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This paper provides an annual review of capillary electrophoresis (CE) technology in 2021. A total of 291 research papers related to CE technology published in 2021 were retrieved from the ISI Web of Science using the keywords, "capillary electrophoresis-mass spectrometry" "capillary isoelectric focusing" "micellar electrokinetic chromatography", or "capillary electrophoresis" (not "capillary electrochromatography" "microchip" and "capillary monolithic column"). In addition, nine research papers related to CE technology in Chinese journals were reviewed: Chinese Journal of Chromatography and Chinese Journal of Analytical Chemistry . This review focused on seven papers published in Coordination Chemistry Reviews , Angewandte Chemie-International Edition , Nature Protocols , TrAC-Trends in Analytical Chemistry , and Signal Transduction and Targeted Therapy with impact factors (IFs) greater than 10.0, as well as 42 papers reported in Analytical Chemistry , Analytica Chimica Acta , Talanta , and Food Chemistry with IFs between 5.0 and 10.0. This review also provides a comprehensive overview of representative CE works in Journal of Chromatography A and Electrophoresis with IFs<5.0, as well as important Chinese journals, Chinese Journal of Chromatography and Chinese Journal of Analytical Chemistry . According to the IF, this paper introduces the representative work of CE-related papers to allow readers to quickly understand the important research progress of CE technology in the past year.

Free-flow electrophoresis (FFE) is an all-liquid-phase electrophoresis technique without any supporting media, which has both analytical and preparative functions. Compared to other electrophoresis techniques, FFE has been used for the separation of peptides, proteins, cells, and microorganisms due to its advantages of mild separation environment, high recovery, and sustainable separation. Both the online detection of the characteristic parameters for each component solution and the real-time control of the progress of the separation experiment are of considerable importance for the study of FFE separation. Since the existing FFE devices do not have the online detection function, there are obvious deficiencies in their practicability. The absence of online detection function not only made it impossible to track the progress of the separation experiment in real time, but also made it difficult to detect the properties of the component solutions, which still require offline testing after separation. In this study, a multi-channel capacitively coupled contactless conductivity detection (MC-C 4 D) device has been developed to solve this problem, and an automatic measurement software has also been developed. The MC-C 4 D device used a parallel time-sharing contactless conductivity detection technique. It consisted of several contactless conductivity detection modules arranged in parallel, which in turn consisted of a number of contactless conductivity cells that were switched on/off by analog multiplexers for detecting the conductivity of the solution flowing through the cells in real time. The number of cells was equal to the number of components of the FFE. The components were connected to each of the FFE flow channels, such that the MC-C 4 D device could be used to measure the conductivity of the solution flowing through each channel in parallel online. To verify the performance of the MC-C 4 D device, calibration was conducted by using potassium chloride (KCl) standard solutions on MC-C 4 D device. The experimental data showed that the detection range of MC-C 4 D was 0.015-2.5 mS/cm, and the limit of detection (LOD) was 0.002 mS/cm. The intra-day relative standard deviation (RSD, n =3) was 2.31%, the measurement relative error (RE) was 3.03%, and the measurement difference between channels was 1.60%. All these data validated that the device had the advantages of wide detection range, low LOD, good repeatability, high accuracy, and low variation between channels. The MC-C 4 D device was also applied to reciprocating free-flow isoelectric focusing (RFFIEF) electrophoresis for real-time online detection of the conductivity of each component solution during protein focusing. At the start of isoelectric focusing, when the ions had not reached equilibrium loading in the electric field and the pH gradient had not yet been fully developed, there was little difference in conductivity between the different channels and the channel conductivity curve was relatively flat. As the experiment progressed, the proteins gradually started to enrich the anodic end. As the proteins accumulated towards the isoelectric point, their own net charge gradually decreased, and thus, the conductivity of the solution in the channels near the anodic region also decreased. Under sufficient isoelectric focusing, protein enrichment was evident. In the focusing region, the conductivity of the solution in the corresponding channel decreased further. There was also an increase in the conductivity of the solution in the corresponding channel due to the accumulation of ions near the electrode ends. These results showed that the MC-C 4 D device not only enabled real-time online detection of the conductivity of each component solution in FFE, but also aided in mastering the progress of separation experiment in RFFIEF, thus improving the practicality of the FFE device. Thus, the MC-C 4 D device, which had the advantages of good performance, small size, simple circuit system, easy installation and commissioning, and low cost, could play an important role in multi-channel measurement, online inspection, and process monitoring.

This paper mainly discussed the dyeing of cashmere fiber at low temperature and isoelectric point, the dyeing of mordant dyes with low chrome in theory and in practice. Contrast experiments were also conducted from in the lab to mass production. It was proved that in the process of semi-worsted cashmere dyeing, the two new methods could ease fiber damage, improve semi-worsted yarn spinnability and yield rate. [ABSTRACT FROM AUTHOR]

Hemoglobin A 1c (HbA 1c ) is a major component of glycated hemoglobin in human red blood cells. It has been proven to be a significant biomarker for the diagnosis of diabetes; its content in fresh red cells in diabetes blood reflects the average level of blood glucose over the previous three months. Thus, HbA 1c level has been used for the assessment of long-term glycemic control in diabetes; the level of 6.5% HbA 1c has been certified as a critical cut-off for the diabetes diagnosis. The current commonly used method for HbA 1c quantification is based on cation-exchange high performance liquid chromatography (CX-HPLC). The method has advantages such as high stability, rapidity, and automation, but there are still some unidentified peaks of Hb species in CX-HPLC (VARIANT Ⅱ system); in particular, the presence of HbA 3 (a glutathiolated Hb) affects the accurate determination of HbA 1c . HbA 3 is usually present in healthy adult blood samples at 2%-4%, but the concentration of HbA 3 increases due to the protection of erythrocytes from oxidation, resulting in decreased HbA 1c . However, the relative location of the HbA 3 peak in the CX-HPLC clinical chromatogram has not been established. To address this issue, we extracted Hb species from fresh blood samples obtained from a hospital in an anaerobic environment to avoid possible redox reactions of Hb and glutathione. After the extraction, the Hb samples were analyzed using two methods: a low-resolution CX-HPLC (5/50 mm column) currently used for diabetes diagnosis and a high-resolution cationic exchange HPLC (Mono-S 5/50 mm column), to identify the peak corresponding to HbA 3 . The CX-HPLC analysis of fresh blood samples indicated that the unknown peak P3 located between HbA 1c and HbA 0 peaks corresponded to the HbA 3 peak between HbA 1c and HbA 0 in the Mono-S-HPLC. Microarray isoelectric focusing (IEF) was used for the micro-preparation of HbA 3 , HbA 1c , and HbA 0 in healthy blood samples; then, the micro-prepared species of HbA 3 , HbA 1c , and HbA 0 were individually identified via Mono-S-HPLC. The results of the CX-HPLC, Mono-S-HPLC, and microarray IEF experiments indicated that the P3 peak might correspond to HbA 3 . To confirm this, glutathiolated Hb samples were synthesized via acetylphenylhydrazine and analyzed using both the Mono-S- and CX-HPLC systems. The results showed that the content of both glutaminated hemoglobin of HbA 3 in Mono-S-HPLC and P3 in CX-HPLC increased, implying the peak of P3 with the retention time of 1.50 min in CX-HPLC was the peak corresponding to HbA 3 in Mono-S-HPLC and microarray IEF. Based on the above experiments and our previous results, the influence of HbA 3 on both the analysis of HbA 1c in blood samples and the diabetes diagnosis needs to be considered and discussed. The study results are significant for the tentative assignment of peak P3 and for offering more information on diabetes diagnosis using CX-HPLC in the clinical setting.

Proteomic analysis plays an important role in basic biological studies and precision medicine. However, real samples contain numerous proteins with a wide dynamic distribution range. Such high complexity of the samples has a drastic effect on the identification coverage of proteins. Consequently, with advancements in mass spectrometry (MS) technology, concomitant improvements in separation technologies for simplifying the sample should be critical. With the advantages of small sample loading volume, high separation efficiency, and high speed, capillary electrophoresis (CE) coupled to MS has been gained much attention in the field of proteomics research. A nanoflow sheath liquid interface and a sheathless interface have been developed and commercialized, boosting the development of the CE-MS technology. Capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and capillary electrochromatography (CEC) have been successfully combined with MS, and CZE-MS has widespread application. In proteomic research, the "bottom-up" strategy, which is based on the separation and identification of enzymatic peptides, is widely applied. With the limit of detection as low as 1 zmol for peptides, CE-MS has been successfully applied to single-cell proteomic analysis. Besides, CE is complementary to reversed-phase liquid chromatography (RPLC), providing a new approach for the separation and identification of peptides with similar hydrophobic properties (especially, post-translational peptides). The "top-down" strategy, which is based on the separation and identification of intact proteins, can directly provide more accurate and complete information about proteins. For protein separation, CE is advantageous in terms of the high separation resolution and high protein recovery, thereby improving the sensitivity and coverage of protein identification. Native MS enables successful identification and characterization of protein complexes under nondenaturing conditions. Because of the good compatibility of CE with MS, attempts have been made to use CE coupled with native MS for the separation and identification of protein complexes. In this review, the development of the CE-MS technology is first reported, including a robust and sensitive CE-MS interface, and a separation mode coupled to MS. Then, the application of the CE-MS technology to "bottom-up", "top-down" and native MS analysis is discussed. The superiority of CE-MS in proteomic analysis is also emphasized. Finally, the promising future prospects of CE-MS are discussed.

A partially filled monolith was prepared by in situ polymerization, and then carrier ampholytes (CAs, pH 3-10) were immobilized on its surface. For effective utilization of capillary isoelectric focusing (cIEF) with the monolithic immobilized pH gradient (M-IPG), a new online platform was established by the introducing of an eight-way injection valve, a three-way valve and a cross-shaped unit. Besides, a capillary coated with hydroxypropyl cellulose (HPC capillary) was prepared and used to determine the isoelectric points (p I ) of trastuzumab and etanercept. In parallel, using the newly built capillary isoelectric focusing platform, the p I values of trastuzumab and etanercept were measured with the M-IPG column, and compared with the results obtained using the HPC capillary. It was found that these two cIEF columns can be effectively used to separate proteins and determine the p I values of monoclonal antibodies and fusion proteins in protein drugs. Moreover, the measured p I values were consistent with those estimated using the HPC capillary.

Therapeutic monoclonal antibodies play an important role in biopharmaceuticals, and gradually become one of the main directions of the development of biological medicine. The quality control of monoclonal antibodies has also become the focus of global biopharma companies and regulatory agencies. Capillary electrophoresis has become an important tool in the analysis of monoclonal antibodies with multi-modes and high resolution. In this paper, we reviewed the application of capillary gel electrophoresis, capillary isoelectric focusing and capillary zone electrophoresis on the analysis of purity, isoelectric point/charge heterogeneity and N -glycan profiling of therapeutic monoclonal antibodies.

Preparative electromigration techniques are a category of separation and preparation techniques based on differential electromigration principle. These techniques are widely applied in biomacromolecule and proteome researches. Related techniques mainly include preparative electrophoresis, preparative electrochromatography, preparative isoelectric focusing and free flow electrophoresis and so on. We review each kind of the preparative electromigration techniques on its apparatus designs, applications, advantages and disadvantages in detail. Microscale preparative electrophoresis is a hot spot in recent years. Its high resolution, high recovery and high efficiency make it a more prominent role in the biological sample analysis. In this review, we focus on some advances in microscale preparative electrophoresis and also give an outlook on preparative electromigration techniques in the future.

Here we discuss whether N terminal sequencing is appropriate as one of the conventional control methods for monoclonal antibody products. We determined the N terminal sequences of two monoclonal antibody products targeting two antigens separately with both Edman degradation and mass peptide spectrometry. We also identified the characteristic peptide fragments with mass spectrometry. Furthermore, we analyzed their heterogeneity with ion exchange chromatography, capillary zone electrophoresis and Imaged Capillary Isoelectric Focusing. Edman degradation method showed that the N terminal 15 amino acids of heavy and light chains of the two monoclonal antibodies were identical. Peptide mass spectrometry demonstrated that T1 peptide fragments of heavy and light chains of the two antibodies were also the same. But in contrast, peptide mapping and the three analytical methods for heterogeneity analysis could effectively identify and differentiate the two antibodies. The N terminal sequences of two monoclonal antibodies are identical because the number of framework sequences of humanized or human monoclonal antibodies is relatively limited, so whether N terminal sequencing analysis could be regulated as one of the practical control methods should be carefully discussed. Our work also proves that the above analytical methods could combinatorially applied to the identification of monoclonal antibody products, and are more objective compared to N terminal sequencing.

Secretome (secreted proteins) has been arousing considerable attention because of its potential to develop novel biomarkers for the early detection and diagnosis of diseases. Like other proteome systems, a secretome generally consists of thousands of proteins with a wide dynamic range of abundance. The effective way to decrease the complexity of the analyte is to combine different prefractionation methods with orthogonal separation capabilities. The newly appeared OFFGEL fractionation system provides the ability to separate the peptides into discrete liquid fractions according to their differences in isoelectric point (pI), and has demonstrated high efficiency and repeatability for the analysis of the complex proteins. Here the OFFGEL fractionation system was evaluated for the separation of peptides digests from secreted proteins of hepatocellular carcinoma cells (HCC), using a 12-wells device encompassing the pH range of 3 - 10. In combination with the online reversed phase liquid chromatography and mass spectrometry, peptides from the digestion of MHCC97L secreted proteins were separated and identified. Preliminary results showed that nearly 80% of the peptides identified were specific to one fraction, and 40% of the proteins were obtained from the fractions 1 -3 from the acid end. The average of peptide pI values distributed in each fraction is consistent with the theoretical pIs, and the pI distribution of the peptides focused on the low pH range showed a smaller deviation. Finally, 2 995 proteins were identified from secretome of MHCC97L cell lines. Thus the data show that the OFFGEL system provides a highly valuable tool to fractionate peptides from complex protein samples.

One-step and two-step capillary isoelectric focusing (clEF) methods were employed the separation and pI determination of proteins and polypeptides. The parameters affecting the analysis efficiency, such as the sample solution, injection volume, focusing voltage, focusing time and driving conditions were optimized. The comparison of the two methods for separation of cytochrome C, hemoglobin, myoglobin, transferrin, bovine serum albumin and six polypeptides showed that the one-step cIEF was simple and fast, which could determine the pI of single component as well as it was rapid for protein and polypeptide separation, but it could not get good resolution or accurate pI of each component in a mixed sample. The two-step cIEF was more complex and needed longer time, however, which could separate and exactly determine the pI of each component in the mixture, and the pI value of each component determined was consistent with that determined using a single sample. The two methods are complementary, and can be widely used in rapid and accurate determination of the pI of amphiphilic biological particles.

Objective: To optimize the two-dimensional electrophoresis (2-DE) method for the proteome analysis of the Cervus nippon antler, and compare the protein maps of different parts of Cervus nippon antler., Methods: The total proteins of Cervus nippon antler were extracted by protein lysate containing 7 mol/L Urea, 4% CHAPS, 2 mol/L Thiourea, 65 mmol/L DTT, 1 mmol/L PMSF and 0.2% Bio-Lyte. The proteins were separated by immobilized pH 3 - 10 linear gradient (IPG), 7 cm strips as the first dimension. Isoelectric focusing conditions were optimized. 12% SDS-PAGE was used as the second dimension electrophoresis. The gels were stained with Coomassie brilliant blue and analyzed by PDQuest analysis software., Results: The contents of total protein and the numbers of protein points of three different parts of Cervus nippon antler reduced gradually from the top to the bottom. Comparing three maps of different parts of Cervus nippon antler, there were 18 different protein points. Isoelectric point, molecular weight and gray value of each different protein point were calculated., Conclusion: An optimized two-dimensional electrophoresis method for the proteome analysis of the Cervus nippon antler is established. The 2-DE profiles of different parts of Cervus nippon antler exist obvious differences. The different protein points can be used as reference for Cervus nippon antler quality control and evaluation.

The pancreas ferritins from chickens, ducks, cattle and pigs were isolated by thermal denaturation, ammonium sulphate fractionation and DEAE-52 cellulose anion exchange chromatography separately, in order to obtain the characteristics both subunit types and isoelectric points. Four ferritins such as chicken pancreas ferritin (ChPF), duck pancreas ferritin (DPF), cattle pancreas ferritin (CaPF), and pig pancreas ferritin (PPF) showed different mobility in polyacrylamide gel electrophoresis (PAGE). The relative molecular masses (M(r)) of these ferritin were indicated to be M(r) (ChPF) > M(r) (DPF) > M(r) (CaPF) > M(r) (PPF), which are all bigger than that in horse spleen ferritin (HSF). Sodium dodecyl sulfate (SDS)-PAGE results indicate that ChPF, DPF, CaPF and PPF consist of H and L subunits, showing different ratios of H/L subunits. Two subunit types in the ferritin were further identified by peptide-mass fingerprinting (PMF) technology. The four ferritins such as ChPF, DPF, CaPF and PPF in denatured-isoelectric focusing (IEF) gel show the subunit polymers containing from 3 to 6 with different pI values, respectively. These phenomena reveal the complicated interactions and different polymers between H and L subunits in the ferritins. There are differences both interaction intensities and polymers in the ferritin subunits coming from different mammals. These heterogeneity may response to the rate of iron release in ferritins and to the detoxification requirement of iron in animals in vivo.

Three Se-containing allophycocyanins (Se-APC) with high purity were purified from Se rich Spirulina platensis (Se-sp.) by hydroxyapatite chromatography, DEAE-52 anion-exchange chromatography and native gel preparative electrophoresis. Their biochemicial properties were explored by spectral scanning and electrophoresis analysis of Native-PAGE, SDS-PAGE and IEF on thin slab gel. Protein molecular weight (MW) of APC aggregation was determined by gel filter on Sephadex G-200 column. Se content of native and denatured Se-APC was detected by 2, 3-DAN fluorocence method. According to visible and fluorescence spectral character, three purified fractions of APC were identified to be APCI, APCII and APCIII. Native-PAGE and SDS-PAGE analysis revealed that they all shaped trimer (alphabeta) 3 of alpha and beta subunit with molecular mass of 18.3kDa and 15.7kDa, whereas APCI contains gamma subunit (about 32kDa) visibly and APCIII maybe contain the linker peptide of L(C)(8 - 10 kDa) based on their MW to be determined of 130.9, 98.1 and 106.30 kDa. IEF detection showed that the pl of Se-APCs was 4.76, 4.85 and 5.02 respectively. Se content of three purified Se-APCs were 316, 273 and 408 microg/g, which decreased about 25% after deaggregation treatment by 0.50 mol/L NaSCN and decreased more than 50% after denaturation treatment by 2-mercaptoethanol and reached to a steady content of 132 microg/g on average. These results indicated that Se incorporation into APC had no influence on function of energy transfer as well as biochemical property of APCs, and Se binding with APCs was highly relevant to its aggregation states whereas Se integrated steadily with its subunits.

Objective: To analyze the zymogram of peroxidase (PER) and phosphoglucose isomerase (PGI) of three species of Sarcocystis., Methods: The collected parasites were homogenized and fragmented by ultrasonication. After centrifugation, the supernatants were analyzed by isoelectric focusing electrophoresis., Results: The isolates of S. cruzi from infected water buffalo and cattle all showed identical enzyme profiles, 7 bands of PER at pH 4.44-6.98 and 6 bands of PGI at pH 4.66-6.53; and same with the isolates of S. hirsuta. 5 bands of PER at pH 4.97-7.15 and 4 bands of PGI at pH 4.70-6.51. The zymograms among S. cruzi, S. hirsuta and S. fusiformis were different considerably., Conclusion: The data support the hypothesis that both water buffalo and cattle are the natural intermediate hosts of S. cruzi and S. hirsuta at the gene level. S. cruzi, S. hirsuta and S. fusiformis are different species.

Objective: To investigate the molecular mechanism of multiple-drug and pan-drug resistance among Acinetobacter species., Methods: Non-repetitive 90 carbapenem-resistant strains of Acinetobacter species were collected in Beijing, Guangzhou, and Fuzhou 1999-2004. The homology of the isolates was determined by both pulsed field gel electrophoresis and randomly amplified polymorphic DNA typing. Seven representative clones were selected from the 90 strains of Acinetobacter isolated from different hospitals to be used for further study. Analytical isoelectric focusing was used to measure the isoelectric point of the beta-lactamase. Plasmid DNA was extracted and purified Genes of different beta-lactamase, including bla(TEM--), bla(SHV-), bla(PER-), blaI(MP-), bla(VIM-), and bla(OXA-) genes, in these clone strains were amplified and sequenced. PCR was used to analyze the integrons., Results: The P clone strain isolated during an outbreak of pan-drug-resistant Acinetobacter species in Peking Union Medical College Hospital 2004 was not susceptible to most common antimicrobial agents tested. The 7 representative clones produced multiple beta-lactamases: TEM-1, high-level AmpC, SHV-type, OXA-23 carbapenemase and IMP-8 and metalloenzyme respectively. One clone produced PER-1 enzyme. These 7 clone strains were resistant to most beta-lactams (including carbapenems), erythromycin, chloramphenicol, and rifampin. Two clone strains were susceptible to cefoperazone/sulbactam and amikacin while 4 clone strains susceptible to levofloxacin. All of the 7 clones were susceptible to minocycline and colistin. Five different integrons were found, harboring the genes mediating the resistance to aminoglycosides, rifampin, chloramphenicol, and carbapenems (bla(IMP-8))., Conclusion: The molecular bases of multiple-drug or pan-drug resistance in Acinetobacter species include production of OXA-23 carbapenemase or IMP type metalloenzyme and integrons with different resistance gene cassettes. Pan-drug-resistant Acinetobacter species are susceptible to old antimicrobials agents, such as colistin and minocycline.

Objective: To study differential proteins and their biological functions associated with colorectal cancer genesis and hepatic metastasis by proteomics and molecular biology techniques., Methods: Isoelectric focusing/SDS acrylamide gel two-dimensional electrophoresis was used to analyse the expression of differential proteins from normal colorectal mucosa, primary cancer lesion and hepatic metastasis region. Liquid chromatography/mass spectrometry (LC-MS/MS) was used to identify the differential proteins. Transfection of colorectal cancer lovo cells was performed with the differential protein cDNA, and the changes of cell biological behavior was observed., Results: Significant difference in protein expression was found on two-dimensional electrophoresis. Thirteen differential protein spots were analysed and identified. Human carbonic anhydrase II was detected in normal colorectal mucosa but not in primary cancer lesion and hepatic metastasisnegion. Phosphoglycerate kinase 1, fumarate hydratase and aldolase A were expressed in primary cancer. Expression of homo sapiens arginase and homo sapiens glutathione S-transferase A3 was found in hepatic metastasisnegion, but not in primary cancer lesion. After transfection with human carbonic anhydrase II cDNA, the lovo cells changed obviously with reduction in invasiveness, chemotaxy motor ability and tolerance., Conclusion: Differential expression of proteins was found between colorectal cancer genesis and hepatic metastasisnegion. No carbonic anhydrase II expression and enhanced expression of sapiens arginase and sapiens glutathione S-transferase A3 are related with biological behavior of colorectal cancer cell and facilitate hepatic metastasis.

Objective: To investigate the molecular mechanisms of tauopathies. Comparative proteomic analysis of brain proteins was employed to study 4 patients with tauopathies as compared with 4 controls., Methods: The brains of subjects who died without clinical or pathological involvement of nervous system and brains of patients with tauopathies were obtained at autopsy. The brain proteins were run by immobilized pH gradient (IPG) isoelectric focusing electrophoresis as the first dimension, and then run by vertical SDS-PAGE as the second dimension. The maps were visualized by silver staining or colloidal coomassie blue and analyzed with Image Master 2D Elite software. The proteins of interest were in-gel digested and identified using MALDI-TOF mass spectrometry or MALDI-TOF/TOF tandem mass spectrometry., Results: 18 protein spots were differentially expressed as compared with age-matched nondemented control brains which were identified as glyceraldehyde 3-phosphate dehydrogenase, uracil DNA glycosylase, human superoxide dismutase, isocitrate dehydrogenase subunit, synaptotagmin I, thioredoxin peroxidase 1, glial fibrillary acidic protein, p25 alpha, enoyl coenzyme A hydratase short chain 1, pyridoxine-5'-phosphate oxidase, Mn-superoxide dismutase and alpha enolase, antioxidant protein 2, ferritin heavy chain, glutamate dehydrogenase precursor, peptidyl-prolyl cis-trans isomerase A, serum albumin precursor and dihydropyrimidinase-related protein 2., Conclusions: We got a number of related-proteins of tauopathies. Some proteins are quite useful for discovering the molecular mechanisms of tauopathies and may be helpful for diagnosis and of treatment tauopathies.

Two-dimensional electrophoresis (2-DE) is the core technology of proteome study. This paper mainly introduces the 2-DE principle, sample preparation and usual methods to improve its resolving power. Furthermore, isoelectric focusing, SDS-PAGE electrophoresis and 2-DE staining methods are also reviewed.

The aim of this study was to explore the effect of different conditions on two-dimensional gel electrophoresis of proteins from human acute promyelocytic leukemia cell line NB4. The 24 cm pH 3-10 linear immobilized pH gradient (IPG) strips were chosen, the isoelectric focusing was carried out by using IPGphor. Then, the second-dimensional SDS-PAGE was performed. After silver staining, the gel was analyzed by ImageMaster 2D Elite. The results showed that low ion intensity sample washing buffer improved the performance of isoelectric focusing. The lysis buffer containing 7 mol/L urea and 40 mmol/L DTT could solubilize the most proteins from NB4 cell line. The rehydration solution containing thiourea and urea increased the low molecular weight protein points to be resolved in the area of basic end. The reasonable sample load and Volt/hour of NB4 were about 100 micro g and 63 200 V/h for the 24 cm pH 3-10 IPG strips. It is concluded that the proteins from NB4 and similar cell line are complicated and affected by many factors, so that, it is very important to select the right methods for sample preparation and the conditions of two-dimensional gel electrophoresis.

Objective: To study differential expression proteins associated with colorectal cancer genesis and hepatic metastasis with proteomic techniques., Methods: Using isoelectric focusing/SDS acrylamide gel two-dimensional electrophoresis to analyse differential expression protein spots among normal colorectal mucosa, primary cancer lesion and hepatic metastasis. Peptide mass fingerprinting was used to identify the differential proteins., Results: Significant differences of protein expression were found on two-dimensional electrophoresis. Nine differential protein spots were analysed and identified. Calmodulin, ribonuclease 6 precursor and protein XP&#95;040720 (mannosidase-alpha) were detected in normal colorectal mucosa, but lost in primary cancer lesion and hepatic metastasis. Proapolipoprotein was expressed progressively from normal mucosa to primary cancer and hepatic metastasis. Expression of beta-globin was found in normal mucosa and hepatic metastasis, but not in primary cancer lesion. Cdc42 was a differential expression protein in hepatic metastasis. Peptide mass fingerprints of differential protein spot C4, M7 and M9 had low homology with database proteins, they were candidates of associated proteins with colorectal cancer genesis and hepatic metastasis., Conclusion: Loss of calmodulin, ribonuclease 6 precursor and mannosidase-alpha expression are associated with colorectal cancer genesis. Enhancement expression of proapolipoprotein is related with colorectal genesis and hepatic metastasis. Cdc42 and beta-globin are associated proteins with hepatic metastasis of colorectal cancer.

Objectives: To analyze human spermatozoa membrane proteins by two-dimensional gel electrophoresis and to provide a basis for drawing the protein map of normal human spermatozoa membrane proteins., Methods: Spermatozoa were purified by Percoll density centrifugation, and spermatozoa membrane proteins were analyzed by two-dimensional gel electrophoresis using isoelectric focusing and polyacrylamide gel electrophoresis., Results: About 800 protein spots could be identified by the imaging analysis system. The molecular weight and isoelectric point of most proteins were 20,000 to 100,000 and 3.0 to 7.0 respectively., Conclusions: There are more than 800 types of proteins in the human spermatozoa membrane. The spermatozoa membrane protein can be identified with a certain precision by two-dimensional gel electrophoresis.

Objective: To investigate the expression of androgen receptor (AR) isoforms in normal human prostate., Methods: Fourteen normal prostatic specimens from donors, aged 25 on average (21-28 yr), were analyzed by high resolution isoelectric focusing (IEF). The expression of AR isoforms was demonstrated in all 14 normal human prostatic tissues., Results: Four types of AR isoforms were detected with isoelectric point value at 6.5, 6.0, 5.8 and 5.3 in 14 prostatic specimens. Binding of 3H-dihydrotestosterone (DHT) to these four AR isoforms was inhibited by the addition of 100-fold excess of DHT and testosterone. No effect of progesterone, estradiol and diethylstilbestrol on tritiated hormone binding was observed., Conclusions: There are four AR isoforms in normal human prostate. The expression of AR isoforms is different from one another.

In this work, the experimental conditions for two-dimensional gel electrophoresis of subretinal fluids (SRF) matrix metalloproteinases were established. The conditions tested included the composition of lysis solution and lysis method, the composition of rehydration solution and isoelectric focusing program (IEF), the composition of equilibration buffer and equilibration process and the composition of incubation solution and incubation methods. The main equipments used were IPGphor isoelectric focusing system from Amersham pharmacia and PROTEAN II xi cell from Bio-Rad, the gel strips used were the 18 cm long, pH 3 - 8 Linear immobiline DryStrips. Among the 9 samples analyzed, 2 were PVR-A, 2 were PVR-C1, 2 were PVR-C2, 2 were PVR-C3 and the remaining one could not be classified definitely. The new 2-DE MMPs method is better than Gelatin SDS-PAGE zymograhpy method, as it is higher in resolution, sensitivity and reproducibility. The experimental results suggested that the four types of MMPs expressed differently at different stages of PVR. Two of the MMPs isomers have same molecular weight (MW) but different in isoelectic points (pI). The four MMPs are determined to be MMP-1, MMP-2, MMP-9 and MMP-9, with MMP-9 has two active forms. In addition, MMP-9 and MMP-1 may be present in PVR-A samples but not in PVR-C samples, whereas MMP-2 is present in PVR-C but not in PVR-A samples. These results revealed the complex profiles of MMPs' expression in PVR. The new method can be applied to test MMPs expression in tissues, cells and other types of samples with a little modification in the protocol, and can be followed by mass spectroscopic analysis of MMPs.

Objective: To understand the phenotype distribution profile of alpha1-antichymotrypsin (alpha1-ACT) in the Han population in Guangzhou municipality so as to decide whether geographical factors affect the distribution of alpha1-ACT phenotypes., Methods: Polyacrylamide gel isoelectric focusing followed by immunofixation techniques was employed for examining the alpha1-ACT phenotypes in 200 unrelated individuals randomly selected from the Guangzhou Han residents., Results: Three phenotypes of alpha1-ACT were identified in this subject cohort, which were identical to those identified in Chongqing but with different distributions of the phenotype frequencies., Conclusion: Geographical factors may affect the distribution of the phenotype frequencies of alpha1-ACT, which is a promising genetic marker for the pursuit of human genetics.

Aim: To study the endogenous mechanism for the inhibition of aquaporin-1 expression in rat renal proximal tubule epithelial cells in response to acetazolamide., Methods: Primary cultured rat renal proximal tubule epithelia cells were divided into two groups: one was subjected to 1 x 10(-5) mol.L-1 acetazolamide, the other served as normal control. When grown to sub-confluency, the cells were disintegrated to perform isoelectrofocusing electrophoresis in order to find the differential proteins induced by the acetazolamide treatment. The differential proteins were defined by peptide mass fingerprinting technology., Results: Two differential proteins were found in the cell disintegrant. The pI 3.8 protein was reduced after treatment, which showed 21.4% similarity with the brush border membrane myosin from rat brain and testis, and 27% with glycogen phosphorylase; The pI 5.5 protein was increased on the contrary, with 20% similarity to phosphatidylinositol transfer protein alpha isoform., Conclusion: Acetazolamide inhibited AQP1 expression probably by affecting the expression of pI 3.8 and pI 5.5 proteins.

Objective: To investigate the androgen receptor isoforms expression and its novel eDNA cloning in prostate tissue., Methods: The tissue from a case of benign prostatic hyperplasia (BPH) was analyzed by high resolution isoelectric focusing (IEF) technique and molecular biological technique of cDNA cloning was used to determine its AR isoforms expression and genes of AR isoforms., Results: From the prostatic specimen, three types of AR isoforms were detected with pI values of 6.5, 6.0 and 5.3 respectively. A novel AR subtype cDNA cloning, named as PHAR-1 and including 1 913 nucleotides was isolated from the same BPH tissue. The GenBank accession number for the nucleotide sequence of this AR cDNA was BankIT372205 AF324243. The comparison between PHAR-1 and AR nucleotide sequence registered in GenBank showed that 961 nucleotides in three part of nucleotide sequence were the same, while the other 952 nucleotides were different., Conclusion: These three AR isoforms may originate from the same gene. The results may be important for the study of BPH, or prostatic growth. It is also valuable in the field of pharmacotherapy for treatment of BPH and prostatic cancer.

Objective: To understand the components of the crude antigens of Echinococcus multilocularis protoscoleces., Methods: Isoelectric focusing was carried out to fractionate the protein components of E. multilocularis protoscoleces. The individual fractions were collected and subjected to SDS-PAGE, the gels were analyzed by a gel analysis system., Results: The pI values of individual fractions varied from 4.0 to 8.5, while the molecule weights of main protein bands were 72/73, 62/63, 42/44, 35/36, 34 and 21/22 kDa., Conclusion: An alternative separation method of crude antigens of Echinococcus multilocularis was established.

Objective: To determine the genetic polymorphisms of serum orosomucoid (ORM) in Han population in Nanjing of China and provide genetic data for studying the individual variance in the plasma protein binding of weak alkaline drugs., Methods: The desialylated sera from 220 unrelated subjects were analyzed by isoelectric focusing on polyacrylamide gels followed by immunoblotting., Results: The band patterns of ORM showed that the polymorphism of the structural locus ORM1 was controlled by three codominant autosomal alleles, ORM1*F1, ORM1*F2 and ORM1*S, which presented five phenotypes, ORM1 F1(47.27%), ORM1 S(5.46%), ORM1 F1F2 (3.18%), ORM1 F1S(43.64%), and ORM1 F2S(0.45%). The allele frequencies were: ORM1*F1=0.7068, ORM1*F2=0.0182, ORM1*S=0.2750., Conclusion: The ORM1 locus is polymorphic and the ORM2 locus is monomorphic in sera from the Han people in Nanjing of China.

A simple, rapid and accurate method for phenotyping sera apoE has been developed. In this method 10 microliters serum or plasma are incubated with Dithiothreitol and Tween-20 for 15 min and then applied to 5% polyacrylamid gel containing pH 4-8 ampholyte and 3 mol/L urea. After 2 h focusing, the apoE bands are transferred to Nitrocellulose membrane. apoE bands are made visible by immunoblotting and TMB is used as the substrate. Identification phenotype is easily accomplished by noting the location and number of protein bands. 56 samples shows apoE 3/3 in 47 cases, apoE 2/3 in 2 cases, and apoE 3/4 in 7 cases. This method is well suited for large-scale population studies and clinical labs.

Objective: To understand the distribution of allele frequencies of blood factor XIIIB subunit(FXIIIB) in Chinese populations and evaluate the genetic polymorphism of FXIIIB for the purposes of population genetics and forensic haemogenetics., Methods: The genetic polymorphism of FXIIIB subunit in seven Chinese populations was investigated. Isoelectrofocusing technique on polyoacrylamid gels followed by immunoblotting was used to determine the phenotype of individuals in each population sample., Results: There were three common alleles in all the seven Chinese populations. The frequency of FXIIIB*3 was the highest, that of FXIIIB*2 was the lowest, and the one of FXXIIIB*1 was at the middle. All of them reached to the polymorphism's level. A rare variant allele was also found in some Chinese populations. Comparison of the constituents of FXIIIB phenotypes in the seven populations showed that there was no significant difference (P>0.05). The distribution of the allele frequencies in these populations reflected most likely the mode of the distribution in all Chinese populations. The phylogenetic tree and genetic distance, based on the allele frequencies of FXIIIB differentiated the populations in the world into the main ethnic groups as what other authors reported., Conclusion: FXIIIB is a useful genetic marker for population genetics and forensic haemogenetics.

The study is to find new genetic markers of simultaneous phenotyping and to raise the determinating power of isoenzymes. Simultaneous electrophoresis of EAP and PGM1 by thin-layer PAG1F combined with reverse zymogram developing was applied and clear isoenzyme bands was observed on both surface of a gel without interference. The method can do typing of the EAP and PGM1 at one gel and the accumulative DP is 0.87. It can be used in paternity test.

Transferrin (Tf) is an important genetic marker for personal identification in forensic science. We examined the subtypes in human semen, semen stain, vaginal fluid and mixed stain using isoelectric focusing and immunoblotting method(IEFIB). The results showed that, Tf subtypes of 8 semen and semen stain samples stored at room temperature for up to 32 weeks were the same as those in sera. Tf subtypes of 17 vaginal fluid and 4 mixed stain containing few semen were not detected. Tf Subtyping of 1 mixed stain containing enough semen was successful by using IEFIB. These results indicate that Tf is stable in semen stain. The Tf subtyping in semen stain and mixed stain containing enough semen can be carried out by using IEFIB method.

The isozyme patterns of malate dehydrogenase (MDH) from six tissues (liver, kidney, eye, muscle, brain and heart) of the Taihe negro fowl (Gallus gallus domesticus Brission) were analysed by thin-layer isoelectric focusing polyacrylamide gel electrophoresis. It was found that there were twelve MDH isozyme bands in the tissues of the fowl, and these isozymes displayed a tissue-specific distribution.

Isoelectric focusing (IEF) followed by immunofixation in thin layer polyacrylamide gels (pH4-6) was used to analyse the Gc subtypes of 1841 individuals from nine Han geographically distributed populations in China. The Gc1F gene frequencies in these Han subpopulations from Lanzhou, Huhhot, Harbin, Xi'an, Zhengzhou, Chengdu, Guiyang, Zhangzhou and Harras in Guangdong Province were 0.3905, 0.4333, 0.4193, 0.4146, 0.4182, 0.4035, 0.4045, 0.4381 and 0.4347 respectively; Gc1S were 0.3071, 0.2190, 0.2734, 0.2261, 0.2182, 0.2871, 0.2889, 0.2738 and 0.2764 respectively; Gc2 were 0.2786, 0.3357, 0.2917, 0.3367, 0.3295, 0.2896, 0.3065, 0.2667, and 0.2814 respectively. GcV were 0.0238, 0.0119, 0.0156, 0.0226, 0.0341, 0.0198, 0.0000, 0.0214 and 0.0075 respectively. The results of present study indicated a clear cline of linear change from north to south China in allelic frequency at the Group-Specific Component (Gc) locus among nine Chinese populations. Three alleles, Gc1F, Gc2 and GcV, showed frequencies varying from 0.3905 to 0.4381, from 0.2786 to 0.3367 and from nil to 0.0341, respectively, over these geographical subpopulations. In addition, some rare variant phenotypes of Gc in Chinese populations were discussed.

We have developed a new technique of complex isoelectric focusing (IEF) by which various proteins with different pHs can be detected out in polyacrylamide gels containing ampholytes with different pH ranges at the same time. The success of phenotyping 12 human proteins by this technique proved it was reliable and efficient. The cumulative Epp and the cumulative Dp reached 0.9 and 0.9999 respectively. It provides a new approach to analysis of genetic markers in the field of forensic haemogenetics.

The genetic polymorphism of properdin factor B(Bf) was first investigated in the Han population in Chengdu by isoelectrofocusing and immunofixation. The results revealed BfSS (129 cases), BfFS (48 cases) and BfFF (9 cases). No Bf variants were noted in this study. The allele frequencies were: Bf * S 0.8226, Bf * F 0.1774.

An isoelectric focusing of Pi pretreated with neuraminidase is reported. Subtyping of PiM in human semen was successfully carried out for the first time. This has provided a new approach for individual identification in sexual crime.

Studying Xanthomonas strains of 4 species and 29 pathovars by isoelectric focusing, it was shown that the protein patterns were very different among the species and pathovars. Through cluster analysis of IEF date, it was found that the protein pattern differences among certain pathovars were no fewer than that among the species. It revealed that some pathovars could become to be species. There were few differences of 6 protein bands among X. campestris pv. cerealis, X. campestris pv. undulosa and X. campestris pv. hordei which cause bacterial black cheff, so the classification of the three pathovars need to be re-considered. It also showed that isoelectric focusing could be used to classify Xanthomonas below species.

In order to study the function of apoA-IV, human serum apoA-IV was isolated and purified. LDS was isolated with NaBr density gradient ultracentrifugation and then incubated with intralipid according to the method of Weinberg and Scanu. Protein complex containing apoA-IV was obtained. The apo-IV was purified from protein complex by preparative SDS-PAGE, electroelution and removal of SDS. ApoA-IV thus prepared gave a single band on SDS-PAGE with a MW of 46 kD. Amino acid composition and isoelectric focusing pI (5.27 and 5.47) of apoA-IV were similar to those reported in the literature. The effects of apoA-IV and liposome (apoA-IV: DMPC) on cholesterol efflux from hum skin fibroblast were studied. There was no significant difference (P > 0.5) between apoA-IV and control but a very significant difference (P < 0.001) between liposome (apoA-IV:DMPC) and control, suggesting that liposomes (apoA-IV:DMPC) play a role in reverse cholesterol transport. The results may offer a new approach for the prevention and treatment of atherosclerotic coronary heart disease.

The genetic polymorphism of transferrin (Tf) and alpha 1-Antitrypsin (alpha 1-AT) in 202 gastric cancer patients and 202 controls in Shanghai Hans were analyzed by isoelectric focusing. In transferrin, the phenotype frequency of C1C1 and gene frequencies of c1 and c2 were 0.3713, 0.5718 and 0.4109 respectively in gastric cancer patients, and 0.5149, 0.6782 and 0.2970 separately in controls. The frequencies of the c1c1 phenotype and the C1 gene increased significantly (p < 0.01) among the controls, while the frequency of the C2 gene greatly increased (p < 0.01) among the patients. The frequencies of the C2C2 in gastric cancer patients and controls were 0.2228 and 0.1436 respectively, showing a significant difference between them (p < 0.05). The genetic polymorphisms of group-specific component (Gc) in 200 gastric cancer patients and 200 controls in Shanghai Hans were studied with isoelectric focusing followed by immunofixation. The Gc1F1F phenotype frequency and 1Fgene frequency were 0.22 and 0.4375 in gastric cancer patients, and 0.14 and 0.3600 in controls, respectively. The frequencies of the Gc1F1F phenotype and the 1F gene were significantly increased (p < 0.05) among the patients. In alpha 1-Antitrypsin, there was no significant difference in phenotype frequencies and gene frequencies between the patients and controls. The data reported here indicate that the gastric cancer is associated positively with TfC2 and Gc1F, and negatively with TfC1. These facts support the notion that cancerogenesis is a multi-step process controlled by multifactorial inheritance.

The distribution of alpha 2HS-glycoprotein (AHSG) phenotype frequencies in the Han population in Chengdu was studied using polyacrylamide gel isoelectric focusing followed by immunofixation with rabbit anti-human AHSG serum. Two hundred eighty-six serum samples collected at random from unrelated individuals were phenotyped for AHSG. The distribution of AHSG phenotype frequencies was found to be AHSG 1 = 47.20%, AHSG 2 = 8.04% and AHSG 2 - 1 = 44.76%. The observed numbers agreed well with the expected numbers calculated on the basis of the Hardy-Weinberg equilibrium. The allele frequencies were estimated to be AHSG.1 = 0.6958 and AHSG.2 = 0.3042. The discrimination probability of AHSG is 0.5704 and the exclusion probability of parentage 0.1669.

The isoenzyme patterns of Paragonimus iloktsuenesis and P. westermani were studied by isoelectric focusing on polyacrylamide gels for PGM, MDH, HK, ME, PO and ACP. Interspecific differences were apparent as shown by the isoenzyme profiles of these enzymes examined. MDH was polymorphic with 2 recognizable phenotypes, designated common type and variant type respectively, in P. i. and with 3 phenotypes, designated MDH1, MDH2 and MDH1-2 respectively, in P. w. The result showed that the two species could be distinguished one from another by such species-specific band patterns of isoenzymes. This suggests that P. w. and P. i. have undergone genetically divergent development and P. i. can be regarded as a valid species different from P. w. In the light of MDH polymorphism, some implications of isoenzyme analysis on taxonomy of the genus Paragonimus were discussed.

Human seminal plasma specific antigen p30, which has been characterized by other authors under the name of prostate-specific antigen or gamma-seminoprotein, has been purified. This communication describes the physico-chemical characterization of p30 demonstrated by various SDS-polyacrylamide gel electrophoresis and isoelectrofocusing as well as amino acid analysis. The results showed that the molecular weight of p30 was 33 kd and its isoelectric point was around pH 6.9. Its amino acid composition was similar to that of prostate-specific antigen. It is concluded that p30 purified by our laboratory and prostate-specific antigen named by Wang are the identical one of isomers.

The polymorphism of C6 in three groups of Han nationality was investigated by using polyacrylamide gel isoelectric focusing and immunoblotting technique. The gene frequencies obtained are as follows: Zhangzhou City (Fujiang Province) C6*A:0.4634, C6*B:0.5000, C6*R:0.0366 (C6*B2:0.0317); Chendu City (Sichuan Province) C6*A:0.4975, C6*B:0.4480, C6*R:0.0545 (C6*B2:0.0396); Harbin City (Heilongjiang Province) C6*A:0.4708, C6*B:0.5219, C6*R:0.0073 (C6*B2:0.0073). The frequency of C6*A in Mongoloid populations is usually lower than 0.5, while that of black people ranges between 0.5 and 0.6 and that of white people is higher than 0.6. The other difference between Caucasian and Mongoloid is that the former has very few C6*B2 while the frequency of C6*B2 in the latter ranges from 0.03 to 0.07.

P2 membrane preparation of rat brain (without cerebellum) was solubilized with CHAPS in Tris containing DTT and trypsin inhibitor. Two opiate ligands, 10b and 10cd, prepared by Liu et al, were applied consecutively in affinity chromatography, from which OPRs were eluted with Nx. The eluate was subsequently passed through a WGA affinity column and the OPR eluted with N-GluNAc. This eluate was further purified and concentrated by preparative granular gel isoelectric focusing on SG200. Two protein peaks appeared separately at pH 5 and pH7.8. The eluates from both peaks were analyzed for protein content by using silver staining method and binding activity was measured by RRA with 3H-etor. The results revealed that both samples contained active OPR purified to over 80,000 fold. The Mr was estimated by gel filtration to be 52 and 42 kD for OPR in the pH 5 and pH 7.8 samples respectively. The OPR in pH 5 sample has been characterized to be of mu-type, by its binding activity with 3H-ohm.

A total number of 231 unrelated Chinese (Han Nationality) were investigated for C2 polymorphism by hemolytic overlay technique after polyacrylamide gel isoelectric focusing on plasmas. The following phenotype distributions were observed: C2C, 216; C2BC, 9; C2AC, 5; and C2A, 1. The gene frequencies calculated from these phenotypes were as follows: C2*A, 0.015; C2*B, 0.019; C2*C, 0.965. The C2 phenotypic frequency distributions were in agreement with those expected from Hardy-Weinberg equilibrium.

Subtyping of Pi (alpha 1-AT) in 90 human bloodstains stored under different conditions was carried out by using ULPAGIF. The detectable time of Pi subtypes of all bloodstains kept at room temperature (12-20 degrees C), 4 degrees C and-15 degrees C was 10 days, 4 weeks and 4 weeks respectively. Beyond this time limit, the detectable rate decreased gradually. The minimal detectable quantity, of fresh bloodstains was 4 microliters. The factors influencing the detection of Pi subtypes in bloodstains were discussed.