Feasibility of nonvolatile buffers in capillary electrophoresis-electrospray ionization-mass spectrometry of proteins (original) (raw)
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Journal of the American Society for Mass Spectrometry, 1998
An in-capillary electrode sheathless interface was applied to the capillary electrophoresis/ electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of mixtures of small peptides, proteins, and tryptic digests of proteins. The effects of different experimental parameters on the performance of this CE/ESI-MS interface were studied. The distance of the in-capillary electrode from the CE outlet and the length of the electrode inside the capillary had no significant effects on the CE separation and ESI behavior under the experimental conditions used. However, significant enhancement of the sensitivity resulted from the use of narrower CE capillaries. Using a quadrupole mass spectrometer, an aminopropylsilane-coated capillary, and a wide scan mass-to-charge ratio range of 500 -1400, detection limits of approximately 4, 1, and 0.6 fmol for cytochrome c and myoglobin were achieved for 75-, 50-, and 30-m inner diameter capillaries, respectively. Approximately one order of magnitude lower detection limits were achieved under the multiple-ion monitoring mode. The application of the in-capillary electrode sheathless interface to real-world samples was demonstrated by CE/ ESI-MS analysis of a human blood sample
Current Protein & Peptide Science, 2014
Proteomics is a growing field that has the potential to be applied to many biology-related disciplines. However, the study of the proteome has proven to be very challenging due to its high level of complexity when compared to genome and transcriptome data. In order to analyse this level of complexity, high resolution separation of peptides/proteins are needed together with high resolution analysers. Currently, liquid chromatography and capillary electrophoresis (CE) are the two most widely used separation techniques that can be coupled on-line with a mass spectrometer (MS). In CE, proteins/peptides are separated according to their size, charge and shape leading to high resolving power. Although further progress in the area of sensitivity, throughput and proteome coverage are expected, MS-based proteomics have developed to a level at which they are habitually applied to study a wide range of biological questions. The aim of this review is to present CE-MS as a proteomic analytical platform for biomarker research that could be used in farm animal and veterinary studies. This is a MS-analytical platform that has been widely used for biomarker research in the biomedical field but its application in animal proteomic studies is relatively novel. The review will focus on introducing the CE-MS platform and the primary considerations for its application to biomarker research. Furthermore, current applications but more importantly potential application in the field of farm animals and veterinary science will be presented and discussed.
Journal of Chromatography A, 1999
A method is described for the direct determination of small inorganic cations in samples containing large amounts of proteins, such as milk or blood plasma. The method is based on electrokinetic injection in a flow injection analysis-capillary electrophoresis (CE) system. The selected CE-electrolyte, containing 5 mM 4-aminopyridine and 7 mM cetyltrimethylammonium bromide at pH 4.5, prevents detrimental protein adsorption on the capillary walls. Therefore, no sample pretreatment, except for dilution, is required. Up to 30 repeated injections in one electrophoretic run can be performed, yielding RSD values of the migration time of less than 1 and 2.5% (n530) for milk and blood plasma samples, respectively.
Clinical Chemistry and Laboratory Medicine, 1990
The reliability of electrolyte determinations with the Ektachem 700 was evaluated by various means including the use of reference method values. The influence of protein concentration, which may alter the viscosity and hence the speed of diffusion, was systematically investigated by using samples of varying protein concentration obtained by ultracentrifugation. Calcium. The mean bias between Ektachem results and reference method values of 9 control sera was-7.3%. In a comparative study with native sera a negative bias was not obtained. The accuracy was independent of the protein concentration. Chloride. The mean bias was-0.1%, compared with reference method values. As might be expected, the difference between chloride values with Ektachem-using a "direct" ion-selective electrode-and determinations by coulometry increased with increasing protein concentrations. Magnesium. A negative bias (-5.0%) was obtained in accuracy control with reference method values, and to a smaller extent in a comparative study with native sera. An influence of the protein concentration on the magnesium determination was not observed. Phosphate. Accuracy control by method-dependent assigned values and a comparative study with native sera showed a positive bias. Ektachem results depend on the protein concentration. At 120 g/1 protein the bias was +13.6%. Potassium. The mean bias, with respect to reference method values, was-1.2%. At high concentrations of proteins of "normal" composition, Ektachem results agree with measurements by flame atomic emission spectrometry; in paraproteinaemic sera the values are higher. Sodium. The mean inaccuracy was 3.3%, compared with reference method values. The dependence on the amount and composition of the total protein was similar to that found for potassium analysis. evaluation protocols based on established methodol-The advantages of multicentre evaluations of clinical ogies. Therefore, in addition to the general evaluation chemical analysers are unquestioned. However, the of the Ektachem system, this study was especially introduction of fundamentally new principles of de-concerned with the possible influence of the protein termination may increase the importance of some concentration, which has not yet been systematically aspects that are ignored or treated only briefly in investigated.
Analytical Chemistry, 2002
To improve the separation efficiency while achieving high sensitivity for the analysis of proteins' microheterogeneity, a segmental-filling technique has been developed and tested in capillary electrophoresis with laser-induced native fluorescence using a pulsed Nd:YAG laser. Using a short plug of SDS applied to the capillary and the anticonvectant poly(ethylene oxide) (PEO), the microheterogeneities of a number of proteins with pI values ranging from 4.5 to 11.1 could be detected. This high resolving power is due to reduced adsorption on the capillary wall, sieving, and the interaction with SDS. Consequently, the length and the concentration of the SDS plug play a significant role in determining the resolution and sensitivity. The method has been applied to the analysis of salivary and cerebrospinal fluid (CSF) samples. Without any sample pretreatment, using a 10-s 1× SDS plug, six r-amylase isoforms in a salivary sample were resolved in 17 min and three more peaks were detected in a CSF sample. With simplicity, high resolving power, and rapidity, the method has shown great potential for proteomics.
Journal of Chromatography A, 1993
Successful separations of alcohol dehydrogenase (pZ= 5.4), /l-amylase (pZ= 5.2) and albumin (pZ= 4.7) by capillary electrophoresis in uncoated fused-silica capillaries are reported. Different electrophoretic conditions, consisting in variation of temperature, applied voltage and ionic strength of the buffer used as electrolyte, were tested in order to compare the separation efficiency, resolution and selectivity of the acidic proteins. The results were compared with those obtained by size-exclusion chromatography. Rinsing of the capillary between runs, in order to eliminate adsorbed proteins, can shorten its useful lifetime.
Journal of Chromatography A, 1997
The use of phytic acid to improve protein analysis by capillary electrophoresis (CE) is becoming more and more popular, Due to its size and number of negative charges (up to 12) it provides a high ionic strength combined with a low conductance resulting in an efficient decrease of wall adsorption for proteins. Because of its twelve acidic groups, phytic acid can be used as a buffer over a wide pH range (pH 2-11). The limited wall adsorption of proteins using phytic acid-containing buffers is observed for buffers with a pH of 5.5 and higher. With a monoprotic buffer, most of the investigated proteins show wall adsorption at the pH values studied. In case of a phytic acid buffer, wall adsorption is reduced by a factor of 2-4. The use of phytic acid both as a modifier and as a pH buffer results in more pronounced differences between the various protein mobilities compared with the use of monoprotic buffers. As a result this feature can be used to improve resolution in protein separations.
Automated Serum Protein Electrophoresis by Capillarys®
Clinical Chemistry and Laboratory Medicine, 2000
In this report, we evaluate automated capillary zone electrophoresis by Capillarys ® (Sebia, France). Withinrun and between-run imprecision for the five electrophoretic fractions was < 2% and < 6%, respectively. Data obtained with Capillarys correlated with results obtained with agarose gel electrophoresis and Paragon CZE ® 2000 (Beckman Coulter, USA). Analysis of serum obtained from patients with inflammation, nephrotic syndrome, bisalbuminemia, and ␣ 1 -antitrypsin deficiency revealed that Capillarys was able to detect these abnormalities. Two hundred thirty eight samples were analyzed by agarose gel electrophoresis, Capillarys, capillary electrophoresis using Paragon CZE 2000 system, and immunofixation. Sample selection was based on the presence of a disturbed morphology (e.g., spike) of the protein profile or hypogammaglobulinemia on agarose gel electrophoresis and/ or Capillarys. Immunofixation revealed the presence of a monoclonal protein, oligoclonal bands, polyclonal pattern, and a normal profile in, respectively, 89, 66, 19, and 64 samples. With Capillarys, Paragon, and agarose gel electrophoresis, a spike and/or disturbed morphology of the profile was found in 222, 182, and 180 samples, respectively. In these samples, immunofixation was negative in 73 (33%), 46 (25%), and 39 (22%) samples, respectively. These data indicate that Capillarys has a lower specificity than agarose gel electrophoresis and Paragon 2000. Of the 89 samples with a monoclonal protein, Capillarys, Paragon, and agarose gel electrophoresis failed to detect, respectively, three, three, and one monoclonal protein(s). Interferences by radio-opaque agents, complement degradation products, fibrinogen, and triglycerides are described. In conclusion, automated capillary zone electrophoresis with Capillarys provides for reproducible, rapid, and reliable serum electrophoresis. Clin Chem Lab Med 2003; 41(5):704 -710
Electrophoresis, 1998
Pressure assisting and pressure programming the inlet of the capillary electrophoresis instrument were used for the analysis of peptide mixtures and protein digests using capillary electrophoresis/electrospray ionizationmass spectrometry (CE/ESI-MS). CEIESI-MS of peptide mixtures and tryptic digests of proteins was studied using three different types of capillary columns: (i) a freshly aminopropylsilane (APS)-treated column, (ii) an untreated column, and (iii) a degraded APS-treated column. To maintain a constant and adequate buffer flow toward the CE capillary outlet for stable CE and ESI operation, low pressure was applied to the inlet of the CE when an untreated or degraded APS capillary was used. By programming the inlet pressure, CE/ESI-MS analysis time was reduced to 1/3 of its original time. The utility of this technique is demonstrated by CE/ESI-MS analysis of a hemoglobin variant (hemoglobin-S) and its tryptic digests. Identification of the mutant peptide in the tryptic digest of hemoglobin-S was achieved by collision-induced dissociation (CID) of the urotein digests using CE/ESI time of flightmass spectrometry (TOF--MS).