Huizhi Fan

Microchip bioreactors based on trypsin-immobilized graphene oxide-poly(urea-formaldehyde) composite coating for efficient peptide mapping

Trypsin was covalently immobilized to graphene oxide (GO)-poly(urea-formaldehyde) (PUF) composite coated on the channel wall of poly(methyl methacrylate) microchips to fabricate microfluidic bioreactors for highly efficient proteolysis. A mixture solution containing urea-formaldehyde prepolymer and GO nanosheets was allowed to flow through the channels. The modification layer on the channel wall could further polycondense to form GO-PUF composite coating in the presence of ammonium chloride. The primary amino groups of trypsin could react with the carboxyl groups of the GO sheets in the coating with the aid of carboxyl activating agents to realize covalent immobilization. The feasibility and performance of the novel GO-based microchip bioreactors were demonstrated by the digestion of bovine serum albumin, lysozyme, ovalbumin, and myoglobin. The digestion time was significantly reduced to less than 5s. The obtained digests were identified by MALDI-TOF MS with satisfactory sequence coverages that were comparable to those obtained by using 12-h in-solution digestion. The present proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.

Immobilization of trypsin on poly(urea-formaldehyde)-coated fiberglass cores in microchip for highly efficient proteolysis.

Trypsin was covalently immobilized on poly(urea‐formaldehyde)‐coated fiberglass cores based on the condensation reaction between poly(urea‐formaldehyde) and trypsin for efficient microfluidic proteolysis in this work. Prior to use, a piece of the trypsin‐immobilized fiber was inserted into the main channel of a microchip under a magnifier to form a core‐changeable bioreactor. Because trypsin was not permanently immobilized on the channel wall, the novel bioreactor was regenerable. Two standard proteins, hemoglobin (HEM) and lysozyme (LYS), were digested by the unique bioreactor to demonstrate its feasibility and performance. The interaction time between the flowing proteins and the immobilized trypsin was evaluated to be less than 10 s. The peptides in the digests were identified by MALDI‐TOF MS to obtain PMF. The results indicated that digestion performance of the microfluidic bioreactor was better than that of 12‐h in‐solution digestion.

Nuclear proteome profile of C57BL/6J mouse liver.

The liver proteome can serve as a reference to better understand both disease mechanisms and possible therapeutics, since the liver is an important organ in the body that performs a large number of tasks. Here we identify the organelle proteome of C57BL/6J mouse liver nuclei as a promising strategy to enrich low abundance proteins, in the sense that analysis of whole liver cells is rather complex for current techniques and may not be suitable for proteins with low abundance. Evaluation of nucleus integrity and purity was performed to demonstrate the effectiveness of the optimized isolation procedure. The extracted nuclear proteins were identified by 2-DE MS analyses, and a total of 748 proteins were identified. Bioinformatic analyses were performed to demonstrate the physicochemical properties, cellular locations and functions of the proteins.

Quantitative proteomic analysis of a paired human liver healthy versus carcinoma cell lines with the same genetic background to identify potential hepatocellular carcinoma markers

To comprehensively measure global changes in protein expression associated with human hepatocellular carcinoma (HCC), comparative proteomic analysis of two cell lines derived from the healthy and carcinoma tissue of a same donor respectively was conducted using quantitative amino acid‐coded mass tagging /stable isotope labeling with amino acids in cell culture‐based LC‐MS/MS approach. Among a total of 501 proteins precisely quantified, the expressions of 128 proteins were significantly altered including 70 proteins up‐regulated and 58 down‐regulated in HCC cells. According to their previously characterized functions, the differentially expressed proteins were found associated with nine functional categories including glycolysis, stress response, cell communication, cell cycle, apoptosis/death, etc. For example, multiple enzymes involving glycolysis pathway were found differentially regulated in HCC cells, illustrating the critical participation of glycolysis in the HCC transformation. The accuracy of certain differentially expressed proteins identified through the amino acid‐coded mass tagging‐based quantification was validated in the paired cell lines, and later their pathological correlations were examined in multiple clinical pairs of normal versus tumor tissues from HCC specimen by using a variety of biological approaches including Western blotting and in situ immunoassays. These consistencies suggested that multiple proteins such as HSP27, annexin V, glyceraldehyde‐3‐phosphate dehydrogenase, nucleolin and elongation factor Tu could be the biomarkers candidates for diagnosis of HCC.

Comprehensive profiling of metastasis-related proteins in paired hepatocellular carcinoma cells with different metastasis potentials

Precise and comprehensive identifications of the proteins associated with metastasis are critical for early diagnosis and therapeutic intervention of hepatocellular carcinoma (HCC). Therefore, we investigated the proteomic differences between a pair of HCC cell lines, originating from the same progenitor, with different metastasis potential using amino acid‐coded mass tagging‐based LC‐MS/MS quantitative proteomic approach. Totally the relative abundance of 336 proteins in these cell lines were quantified, in which 121 proteins were upregulated by >30%, and 64 proteins were downregulated by >23% in the cells with high metastasis potential. Further validation studies by Western blotting in a series of HCC cell types with progressively increasing trend of metastasis showed that peroxiredoxin 4, HSP90β and HSP27 were positively correlated with increasing metastasis while prohibitin was negatively correlated with metastasis potential. These validation results were also consistent with that obtained from comparative analysis of clinic tissues samples. Function annotations of differentially expressed HCC proteome suggested that the emergence and development of high metastasis involved the dysregulation of cell migration, cell cycle and membrane traffics. Together our results revealed a much more comprehensive profile than that from 2‐DE‐based method and provided more global insights into the mechanisms of HCC metastasis and potential markers for clinical diagnosis.

Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusing.

In this study, ampholyte‐free liquid‐phase IEF (LIEF) was combined with narrow pH range 2‐DE and SDS‐PAGE RP‐HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2‐DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2‐DE gels following LIEF prefractionation into a single virtual 2‐DE gel. Furthermore, the pH 3–5 LIEF fraction and the unfractionated sample were separated by pH 3–6 2‐DE and identified by MALDI‐TOF/TOF MS, respectively. In parallel, the pH 3–5 LIEF fraction was also analyzed by SDS‐PAGE RP‐HPLC MS/MS. LIEF‐2‐DE and LIEF‐HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low‐abundance proteins and proteins with extreme physicochemical characteristics or post‐translational modifications compared to conventional 2‐DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large‐scale datasets. It was observed that the combination of LIEF‐2‐DE and LIEF‐HPLC was effective in promoting MS‐based liver proteome profiling and could be applied on similar complex tissue samples.

Quantitative research of histone H3 acetylation levels of human hepatocellular carcinoma cells

BACKGROUND Core histone H3 is a highly conserved protein in the cell nucleus, it goes through various post-translational modifications easily, and the state of the acetylation has clinical diagnostic significance in prostate cancer, breast cancer, lung cancer and other diseases. RESULTS In this work, the combinatorial method of chromatographic separation, methylation isotope labeling and LTQ-Orbitrap(®) MS was employed to quantify the acetylation sites of histone H3 separately within normal liver cells L02 and hepatocellular carcinoma (HCC) cells HepG2, HCC metastasis cells 97H and HCC cells HepG2, high HCC metastasis potential cells LM3 and low HCC metastasis potential cells 97L. In comparison with the quantitative results of HepG2 and L02, the amounts of five acetylated and methylated peptides were found decreased. Similarly, when comparing the 97H with HepG2, the amounts of eight acetylated and methylated peptides were found decreased, and when comparing the LM3 with 97L, the amounts of six acetylated and methylated peptides were found decreased. CONCLUSION These results provide some fundamental reference information for the research into post-translational modifications of histones in human liver cancer and other related diseases.

Impact of glutathione-HbA1c on HbA1c measurement in diabetes diagnosis via array isoelectric focusing, liquid chromatography, mass spectrometry and ELISA

Hemoglobin A1c (HbA1c) has been proven to be a key biomarker for diabetes screening, and glutathiolation of HbA1c (viz., GSS-HbA1c) has been identified. However, the impact of GSS-HbA1c on the measurement of HbA1c for diabetes screening has not been quantitatively assessed yet. To address the issue, the micropreparative capillary isoelectric focusing (cIEF) developed in our previous work was used for the high resolution separation and purification of hemoglobin (Hb) species. The main fractions of HbA0, HbA3 and HbA1c extracted from the developed cIEF were identified by validated Mono S method. The proposed GSS-HbA1c fractions in the cIEF were pooled and identified by electrospray ionization mass spectrometry (ESI-MS). The HbA1c enzyme-linked immunosorbent assay (ELISA) kit was employed for further quantitative analysis of GSS-HbA1c. A total of 34 blood samples with HbA1c levels from 4.2% to 13.4% were assessed via the above comprehensive strategy of IEF-HPLC-MS-ELISA. It was demonstrated that the HbA1c levels detected by cation exchange LC were considerably influenced by the glutathiolation of Hb and the range of detected GSS-HbA1c values was between 0.23% and 0.74%. The results and developed cIEF methods have considerable significances for investigation of diabetes and clinical diagnosis.

Peptide-tight ESI/MSn analysis with segment of liquid chromatography effluent

A segment flow protocol has been proposed for coupling nano-liquid chromatography (nano-LC) with mass spectrometry (MS) in peptide-based proteomics research. LC effluent was segmented by perfluorodecalin oil and stored as segments in sequence to carry out the following off-line MSn analysis. In this way, incomplete identification of co-eluted peptides resulting from the MS scan rate, relatively slower than the LC elution rate, was solved to some extent for on-line coupling LC-MSn analysis. The complex proteins from a sample of T. tengcongensis were successfully identified with one run using segment as the interface between nano-LC and MSn analysis, compared to the total identified proteins with six runs using routine on-line nano-LC-MSn analysis. This segment flow approach could eliminate detection loss and sample cross-contamination, providing a new method for peptide-based proteomic research, especially when the amount of sample is limited.

Detection and quantification of lysine acetyl-alteration using antibody microarray

BACKGROUND Lysine acetylation is a reversible and dynamic post-translational modification on proteins, and plays an important role in diverse biological processes. Technological limitations have so far prevented comparative quantification of lysine acetylation in different samples. RESULTS We developed a method to efficiently study lysine acetylation on individual proteins from complex mixtures, using antibody microarrays to capture individual proteins followed by detection with lysine acetyl antibody. By profiling both protein and acetylation variations in multiple samples using this microarray, we found cancer-associated lysine acetylation alteration on VEGF in the serum of hepatocellular carcinoma patients. CONCLUSION Microarrays of lysine acetylation are highly effective for detecting acetylation, and should be useful in identifying and validating disease-associated acetylation alterations as biomarkers under both normal and pathological circumstances.