Mingxia Gao

Recent development of multi-dimensional chromatography strategies in proteome research.

Abstract As a complementary approach to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), multi-dimensional chromatography separation methods have been widely applied in all kinds of biological sample investigations. Multi-dimensional liquid chromatography (MDLC) coupled with bio-mass spectrometry (MS) is playing important roles in proteome research due to its high speed, high resolution and high sensitivity. Proteome analysis strategies mainly include bottom-up and top-down approaches which carry out biological sample separation based on peptide and protein levels, respectively. Electrophoretic methods combined with liquid chromatography like IEF-HPLC and HPLC-SDS-PAGE have been successful applied for protein separations. As for MDLC strategy, ion-exchange chromatography (IEX) together with reversed phase liquid chromatography (RPLC) is still a most widely used chromatography in proteome analysis, other chromatographic methods are also frequently used in protein pre-fractionations, while affinity chromatography is usually adopted for specific functional protein analysis. Recent MDLC technologies and applications to variety of proteome analysis have been achieved great development. A digest peptide-based approach as so-called “bottom-up” and intact protein-based approach “top-down” analysis of proteome samples were briefly reviewed in this paper. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as high-abundance protein depletion and chromatography array were also included in this review.

Large scale depletion of the high-abundance proteins and analysis of middle- and low-abundance proteins in human liver proteome by multidimensional liquid chromatography

An unbiased method for large‐scale depletion of high‐abundance proteins and identification of middle‐ or low‐abundance proteins by multidimensional LC (MDLC) was demonstrated in this paper. At the protein level, the MDLC system, coupling the first dimensional strong cation exchange (SCX) chromatography with the second dimensional RP‐HPLC, instead of immunoaffinity technology, was used to deplete high‐abundance proteins. Sixty‐two fractions from SCX were separated further by RPLC. UV absorption spectra were observed to differentiate high‐abundance proteins from middle‐ or low‐abundance proteins. After the depletion of high‐abundance proteins, middle‐ or low‐abundance proteins were enriched, digested, and separated by online 2D‐micro‐SCX/cRPLC. The eluted peptides were deposited on the MALDI target and detected by MALDI‐TOF/TOF MS. This depletion strategy was applied to the proteome of the normal human liver (NHL) provided by the China Human Liver Proteome Project (CHLPP). In total, 58 high‐abundance proteins were depleted in one experiment. The strategy increases greatly the number of identified proteins and around 1213 proteins were identified, which was about 2.7 times as that of the nondepletion method.

High throughput identification of components from traditional Chinese medicine herbs by utilizing graphene or graphene oxide as MALDI-TOF-MS matrix

In this work, graphene or graphene oxide was utilized, for the first time, to identify small molecular components from traditional Chinese medicine (TCM) herbs, by acting as matrix of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Due to the large surface area of graphene or graphene oxide, the analytes were trapped tightly to the matrix, which avoids the contamination of the ion source and vacuum system. Besides, their excellent electronic, thermal and mechanical properties make them desired matrices for MALDI-TOF-MS. Stable analysis was achieved with no background inference even at the concentration of 100 nM. Moreover, the limit of detection (LOD) could be greatly lowered by utilizing graphene or graphene oxide as a pre-enrichment adsorbent. In summary, the promoted MALDI-TOF-MS methodology was demonstrated to be simple, sensitive, fast, cost effective and, most importantly, high throughput.

Multilayer Hydrophilic Poly(phenol-formaldehyde resin)-Coated Magnetic Graphene for Boronic Acid Immobilization as a Novel Matrix for Glycoproteome Analysis

Capturing glycopeptides selectively and efficiently from mixed biological samples has always been critical for comprehensive and in-depth glycoproteomics analysis, but the lack of materials with superior capture capacity and high specificity still makes it a challenge. In this work, we introduce a way first to synthesize a novel boronic-acid-functionalized magnetic graphene@phenolic-formaldehyde resin multilayer composites via a facile process. The as-prepared composites gathered excellent characters of large specific surface area and strong magnetic responsiveness of magnetic graphene, biocompatibility of resin, and enhanced affinity properties of boronic acid. Furthermore, the functional graphene composites were shown to have low detection limit (1 fmol) and good selectivity, even when the background nonglycopeptides has a concentration 100 fold higher. Additionally, enrichment efficiency of the composites was still retained after being used repeatedly (at least three times). Better yet, the practical applicability of this approach was evaluated by the enrichment of human serum with a low sample volume of 1 μL. All the results have illustrated that the magG@PF@APB has a great potential in glycoproteome analysis of complex biological samples.

Comparison of 2-D LC and 3-D LC with post- and pre-tryptic-digestion SEC fractionation for proteome analysis of normal human liver tissue

The current “shotgun” proteomic analysis, strong cation exchange‐RPLC‐MS/MS system, is a widely used method for proteome research. Currently, it is not suitable for complicated protein sample analysis, like mammal tissues or cells. To increase the protein identification confidence and number, an additional separation dimension for sample fractionation is necessary to be coupled prior to current multi‐dimensional protein identification technology (MudPIT). In this work, SEC was elaborately selected and applied for sample prefractionation in consideration of its non‐bias against sample and variety of choice of mobile phases. The analysis of the global lysate of normal human liver tissue sample provided by the China Human Liver Proteome Project, were performed to compare the proteome coverage, sequence coverage (peptide per protein identification) and protein identification efficiency in MudPIT, 3‐D LC‐MS/MS identification strategy with preproteolytic and postproteolytic fractionation. It was demonstrated that 3‐D LC‐MS/MS utilizing protein level fractionation was the most effective method. A MASCOT search using the MS/MS results acquired by QSTARXL identified 1622 proteins from 3‐D LC‐MS/MS identification approaches. A primary analysis on molecular weight, pI and grand average hydrophobicity value distribution of the identified proteins in different approaches was made to further evaluate the 3‐D LC‐MS/MS analysis strategy.

Recent developments and contributions from Chinese scientists in multidimensional separations for proteomics and traditional Chinese medicines.

Abstract The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists in protein separations performed by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). Yet the 2‐D PAGE‐mass spectrometry (MS) approach has its drawbacks with regard to automation, sensitivity, and throughput. Consequently, considerable effort has been devoted to the development of non‐gel‐based proteome separation technologies in an effort to alleviate the shortcomings of 2‐D PAGE. In addition, traditional Chinese medicines (TCMs), due to their long period of clinical testing and reliable therapeutic efficacy, are attracting increased global attention. However, hundreds or even thousands of components are usually present in TCMs, which results in great difficulties of separation. As a mainstream separation tool, multidimensional liquid separation systems have shown powerful separation ability, high peak capacity, and excellent detectability in the analysis of complex samples including biological samples and TCMs, etc. Therefore, this review emphasizes the most recent advances in multidimensional liquid chromatography and capillary electrophoresis‐based separation techniques, and the corresponding applications in proteomics and TCMs. In view of the significant contributions from Chinese scientists, this review focuses mainly on the work of Chinese scientists in the above fields.

Development of Versatile Metal–Organic Framework Functionalized Magnetic Graphene Core–Shell Biocomposite for Highly Specific Recognition of Glycopeptides

Protein N-glycosylation is a ubiquitous and important post-translational modification that has been involved in the development and progression of a series of human-related diseases. Until recently, the highly selective capturing of glycopeptides from complex biosamples was still significant and challenging work due to their changeable structures, ultralow abundance, and strong ion-suppressing effect. Here we first report the preparation and characterization of a novel, hydrophilic, porous biocomposite composed of magnetic graphene functionalized with metal-organic frameworks (MOFs) (MG@Zn-MOFs) able to recognize glycopeptides. Thanks to its strong magnetic responsiveness, large specific surface area, excellent biocompatibility, and unique size-exclusion effect, the MG@Zn-MOFs showed outstanding sensitivity and selectivity and good recyclability in glycopeptides analysis. More excitingly, in practical application, 517 N-glycopeptides within 151 unique glycoproteins were clearly identified from human serum (1 μL) treated with the MG@Zn-MOFs, which is the best result among published reports so far. All the results demonstrate the promising commercialized usage of the biocomposite for the enrichment of glycopeptides in complex samples through a convenient and efficient process. Furthermore, it is anticipated that our strategy may offer promising guidance to develop new biocomposites functionalized with bio-MOFs for glycoproteomic applications.

Magnetic nanoparticles-based digestion and enrichment methods in proteomics analysis.

In proteome research, rapid and effective proteolysis and enrichment strategies are essential for successful protein identification. Functionalized magnetic microspheres of micro- and nano-meter size are gaining increasing attention due to their easy manipulation and recovery, great specific surface areas and high surface activity. The introduction of magnetic nanoparticles into the field of proteomics study has accelerated the development of digestion and enrichment methods. In this article, we mainly focus on recent developments of using different functionalized magnetic nanoparticles for rapid digestion and preconcentration of low-abundance peptides/proteins, including those containing post-translational modifications, such as phosphorylation and glycosylation, prior to mass spectrometric analysis.

Development of multidimensional liquid chromatography and application in proteomic analysis

As a complementary approach to 2D-PAGE, multidimensional liquid chromatography (MDLC) separation methods have been widely applied in all kinds of biological sample investigations. MDLC coupled with mass spectrometry is playing an important role in proteome research owing to its high speed, high resolution and high sensitivity. Among MDLC strategies, ion-exchange chromatography together with reversed-phase LC is still a most widely used chromatography in proteome analysis; other chromatographic methods are also frequently used in protein prefractionations. Recent MDLC technologies and applications to a variety of proteome analyses have achieved great development. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as ultra-pressure system, array-based separation and monolithic material are also included in this article.

Ultrasensitive Proteome Profiling for 100 Living Cells by Direct Cell Injection, Online Digestion and Nano-LC-MS/MS Analysis

Single-cell proteome analysis has always been an exciting goal because it provides crucial information about cellular heterogeneity and dynamic change. Here we presented an integrated proteome analysis device (iPAD) for 100 living cells (iPAD-100) that might be suitable for single-cell analysis. Once cells were cultured, the iPAD-100 could be applied to inject 100 living cells, to transform the living cells into peptides, and to produce protein identification results with total automation. Due to the major obstacle for detection limit of mass spectrometry, we applied the iPAD-100 to analyze the proteome of 100 cells. In total, 813 proteins were identified in a DLD-cell proteome by three duplicate runs. Gene Ontology analysis revealed that proteins from different cellular compartments were well-represented, including membrane proteins. The iPAD-100 greatly simplified the sampling process, reduced sample loss, and prevented contamination. As a result, proteins whose copy numbers were lower than 1000 were identified from 100-cell samples with the iPAD-100, showing that a detection limit of 200 zmol was achieved. With increased sensitivity of mass spectrometry, the iPAD-100 may be able to reveal bountiful proteome information from a single cell in the near future.