Biomedical subjects

Xue Gu

Publications and source records attributed to Xue Gu.

6 recordsLinked to original sources

PubMed2006

Capillary array reversed-phase liquid chromatography-based multidimensional separation system coupled with MALDI-TOF-TOF-MS detection for high-throughput proteome analysis.

A high-throughput on-line capillary array-based two-dimensional liquid chromatography (2D-LC) system coupled with MALDI-TOF-TOF-MS proteomics analyzer for comprehensive proteomic analyses has been developed, in which one capillary strong-cation exchange (SCX) chromatographic column was used as the first separation dimension and 18 parallel capillary reversed-phase liquid chromatographic (RPLC) columns were integrated as the second separation dimension. Peptides bound to the SCX phase were "stepped" off using multiple salt pulses followed by sequentially loading of each subset of peptides onto the corresponding precolumns. After salt fractionation, by directing identically split solvent-gradient flows into 18 channels, peptide fractions were concurrently back-flushed from the precolumns and separated simultaneously with 18 capillary RP columns. LC effluents were directly deposited onto the MALDI target plates through an array of capillary tips at a 15-s interval, and then alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution was added to each sample spot for subsequent MALDI experiments. This new system allows an 18-fold increase in throughput compared with serial-based 2D-LC system. The high efficiency of the overall system was demonstrated by the analysis of a tryptic digest of proteins extracted from normal human liver tissue. A total of 462 proteins was identified, which proved the system's promising potential for high-throughput analysis and application in proteomics.

Capillary Action↗

PubMed2005

Protein identification assisted by the prediction of retention time in liquid chromatography/tandem mass spectrometry.

Two-dimensional liquid chromatography (2D-LC) coupled on-line with electrospray ionization tandem mass spectrometry (2D-LC-ESI-MS/MS) is a new platform for analysis and identification of proteome. Peptides are separated by 2D-LC and then performed MS/MS analysis by tandem MS/MS. The MS/MS data are searched against database for protein identification. In one 2D-LC-ESI-MS/MS run, we obtained not only the structural information of peptides directly from MS/MS, but also the retention time of peptides eluted from LC. Information on the chromatographic behavior of peptides can assist protein identification in the new platform for proteomics. The retention time of the matching peptides of the identified protein was predicted by the hydrophobic contribute of each amino acid on reversed-phase liquid chromatography (RPLC). By using this strategy proteins were identified by four types of information: peptide mass fingerprinting (PMF), sequence query, and MS/MS ions searched and the predicted retention time. This additional information obtained from LC could assist protein identification with no extra experimental cost.

Amino Acid Sequence↗

PubMed2005

Large-bore particle-entrapped monolithic precolumns prepared by a sol-gel method for on-line peptides trapping and preconcentration in multidimensional liquid chromatography system for proteome analysis.

The present report describes the preparation and characterization of large-bore particle-entrapped monolithic precolumns, which are suitable for incorporation into a two-dimensional liquid chromatography (2D-LC) system for proteome analysis. The fritless precolumns with different inner diameter (i.d.) (320 and 530 microm) were rapidly and successfully prepared by entrapping octadecylsilica (ODS) particles (5 microm, 300 A) prepacked into fused silica capillaries with a sol-gel network, which was formed by hydrolysis and polycondensation of methyltriethoxysilane (MTES). By optimizing the composition of the sol solution, the resulting large-bore monolithic precolumns of 5 mm length allow a flow rate of 20 microL/min loading buffer at a reasonable low back pressure of 25 bar or less and are capable of withstanding up to 300 bar inlet pressure. Scanning electron micrograms of the precolumns profile showed that the evolving sol-gel network joined particles to each other and onto the column wall, and no cracking or shrinkage of the column bed was observed even in 530 microm-i.d. capillary. The performance of the particle-entrapped monolithic precolumns used for preconcentration and desalting of proteolytic digest was evaluated by on-line coupling the large-bore precolumns with a capillary reversed-phase liquid chromatographic (RPLC) column followed by UV detection. The laboratory-made monolithic precolumns with 320 and 530 microm i.d. were characterized by using BSA tryptic digest or peptide standards as the analytes with respect to sample loading capacity, linearity, recovery and reproducibility, etc. The results indicate that the large-bore and short precolumns (5 mm x 320 microm i.d. or 5 mm x 530 microm i.d.) allow sample fast loading at a flow rate of 30 or 60 microL/min. The precolumns also have a mass loading capacity for BSA peptides of about 70 microg and for standard peptides of about 80 microg. Good linear calibration curves (R2 > 0.99) were obtained and the limits of detection (signal-to-noise ratio, S/N = 3) were improved by more than 60-fold and were between 0.53 and 1.32 ng/microL even with a UV absorbance detector. The total recovery was found to be approximately 90-100% for BSA digest and standard peptides. The day-to-day relative standard deviation (RSD) values for recoveries of BSA peptides on a single precolumn ranged from 4.66 to 7.56% and 2.68 to 3.05% for precolumn back pressure, while the column-to-column RSD values were 3.51-6.13% and 1.22-1.26% for recoveries of BSA peptides and precolumn back pressure, respectively. With good precolumn reproducibility, no significant degradation or decrease in precolumn performance was showed even after approximately 150 preconcentration/desorption cycles. The precolumns also proved to be resistant to salt buffer with high concentration and low-pH mobile phase. The large-bore particle-entrapped monolithic precolumns will be further used in a high-throughput 2D-LC array system coupled with tandem matrix assisted laser desorption/ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS) detection for proteome analysis.

Calibration↗

PubMed2005

[Nano flow multidimensional liquid chromatography for proteome analysis of rat liver].

A nano flow comprehensive multidimensional liquid chromatography was developed. By using this separation platform, complex samples can be injected, desalted, separated and analyzed in complete automatization. Coupling of the strong cation-exchange (SCX) with capillary reversed-phase liquid chromatography (cRPLC) is described for proteomics of rat liver. A step gradient was applied for SCX column, and a linear gradient was applied for RPLC. The flow rate through the nano scale analytical column during the separation process was maintained at 200 nL/min. The peak capacity for the nano-2D-LC was up to 620 under these conditions.

Animals↗

PubMed2004

[Study on the relationship between polymorphism of insulin-receptor gene EXON2-2257 and insulin resistance in Chinese people].

OBJECTIVE: To understand the role of insulin-receptor gene in the development of insulin resistance on a population-based study in China. METHODS: Polymerase chain reaction (PCR) was used to amplify the EXON2 of the insulin-receptor gene and all amplified products were analyzed by direct sequencing. RESULTS: Three genotypes of single nucleotide at the site of 2257 in EXON2 of the insulin-receptor gene were identified. In 237 of the 345 cases (68.7%), homozygote genotype of CC phenotype was found with a gene frequency of 0.825; 13 cases (3.77%) showed homozygote genotype of TT phenotype with a gene frequency of 0.175 and the rest 95 cases (27.54%) showed heterozygote genotype of CT phenotype. Data were in agreement with the test of Hardy-Weinberg balance (chi(2) = 0.2898, upsilon = 3 - 2 = 1, 0.5 < P < 0.75). The serum level of triglyceride and the HOMA-IR index, the status of insulin resistance assessed by homeostasis model assessment (HOMA), were lower in the TT or CT genotype group than that in the CC genotype group (P < 0.05). Comparing with CC genotype, the proportion of CT genotype (18.4%) in the insulin resistance group (large than 75th percentile of HOMA-IR) was significantly lower than that in the control group (30.6%) (P = 0.022, OR = 0.493). The proportion of TT genotype (2.3%) in insulin resistance group was lower than that in the control group (4.3%, P = 0.297). logistic analysis revealed that after adjusting possible confounding factors such as taking anti-hypertensive and anti-diabetic medications, serum triglyceride and high density lipoprotein cholesterol (HDL-C), hypertension, BMI, smoking history, the OR value of people in the insulin resistance group with CT genotype was 0.448 (95% CI: 0.214 to 0.940) compared to the group with CC genotype. CONCLUSION: The hybridization CT genotype at the site of 2257 in EXON2 of insulin-receptor gene might have a candidate gene to serve as a protective factor for insulin resistance.

Adult↗

PubMed2003

[Prevalence of vitamin A deficiency in children under six years of age in Tibet, China].

OBJECTIVE: To understand the prevalence of vitamin A deficiency (VAD) among children under six years of age in Tibet, China. METHODS: Totally, 1 257 children under six years of age were selected from two cities, two farming counties, two semi-farming counties and two livestock farming counties with stratified cluster sampling to asses VAD status in Tibet. Family information, children's feeding and disease history in the previous two weeks were collected by questionnaire. Blood specimen was collected from each child and serum was separated for detection of vitamin A concentration with microfluorescent spectrophotometry. RESULTS: Totally, 1 257 children under six years of age were surveyed, with 635 boys, 622 girls, 862 aged over two years, and 98.5% of Tibet nationality. Six cases of night blindness and two cases of xerophthalmia were detected from them, with prevalence of clinical VAD of 0.96%. Eighteen of 1071 mothers with children under six years of age were found suffering from night blindness, accounting for 1.7%. Clinical cases of VAD both in children and mothers came from all four sampling strata. Average serum concentration of vitamin A and prevalence of subclinical VAD (serum vitamin A lower than or equal to 0.70 micromol/L) was 1.15 micromol/L and 5.4% and 1.12 micromol/L and 4.7% in cities and livestock farming counties, respectively, significantly higher than those in farming (1.04 micromol/L and 11.0%) and semi-farming counties (1.05 micromol/L and 12.3%), respectively, as compared to average levels of 1.09 micromol/L and 8.4% in the autonomous region as a whole. Prevalence of subclinical VAD in children under six months and those aged six to eleven months were 22.2% and 13.3%, respectively, significantly higher than those in children aged one year (8.5%), two to three years (5.4%) and four to five years (7.9%), respectively. There was also significant difference in serum level of vitamin A between children at varied ages, but no significant difference both in serum level of vitamin A and prevalence of subclinical VAD between gender was found. CONCLUSIONS: In general, status of VAD in children of Tibet was milder than that at national level. But, moderate subclinical VAD in some areas, such as farming and semi-farming counties, did exist, so vitamin A supplementation aiming to children, especially those under one year of age, in those areas should be urged.

Age Factors↗