On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ∼10 s peak width. For 0.1 mg mL⁻¹ bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.     

Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry

On-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization-mass spectrometry (IMAC/CE/ESI-MS) offers selective preconcentration of phosphorylated peptides with identification of the phosphorylated amino acid(s). The preconcentration provides low concentration limits of detection and capillary electrophoresis separates the peptides. Recently, we reported a fast, simple, and sensitive on-line IMAC/CE/ESI-MS/MS method for the determination of phosphopeptides at low-pmole levels. That work is expanded here by use of multiple stage tandem mass spectrometry (MS(n), n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI-MS(n) is demonstrated by the analysis of tryptic digests of alpha- and beta-casein and in-gel tryptic digests of beta-casein.     

Preparative separation of immunoglobulins from bovine colostrum by continuous divergent-flow electrophoresis

Immunoglobulins in bovine colostrum were separated and fractionated from other proteins using the method and instrumentation developed in our laboratory. The proposed separation was based on bidirectional isotachophoresis/moving boundary electrophoresis with electrofocusing of the analytes in a pH gradient from 3.9 to 10.1. The preparative instrumentation included the trapezoidal non-woven fabric that served as separation space with divergent continuous flow. The defatted and casein precipitate-free colostrum supernatant was loaded directly into the instrument without any additional colostrum pre-preparation. Immunoglobulin G was fractionated from other immune proteins such as bovine serum albumin, β-lactoglobulin, and α-lactalbumin, and was continuously collected in separated fractions over 3 h. The fractions were further processed, and isolated immunoglobulin G in the liquid fractions was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by re-focusing in gel isoelectric focusing. Separated immunoglobulin G was detected in seven fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a gradually decreased concentration in the fractions. Re-focusing of the proteins in the fractions by gel isoelectric focusing revealed multiple separated zones of immunoglobulin G with the isoelectric point values covering the range from 5.4 to 7.2. Each fraction contained distinct zones with gradually increased isoelectric point values and decreased concentrations from fraction to fraction.     

Capillary electrophoretic analysis of mu- and m-calpain using fluorescently labeled casein substrates

Calpains are unique calcium-dependent thiol proteases that have been proposed to participate in a number of physiological processes including signal transduction and protein turnover in skeletal muscle. Calpains exist in two major forms. Interestingly, the two forms of protease show no significant difference in their action on various substrates. The only demonstrable difference in their activity involves the concentration of calcium required for activation. Both mu- and m-calpains typically achieve half maximal activation at 50 microM and 0.7 mM calcium, respectively. The focus of this study was to examine the action of both forms of calpain on casein substrates and assess whether any differences could be observed in the resulting peptide finger print using capillary electrophoresis. Purified mu- and m-calpain were incubated for various lengths of time with Oregon Green labeled alphas- and beta-casein. The reactions were stopped with sodium dodecyl sulfate (SDS) and products separated by capillary electrophoresis in micellar electrokinetic capillary chromatography (MEKC) mode using laser-induced fluorescence (LIF) detection. Comparison of the electropherograms showed no difference in the peptide profile for either enzyme. However, it was found that beta-casein was hydrolyzed more extensively than alphas-casein, by both enzymes. Capillary electrophoresis was found to be a very sensitive technique for detection of calpain activity. Using beta-casein as substrate, the CE approach was able to detect 2-3 ng of calpain activity. The results also suggest that capillary electrophoresis is a useful tool for proteolytic investigations of protein structure.     

Analysis of α-keratins in the horns of rhinoceros and buffalo by non-native capillary isoelectric focusing

Summary A capillary isoelectric focusing (CIEF) method under denaturing condition has been developed for separation of α-keratins, highly cross-linked biomacromolecules, in horns of rhinoceros and buffalo. The α-keratins were denatured in 8 M urea and separated in the presence of ampholytes with applicable pH ranges of 3–7 or 3–10. In the preliminary results, it was demonstrated that the α-keratins in the horns of buffalo or rhinoceros might have their own unique isoelectric focusing profiles. The analyses of more samples from different rhinoceros and buffalo are presently underway, with the final goal to establish a fast and convenient method for identification of the source (buffalo or rhinoceros) of an unknown powdered sample.     

Analysis of genetic variants of transferrin in human serum after desialylation by capillary zone electrophoresis and capillary isoelectric focusing

Capillary electrophoresis analysis of transferrin in human serum is used to assess genetic variants after desialylation with neuraminidase and iron saturation to reduce the complexity of the transferrin pattern and thus facilitate the recognition of transferrin polymorphisms. Asialo‐transferrin forms are analyzed by capillary zone electrophoresis using assay conditions as for the monitoring of carbohydrate‐deficient transferrin or by capillary isoelectric focusing in a pH 5–8 gradient which requires immunoextraction of transferrin prior to analysis. With the carrier ampholytes used, peaks for iron saturated and iron depleted transferrin are monitored which indicates complexation of iron ions by carrier ampholytes. For BC, CD, and BD genetic variants, the expected peaks for B, C, and D forms of transferrin were detected with both methods. Monitoring of CC patterns revealed three cases, namely those producing double peaks in both methods, a double peak in capillary isoelectric focusing only and a double peak in capillary zone electrophoresis only. For all samples analyzed, data obtained by capillary isoelectric focusing could be confirmed with gel isoelectric focusing. The two capillary electrophoresis methods are shown to represent effective tools to assess unusual transferrin patterns, including genetic variants with dissimilar abundances of the two forms.     

Validation of capillary electrophoresis in the analysis of ewe's milk casein

Recent investigations have shown that capillary electrophoresis (CE) can be an alternative to other techniques such as polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the qualitative analysis and separation of the different casein fractions in cow's and ewe's milk. However, past work has not yet clarified whether that method can achieve good quantifications. The present study has used a commercial whole ovine casein standard and a mixture of the standard and whole casein extracted from ewe's milk cheese to test the reliability of the technique. The results show that CE was able to quantify the ewe's milk caseins. The areas under four of the most representative peaks on the electrophoretogram for two alpha and two beta-caseins (designated alpha-casein1CE, alpha-casein2CE, beta-casein1CE, and beta-casein2CE in order of elution) were used to validate the method. In relation to linearity, coefficient of determination (r2) values greater than 99% were obtained for the regressions of each of the caseins. Moreover, each casein yielded response factors with a relative standard deviation (R.S.D.) of less than or equal to 5. The coefficients obtained in the day-to-day reproducibility analysis were higher than those for the same-day repeatability, but all the values were within acceptable limits. In the study of accuracy, the percentage recovery rates for the alpha-casein fractions were higher than those for the beta-casein fractions, hence quantification of the latter using this technique would appear to be more accurate under the conditions employed.     

Specific capture of phosphopeptides on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry targets modified by magnetic affinity nanoparticles

Specific capture of phosphopeptides from protein digests is a critical step for identification of phosphoproteins by mass spectrometry. In this study, we report a novel phosphopeptide-capture approach based on the specific interaction of phosphopeptides with a stainless steel target modified with magnetic affinity nanoparticles. The modification which was carried out by loading the suspension of nanoparticles into sample wells of the target did not require any pretreatment procedure to the target and did not involve chemical binding reactions. To isolate phosphopeptides, digests were loaded into the wells of the modified target for 10 min incubation, followed by rinsing with washing buffer to remove unbound species; matrix was then added to the captured phosphopeptides prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Capturing the phosphopeptides on the modified target simplified significantly analytical operations and reduced sample loss. This approach has been applied to solution digests of alpha-casein, beta-casein, and a mixture of five proteins; a number of phosphopeptides were confidently detected. Phosphopeptides from digests of 10 fmol beta-casein could be isolated and detected by MALDI-TOFMS with this method. In addition, this approach has been applied successfully to the isolation of phosphopeptides from in-gel digestive products of sub-pmol phosphoproteins after separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE).     

Effect of pH and ionic strength on competitive protein adsorption to air/water interfaces in aqueous foams made with mixed milk proteins

Quantitative analysis of competitive milk protein adsorption to air/water interfaces in aqueous foam was performed by capillary electrophoresis (CE). Foams were made by whipping protein solutions, in which skim milk powder (SMP) and whey protein isolate (WPI) were mixed at 0.5% protein in different proportions at different pH values and NaCl concentrations. Preferential adsorption of beta-casein into foam phases occurred under most solution conditions, if partial dissociation of the casein micelles had occurred. Preferential adsorption of beta-casein was not observed with added Ca2+, due to the re-association of casein micelles. Enrichment of caseins into the foam phase was more apparent than that of whey proteins. The foamability of SMP demonstrated a continuous improvement due to the gradually increasing dissociation of casein micelles when the concentration of NaCl increased from 0 to 0.8 M. The foamability of WPI increased when NaCl concentration rose from 0 to 0.1 M, and decreased with further increase in NaCl concentration. NaCl at low concentration (I < or = 0.4) did not show a significant effect on the competitive adsorption among milk proteins, indicating that electrostatic interactions do not play a key role in competitive adsorption. NaCl at higher concentration, e.g., 0.6 M, caused less whey protein to be adsorbed to the air/water interfaces. The whippability of WPI was highest at pH 4.5 and lowest at pH 3, and that of SMP was the opposite. The proportions of beta-lactoglobulin and alpha-lactalbumin in the foam phase were lower at acidic pH and higher at basic pH, compared with that at natural pH of WPI.     

Miniaturized two-dimensional capillary electrophoresis on a microchip for analysis of the tryptic digest of proteins

A two-dimensional capillary electrophoresis platform, combining isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), was established on a microchip with the channel width and depth as 100 mum and 40 mum, respectively. With polyacrylamide as permanent coating, EOF in the microchannel, which could impair the separation, was decreased to 3.4x10(-9)m(2).V(-1).s(-1), about 1/10 of that obtained in the uncoated set-up. During the separation, peptides were first focused by IEF in the first dimensional channel, and then directly driven into the perpendicular channel by controlling the applied voltages, and separated by CZE. Effects of various experimental parameters, including the electric field strength, channel length, and injection frequency from the first to the second dimensional separation channel, were studied. Under optimized condition, the digests of BSA and proteins extracted from E. coli were separated, and a peak capacity of 540 was obtained, which was far greater than that obtained by each single dimensional separation. All these results showed the promise of multidimensional separation on a microchip for the high-throughput and high-resolution analysis of complex samples.     

Capillary electrophoresis mass spectrometry and its application to the analysis of biological mixtures

Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex biological mixtures. The highest sensitivities and separation efficiencies are usually achieved by using narrow capillaries (5-50 micro m i.d.) and by using sheathless CE-to-MS interfaces. The difficulties in CE-to-MS interfacing and the limited loadability of these narrow columns, however, have prevented CE-MS from becoming a widely used analytical technique. To remedy these limitations, several CE-MS interfacing techniques have recently been introduced. While electrospray ionization is the most commonly used ionization technique for interfacing CE-to-MS, matrix assisted laser desorption ionization has also been used, using both on-line and off-line techniques. Moreover, the high concentration detection limit of CE has been addressed by development of several sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis have now been interfaced to MS. These advances have resulted in a rapid increase in the use of CE-MS in the analysis of complex biological mixtures. CE-MS has now been successfully applied to the analysis of a wide variety of compounds including amino acids, protein digests, protein mixtures, single cells, oligonucleotides, and various small molecules relevant to the pharmaceutical industry.     

On-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis: a two-dimensional separation system for proteomics

A microdialysis junction is employed as the interface for on-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis in a two-dimensional separation system. Capillary isoelectric focusing not only provides high-resolution separation of tryptic peptides based on their differences in isoelectric point, but also potentially allows the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. Carrier ampholytes, employed for the creation of a pH gradient during focusing, are further utilized as the leading electrolyte in the second separation dimension, transient isotachophoresis-zone electrophoresis. Many peptides which have the same isoelectric point would most likely have different charge-to-mass ratios, and thus different electrophoretic mobilities in zone electrophoresis. Two-dimensional separation of proteolytic peptides is demonstrated using standard proteins, including cytochrome c, ribonuclease A, and carbonic anhydrase II. The maximum peak capacity is estimated to be around approximately 1600 and can be significantly increased by simply increasing the capillary column length and manipulating the range of pH gradient in isoelectric focusing. In addition to enhanced separation efficiency and resolution, this two-dimensional electrokinetic separation system permits sensitive and comprehensive analysis of peptide fragments, especially when integrated with electrospray ionization mass spectrometry for peptide/protein identification.     

Carrier ampholyte‐free isoelectric focusing on a paper‐based analytical device for the fractionation of proteins

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte‐free isoelectric focusing on paper‐based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometry. This paper‐based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost‐effective protein sample clean‐up method for target protein analysis with mass spectrometry.     

应用双向电泳和质谱联用技术研究乳源蛋白的差异性

应用双向电泳和质谱联用技术,对不同乳源蛋白的差异性进行了研究。根据ImageMaster 2DPlatinum图像分析软件对不同乳源酪蛋白和乳清蛋白的双向电泳(2-DE)图谱进行蛋白斑点的匹配分析,获得21个存在于水牛奶中主要分布在低丰度蛋白区的酪蛋白差异蛋白点和24个存在于水牛奶中乳清蛋白差异蛋白点。这些差异蛋白点经质谱鉴定分析,得到4个属于水牛奶酪蛋白的主要组分和2个与水牛奶中酪蛋白有较高同源性的新组分,同时获得4个属于水牛奶乳清蛋白的主要组分和3个与水牛奶中乳清蛋白有较高同源性的组分。     

Preparative isoelectric focusing in a cellulose‐based separation medium

An improved preparative method based on isoelectric focusing of analytes in a cellulose‐based separation medium is described in this study. Cellulose is suspended in an aqueous solution of simple buffers, ethylene glycol, glycerol, nonionic surfactant, and colored pI markers. Water partially evaporates during focusing run and the separation takes place in an in situ generated layer of cellulose, which has a gel‐like appearance at the end of analysis. Final positions of analytes are indicated by the positions of zones of focused pI markers. Fractions, segments of the separation medium with analytes, can be simply collected by spatula and analyzed by downstream analytical methods. Good focusing ability of the new method and almost quantitative recovery of model proteins, cytochrome c and bovine serum albumin, was verified by gel electrophoresis and capillary isoelectric focusing of the collected fractions.     

Quality control in speciation investigations

Information is provided about interferences in speciation investigations. Reasons for contamination and losses, stability problems, metal transfer effects or identification problems are ruled out and possible consequences are named. Logically, solutions and methods for quality assurance and quality control are described. The results prove the reliability of these methods, which should be the basis for further speciation investigations.Abbreviations Cas casein - Cas(+Zn) zinc containing casein - Cas(-Zn) zinc stripped casein - CZE capillary zone electrophoresis - IEF isoelectric focusing - LPLC low pressure liquid chromatography - Mt metallothionein - Mt(+Zn) zinc containing Mt - Mt(-Zn) zinc stripped Mt - SEC size exclusion chromatography     

毛细管电泳-电喷雾质谱联用技术及其在蛋白质分析领域中的应用

综述了毛细管电泳与电喷雾质谱联用的接口技术、分离模式及其在蛋白质分析领域中的应用，特别是毛细管等电聚焦与电喷雾质谱联用在蛋白质组学中研究进展。     

Characterization of cationic acid phosphatase isozyme from rat liver mitochondria.

Acid phosphatase isozyme was highly purified from rat liver mitochondrial fraction. The enzyme showed an isoelectric point value of above 9.5 on isoelectric focusing, and the apparent molecular weight was estimated to be 32000 by Sephadex G-100 gel filtration or 16000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, thiamine pyrophosphate, inorganic pyrophosphate, and phosphoprotein such as casein and phosvitin, but not of several phosphomonoesters, except for p-nitrophenyl phosphate and o-phosphotyrosine. The enzyme was not inhibited by L-(+)-tartrate, and was significantly activated by Fe2+ and reducing agents such as ascorbic acid, L-cysteine,and dithiothreitol. The enzyme was found to be distributed in various rat tissues including liver, spleen, kidney, small intestine, lung, stomach, brain and heart, but not in skeletal muscle.     

Monitoring of transferrin isoforms in biological samples by capillary electrophoresis

Work dealing with the monitoring of transferrin isoforms in human serum and other body fluids by capillary electrophoresis is reviewed. It comprises capillary zone electrophoresis and capillary isoelectric focusing efforts that led to the exploration and use of assays for the determination of carbohydrate‐deficient transferrin as a marker for excessive alcohol intake, genetic variants of transferrin, congenital disorders of glycosylation and β‐2‐transferrin, which is a marker for cerebrospinal fluid leakage. This paper provides insight into the development, specifications, strengths, weaknesses, and routine use of the currently known capillary electrophoresis based assays suitable to detect transferrin isoforms in body fluids. The achievements reached so far indicate that capillary zone electrophoresis is an attractive technology to monitor the molecular forms of transferrin in biological specimens as the assays do not require an elaborate sample pretreatment and thus can be fully automated for high‐throughput analyses on multicapillary instruments. Assays based on capillary isoelectric focusing are less attractive. They require immunoextraction of transferrin from the biological matrix and mobilization after focusing if instrumentation with a whole‐column imaging detector is not available. Interactions of the carrier ampholytes with the iron of transferrin may prevent iron saturation and thus provide more complicated isoform patterns.     

毛细管电泳技术在单克隆抗体药物分析中的应用

单克隆抗体药物在生物制药行业占有重要地位,是生物医药领域发展的主要方向。因此,单克隆抗体药物的质量控制已成为全球生物制药企业及法规机构关注的热点,对单克隆抗体药物精确表征的需求日益增加。毛细管电泳技术具有分离效率高、分析速度快、分离模式多、样品用量少等特点,已成为单克隆抗体药物分析和质量控制的重要手段。该文对毛细管凝胶电泳、毛细管等电聚焦、毛细管区带电泳等模式在单克隆抗体药物的纯度分析、等电点测定、电荷异质性分析和N-寡糖分析的应用进行综述,以期为国内单克隆抗体研究开发和生产的企事业单位提供技术参考。