transgenic rice harbouring aequorin showed strong luminescence in roots when treated with exogenous Ca(2+). Considering the histological differences in roots between rice and Arabidopsis, we reappraised the discharging solution, and suggested that the percentage of ethanol should be 25%. Different concentrations of NaCl induced immediate [Ca(2+)]i spikes with the same durations and phases. In contrast, H2O2 induced delayed [Ca(2+)]i spikes with different peaks according to the concentrations of H2O2. According to the Ca(2+) inhibitor research, we also showed that the sources of Ca(2+) induced by NaCl and H2O2 are different. Furthermore, we evaluated the contribution of [Ca(2+)]i responses in the NaCl- and H2O2-induced gene expressions respectively, and present a Ca(2+)- and H2O2-mediated molecular signalling model for the initial response to NaCl in rice.

Improved ion optics for introduction of ions into a 9.4-T Fourier transform ion cyclotron resonance mass spectrometer.

Enhancements to the ion source and transfer optics of our 9.4 T Fourier transform ion cyclotron resonance (ICR) mass spectrometer have resulted in improved ion transmission efficiency for more sensitive mass measurement of complex mixtures at the MS and MS/MS levels. The tube lens/skimmer has been replaced by a dual ion funnel and the following octopole by a quadrupole for reduced ion cloud radial expansion before transmission into a mass-selective quadrupole. The number of ions that reach the ICR cell is increased by an order of magnitude for the funnel/quadrupole relative to the tube lens/skimmer/octopole.

Separation of PEGylated variants of ribonuclease A and apo-α-lactalbumin via reversed phase chromatography.

The covalent attachment of polyethylene glycol (PEG) molecules to pharmaceutical proteins, "PEGylation", often results in a population of conjugate species that includes differing numbers and locations of attached PEG chains. As some portion of this population may be biologically inactive, a challenging separation problem arises. An interesting alternative to the size-based resolution of these conjugates involves the use of reversed phase chromatography (RPC), treating the PEG moieties as hydrophobic purification tags. The use of RPC raises concerns about protein denaturation in the mobile and on the stationary phase. Here, the potential dual role of conjugated PEG chains as both group-specific separation tags and as steric or structural stabilizers in RPC was explored. In this work, RPC with C18-based media was used to resolve PEGylation number variants of ribonuclease A (RNase A) and apo-α-lactalbumin (apo-αLac) in a neutral pH mobile phase. While the attachment of 20kDa PEG molecules did not modify the structures of RNase A and apo-αLac, as confirmed by structural analysis using circular dichroism, exposure to the mobile phase modifier, acetonitrile, and to the C18 media during separation resulted in perturbations to both the secondary and tertiary structures of all species studied. RNase A experienced small perturbations that were mediated to some extent by PEGylation; these results were consistent with activity assays which showed that PEGylated RNase A species retained native-like activity after RPC separation. Apo-αLac, a more hydrophobic and less stable protein than RNase A, experienced extensive structural perturbations regardless of PEGylation state. The temperature of the mobile phase was found to strongly influence chromatographic separation of PEG-conjugates with conjugate species becoming more strongly retained with increasing temperature. This work shows that it is feasible to employ RPC with neutral pH mobile phases to resolve PEG conjugate number heterogeneity.

Submillisecond protein folding events monitored by rapid mixing and mass spectrometry-based oxidative labeling.

Kinetic measurements can provide insights into protein folding mechanisms. However, the initial (submillisecond) stages of folding still represent a formidable analytical challenge. A number of ultrarapid triggering techniques have been available for some time, but coupling of these techniques with detection methods that are capable of providing detailed structural information has proven to be difficult. The current work addresses this issue by combining submillisecond mixing with laser-induced oxidative labeling. Apomyoglobin (aMb) serves as a model system for our measurements. Exposure of the protein to a brief pulse of hydroxyl radical (·OH) at different time points during folding introduces covalent modifications at solvent accessible side chains. The extent of labeling is monitored using mass spectrometry-based peptide mapping, providing spatially resolved measurements of changes in solvent accessibility. The submillisecond mixer used here improves the time resolution by a factor of 50 compared to earlier ·OH labeling experiments from our laboratory. Data obtained in this way indicate that early aMb folding events are driven by both local and sequence-remote docking of hydrophobic side chains. Assembly of a partially formed A(E)G(H) scaffold after 0.2 ms is followed by stepwise consolidation that ultimately yields the native state. Major conformational changes go to completion within 0.1 s. The technique introduced here is capable of providing in-depth structural information on very short time scales that have thus far been dominated by low resolution (global) spectroscopic probes. By employing submillisecond mixing in conjunction with slower mixing techniques, it is possible to observe complete folding pathways, from fractions of a millisecond all the way to minutes.

A general protease digestion procedure for optimal protein sequence coverage and post-translational modifications analysis of recombinant glycoproteins: application to the characterization of human lysyl oxidase-like 2 glycosylation.

Using recombinant DNA technology for expression of protein therapeutics is a maturing field of pharmaceutical research and development. As recombinant proteins are increasingly utilized as biotherapeutics, improved methodologies ensuring the characterization of post-translational modifications (PTMs) are needed. Typically, proteins prepared for PTM analysis are proteolytically digested and analyzed by mass spectrometry. To ensure full coverage of the PTMs on a given protein, one must obtain complete sequence coverage of the protein, which is often quite challenging. The objective of the research described here is to design a protocol that maximizes protein sequence coverage and enables detection of post-translational modifications, specifically N-linked glycosylation. To achieve this objective, a highly efficient proteolytic digest protocol using trypsin was designed by comparing the relative merits of denaturing agents (urea and Rapigest SF), reducing agents [dithiothreitol (DTT) and tris(2-carboxyethyl)phophine (TCEP)], and various concentrations of alkylating agent [iodoacetamide (IAM)]. After analysis of human apo-transferrin using various protease digestion protocols, ideal conditions were determined to contain 6 M urea for denaturation, 5 mM TCEP for reduction, 10 mM IAM for alkylation, and 10 mM DTT, to quench excess IAM before the addition of trypsin. This method was successfully applied to a novel recombinant protein, human lysyl oxidase-like 2. Furthermore, the glycosylation PTMs were readily detected at two glycosylation sites in the protein. These digestion conditions were specifically designed for PTM analysis of recombinant proteins and biotherapeutics, and the work described herein fills an unmet need in the growing field of biopharmaceutical analysis.

Evaluation of the effects of 20 nonsynonymous single nucleotide polymorphisms of CYP2C19 on S-mephenytoin 4'-hydroxylation and omeprazole 5'-hydroxylation.

CYP2C19 is a highly polymorphic enzyme that affects the metabolism of a wide range of therapeutic drugs. Almost all the identified alleles of CYP2C19 are derived from nonsynonymous single nucleotide polymorphisms (nsSNPs). The objective of this study was to functionally characterize 20 nsSNPs of CYP2C19, distributed throughout the entire coding region, most of which have not been thoroughly characterized. cDNAs of these variants were constructed and expressed in yeast cells. All variants had similar levels of apoprotein and holoprotein expression, except for CYP2C19.16 and D360N, which had significantly lower holoprotein levels than the wild-type (WT) CYP2C19 enzyme, and CYP2C19.5B, which showed only apoprotein. The activity of the CYP2C19 variants was investigated using two substrates, S-mephenytoin and omeprazole, and six different kinetic parameters were measured. CYP2C19.5B, CYP2C19.6, and CYP2C19.8 were found to be catalytically inactive. The entire dataset of the remaining 17 variants, together with the WT, was analyzed by multivariate analysis. This analysis indicated that CYP2C19.9, CYP2C19.10, CYP2C19.16, CYP2C19.18, CYP2C19.19, A161P, W212C, and D360N were substantially altered in catalytic properties in comparison with the WT, with each of these variants exhibiting either dramatically decreased catalytic activities or higher K(m) values. These results not only generally confirmed the function of previously reported variants but also identified additional reduced-function variants. These findings will greatly extend our understanding of CYP2C19 genetic polymorphisms in humans as well as facilitate the structure-function study of the CYP2C19 protein.

New factors of cardiometabolic risk in severely obese children: influence of pubertal status / Nuevos factores de riesgo cardiometabólico en niños con obesidad severa: influencia del estado puberal

The aim of this prospective study was to evaluate the utility of new biochemical markers to assess cardiometabolic risk in severely obese children and adolescents. A total of 107 subjects aged 7 to 14 years, were clinically assessed and anthropometric measures and percentage of fat mass by single frequency bioimpedance analysis were recorded. Of these, 44 were non-overweight and 63 severely obese (body mass index Z-score >2.5) which were stratified by Tanner stages. To estimate the metabolic risk the following variables were considered for analysis: Waist circumference/height >0.5, fasting glucose >100 mg/dL, triglycerides >110 mg/dL, HDL-C <40 mg/dL, and systolic or diastolic blood pressure >95th percentile for age and gender. Fasting insulinemia, apoprotein A1 and B, high-sensitive C-reactive protein, alanine aminotransferase, homocysteine, and folic and uric acids were determined. In severely obese children, metabolic risk was present more frequently in mid puberty. The normalized anthropometric parameters with respect to 50th percentile for age and gender did not differ in the presence of metabolic risk. Insulin resistance was an independent determinant of metabolic risk, adjusted by Tanner stages. Elevated high-sensitive C-reactive protein was noted without any effect of metabolic risk or pubertal stage. Homocysteine, apoprotein B, and alanine aminotransferase values increased with metabolic risk and were not influenced by puberty. Although insulin resistance remains the main factor influencing metabolic risk, biochemical markers as homocysteine, apoprotein B, and alanine aminotransferase, may be useful for identifying severe obese pubertal subjects particularly prone to comorbidities (AU)

El objetivo de este estudio prospectivo ha sido evaluar la utilidad de nuevos marcadores bioquímicos para evaluar el riesgo cardiometabólico en niños y adolescentes extremadamente obesos. Un total de 107 sujetos de entre 7 a 14 años, se valoraron clínicamente registrando sus medidas antropométricas y el porcentaje de masa grasa mediante bioimpedancia. De ellos, 44 presentaban un peso normal para su edad y género y 63 estaban gravemente obesos (puntuación Z del índice de masa corporal > 2,5), los cuales fueron estratificados por estadios de Tanner. Para valorar el riesgo metabólico se consideraron las siguientes variables: Circunferencia cintura/altura> 0,5, glucosa en ayunas >100 mg/dL, triglicéridos >110 mg/dL, HDL-C <40 mg/dL y presión arterial sistólica o diastólica > percentil 95 para edad y género. Se determinaron la insulinemia en ayunas, apoproteinas A1 y B, proteína C reactiva ultrasensible, alaninaminotransferasa, homocisteína y ácidos fólico y úrico. En los niños obesos severos, la presencia de factores de riesgo metabólico se producía con más frecuencia en la pubertad. Los parámetros antropométricos normalizados con respecto al percentil 50 para la edad y género no se diferenciaban según existiera o no mayor riego metabólico. La resistencia a la insulina fue un factor independiente de riesgo metabólico, ajustado por etapas de Tanner. La proteína C reactiva ultrasensible estaba elevada, sin relación con la mayor presencia de factores de riesgo del síndrome metabólico o la etapa puberal. La homocisteína, apoproteína B, y la alaninaminotransferasa se incrementaron con el riesgo metabólico y no fueron influidos por la pubertad. Aunque la resistencia a la insulina sigue siendo el principal factor que influye en el riesgo metabólico, los marcadores bioquímicos como la homocisteína, apoproteína B, y la alaninaminotransferasa, pueden ser útiles para identificar individuos con obesidad grave en etapa puberal predispuestos a padecer enfermedades relacionadas (AU)

Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.

Ca(2+) channels and calmodulin (CaM) are two prominent signalling hubs that synergistically affect functions as diverse as cardiac excitability, synaptic plasticity and gene transcription. It is therefore fitting that these hubs are in some sense coordinated, as the opening of Ca(V)1-2 Ca(2+) channels are regulated by a single CaM constitutively complexed with channels. The Ca(2+)-free form of CaM (apoCaM) is already pre-associated with the isoleucine-glutamine (IQ) domain on the channel carboxy terminus, and subsequent Ca(2+) binding to this 'resident' CaM drives conformational changes that then trigger regulation of channel opening. Another potential avenue for channel-CaM coordination could arise from the absence of Ca(2+) regulation in channels lacking a pre-associated CaM. Natural fluctuations in CaM concentrations might then influence the fraction of regulable channels and, thereby, the overall strength of Ca(2+) feedback. However, the prevailing view has been that the ultrastrong affinity of channels for apoCaM ensures their saturation with CaM, yielding a significant form of concentration independence between Ca(2+) channels and CaM. Here we show that significant exceptions to this autonomy exist, by combining electrophysiology (to characterize channel regulation) with optical fluorescence resonance energy transfer (FRET) sensor determination of free-apoCaM concentration in live cells. This approach translates quantitative CaM biochemistry from the traditional test-tube context into the realm of functioning holochannels within intact cells. From this perspective, we find that long splice forms of Ca(V)1.3 and Ca(V)1.4 channels include a distal carboxy tail that resembles an enzyme competitive inhibitor that retunes channel affinity for apoCaM such that natural CaM variations affect the strength of Ca(2+) feedback modulation. Given the ubiquity of these channels, the connection between ambient CaM levels and Ca(2+) entry through channels is broadly significant for Ca(2+) homeostasis. Strategies such as ours promise key advances for the in situ analysis of signalling molecules resistant to in vitro reconstitution, such as Ca(2+) channels.

A new generation of cross-linkers for selective detection by MALDI MS.

We designed a new cross-linker bearing a CHCA moiety. The use of the CHCA-tagged cross-linker JMV 3378 in conjunction with a neutral MALDI matrix alpha-cyano-4-hydroxycinnamic methyl ester enabled specific signal enhancement in MALDI-TOF MS of cross-link containing peptides. Discrimination between modified and non-modified peptides can be achieved by comparison of two spectra, one using CHCA and the other using the alpha-cyano-4-hydroxycinnamic methyl ester matrix. The methodology was validated using cytochrome c and apo-myoglobine as model proteins.

Metastatic uterine cervical cancer originating in the lung: a case report.

We report a case of primary pulmonary adenocarcinoma which metastasized to the uterine cervix. A 69-year-old postmenopausal Japanese female was admitted to our hospital because of general fatigue and atypical genital bleeding. Four years before, she had undergone video-assisted thoracoscopic right upper lobectomy, for primary lung cancer (adenocarcinoma), stage IIIb, pT3N1M0. Gynecologic investigation showed a cauliflower-like tumor in the uterine cervix and parametrial invasion towards the bilateral pelvic wall. Metastasis of extragenital carcinoma to the cervix uteri is rare. Most such reported cases originated in the breast and gastrointestinal tract. In this case, cervical biopsy specimens were revealed to be adenocarcinomatous, similar in pathological features to the previously resected lung cancer. Immunohistochemical staining was positive for thyroid transcription factor-1 and pulmonary surfactant apoprotein A and negative for CA125 and thyroglobulin. Although rare, the respiratory tract should be considered as a possible primary site of uterine cervical metastatic carcinoma.

Development of an aequorin luminescence calcium assay for high-throughput screening using a plate reader, the LumiLux.

A luminescence assay using a new plate reader, the LumiLux (PerkinElmer, Waltham, MA), has been validated for high-throughput screening (HTS). In this study, we compared the aequorin luminescence-based calcium mobilization assay to the fluorescence-based calcium assay. A cell line stably co-expressing apo-aequorin, a chimeric G-protein, and a G-protein-coupled dopamine receptor was used to screen a collection of 8,106 compounds using the Hamamatsu Photonics (Bridgewater, NJ) FDSS6000 and LumiLux as the plate readers. The assay parameters evaluated included hit rate correlation, signal-to-noise ratio, and overall assay performance calculated by Z and standard deviation. The average Z values and hit rates were comparable between assay platforms;however, the standard deviation for the agonist aequorin assay was significantly smaller. There was also a significant decrease in the number of false-positives with the aequorin assay. These results suggest that the aequorin assay in combination with the new plate reader, LumiLux, provides a simple, cost-effective, robust, and sensitive assay for HTS

A multiway approach for classification and characterization of rabbit liver apothioneins by CE-ESI-MS.

We applied a multiway approach to extract information from the analysis of protein isoforms by CE-ESI-MS. Metallothioneins (MT) are low-molecular-weight proteins (6-7 kDa) with a strong affinity for heavy-metal ions. Rabbit liver MT-I and MT-II fractions are purified from MT samples. At low pH, the bound metal ions were released from the amino acid structures, giving rise to apothioneins. MT-I, MT-II and MT apothioneins, which are complex mixtures of protein isoforms, were analyzed by CE-ESI-MS. After data pre-processing, parallel factor analysis (PARAFAC) and multivariate curve resolution-alternating least squares (MCR-ALS) were applied to the data sets. In both cases, the models enabled classification of the protein samples and identification of their characteristic sub-isoforms using a set of three components. MCR-ALS required an initial estimate of the pure mass spectra of the three components. Thus, PARAFAC loadings were used to initialize the MCR-ALS optimization. The classifications obtained with MCR-ALS were slightly better than those obtained with PARAFAC, probably because MCR-ALS was less affected by the small migration time shifts of the pre-processed electropherograms. However, no differences were found between the pure mass spectra of the three components in either model. Finally, MCR-ALS allowed us to obtain an individual electrophoretic profile of each of the three components for each of the samples analyzed, which proved valuable for characterization and quantification purposes.

Analysis of intact erythropoietin and novel erythropoiesis-stimulating protein by capillary electrophoresis-electrospray-ion trap mass spectrometry.

Recombinant human erythropoietin (rHuEPO) and novel erythropoiesis-stimulating protein (NESP) were analyzed by CE-ESI-MS using an IT as analyzer. The IT parameters were optimized by direct infusion of solutions of different intact proteins (myoglobin, transferrin, alpha1-acid glycoprotein and fetuin) with different degrees of glycosylation (from 0 to 35% w/w). Two physically adsorbed capillary coatings from UltraTol Pre-Coats (low normal (LN) and high reverse (HR)) were evaluated for the separation of rHuEPO and NESP glycoforms by CE-ESI-IT-MS. The results obtained with the neutral LN coating suggest that an IT mass spectrometer enables identification of the main glycoforms of a complex glycoprotein such as rHuEPO. Although LN provided acceptable glycoform resolution for rHuEPO, the separation obtained for NESP was less significant due to the higher microheterogeneity of this glycoprotein. Reproducibility studies confirmed the lack of stability and bleeding of the LN coating, which caused problems with MS detection, such as a dramatic loss of sensitivity and the presence of peaks in the mass spectra corresponding to molecular ions in the coating. In contrast, the cationic HR coating gave faster but poorer glycoform separations due to the presence of an anodal EOF. However, the positive charge of the coating provided enhanced hydrolytic stability, making it more suitable than the LN coating for the on-line MS coupling.