Journal of Chromatography B (v.866, #1-2)

This review article deals with preparation methods for spherical and monodispersed molecularly imprinted polymers (MIPs) in micrometer sizes. Those methods include suspension polymerization in water, liquid perfluorocarbon and mineral oil, seed polymerization and dispersion/precipitation polymerization. The other methods are the use of beaded materials such as a spherical silica or organic polymer for grafting MIP phases onto the surfaces of porous materials or filling the pores of silica with MIPs followed by dissolution of the silica. Furthermore, applications of MIP microspheres as affinity-based chromatography media, HPLC stationary phases and solid-phase extraction media, will be discussed for pharmaceutical, biomedical and environmental analysis.
Keywords: Molecularly imprinted polymer; Monodispersed MIP; MIP microsphere; Affinity-based media; Pharmaceutical analysis; Environmental analysis; Bioanalysis;

Microfluidic immunoaffinity separations for bioanalysis by Michael C. Peoples; H. Thomas Karnes (14-25).
Microfluidic devices often rely on antibody–antigen interactions as a means of separating analytes of interest from sample matrices. Immunoassays and immunoaffinity separations performed in miniaturized formats offer selective target isolation with minimal reagent consumption and reduced analysis times. The introduction of biological fluids and other complicated matrices often requires sample pretreatment or system modifications for compatibility with small-scale devices. Miniaturization of external equipment facilitates the potential for portable use such as in patient point-of-care settings. Microfluidic immunoaffinity systems including capillary and chip platforms have been assembled from basic instrument components for fluid control, sample introduction, and detection. The current review focuses on the use of immunoaffinity separations in microfluidic devices with an emphasis on pump-based flow and biological sample analysis.
Keywords: Immunoaffinity; Microfluidic; Immunoassay; Antibodies; Capillary:microchip;

This review discusses the current trends in molecular profiling for the emerging systems biology applications. Historically, the methodological developments in separation science were coincident with the availability of new ionization techniques in mass spectrometry. Coupling miniaturized separation techniques with technologically-advanced MS instrumentation and the modern data processing capabilities are at the heart of current platforms for proteomics, glycomics and metabolomics. These are being featured here by the examples from quantitative proteomics, glycan mapping and metabolomic profiling of physiological fluids.
Keywords: Systems biology; Metabolomic profiling; Quantitative proteomics; Glycomic profiling; Biodiversity; Animal models; Bioinformatics; Stable-isotope labeling; Metabolic stable-isotope labeling; LC–MS; GC–MS;

Highly efficient peptide separations in proteomics by Koen Sandra; Mahan Moshir; Filip D’hondt; Katleen Verleysen; Koen Kas; Pat Sandra (48-63).
Sample complexity and dynamic range constitute enormous challenges in proteome analysis. The back-end technology in typical proteomics platforms, namely mass spectrometry (MS), can only tolerate a certain complexity, has a limited dynamic range per spectrum and is very sensitive towards ion suppression. Therefore, component overlap has to be minimized for successful mass spectrometric analysis and subsequent protein identification and quantification. The present review describes the advances that have been made in liquid-based separation techniques with focus on the recent developments to boost the resolving power. The review is divided in two parts; the first part deals with unidimensional liquid chromatography and the second part with bi- and multidimensional liquid-based separation techniques. Part 1 mainly focuses on reversed-phase HPLC due to the fact that it is and will, in the near future, remain the technique of choice to be hyphenated with MS. The impact of increasing the column length, decreasing the particle diameter, replacing the traditional packed beds by monolithics, amongst others, is described. The review is complemented with data obtained in the laboratories of the authors.
Keywords: Proteomics; Peptides; Liquid chromatography; High efficiency; Ultra-high pressure; Elevated temperature; Column formats;

LC–MS-based metabonomics analysis by Xin Lu; Xinjie Zhao; Changmin Bai; Chunxia Zhao; Guo Lu; Guowang Xu (64-76).
Metabonomics aims at the comprehensive and quantitative analysis of wide arrays of metabolites in biological samples. It has shown particular promise in the areas of toxicology and drug development, functional genomics, systems biology, and clinical diagnosis. Comprehensive metabonomics investigations are primarily a challenge for analytical chemistry. High-performance liquid chromatography–mass spectrometry (HPLC–MS) is an established technology in drug metabolite analysis and is now expanding into endogenous metabolite research. Its main advantages include wide dynamic range, reproducible quantitative analysis, and the ability to analyze biofluids with extreme molecular complexity. The aims of developing HPLC–MS for metabonomics range from understanding basic biochemistry to biomarker discovery and the structural characterization of physiologically important metabolites. In this review, the strategy and application of HPLC–MS-based metabonomics are reviewed.
Keywords: HPLC–MS; Metabonomics; Metabolic profiling; Metabolomics;

Statistical data processing in clinical proteomics by Suzanne Smit; Huub C.J. Hoefsloot; Age K. Smilde (77-88).
This review discusses data analysis strategies for the discovery of biomarkers in clinical proteomics. Proteomics studies produce large amounts of data, characterized by few samples of which many variables are measured. A wealth of classification methods exists for extracting information from the data. Feature selection plays an important role in reducing the dimensionality of the data prior to classification and in discovering biomarker leads. The question which classification strategy works best is yet unanswered. Validation is a crucial step for biomarker leads towards clinical use. Here we only discuss statistical validation, recognizing that biological and clinical validation is of utmost importance. First, there is the need for validated model selection to develop a generalized classifier that predicts new samples correctly. A cross-validation loop that is wrapped around the model development procedure assesses the performance using unseen data. The significance of the model should be tested; we use permutations of the data for comparison with uninformative data. This procedure also tests the correctness of the performance validation. Preferably, a new set of samples is measured to test the classifier and rule out results specific for a machine, analyst, laboratory or the first set of samples. This is not yet standard practice. We present a modular framework that combines feature selection, classification, biomarker discovery and statistical validation; these data analysis aspects are all discussed in this review. The feature selection, classification and biomarker discovery modules can be incorporated or omitted to the preference of the researcher. The validation modules, however, should not be optional. In each module, the researcher can select from a wide range of methods, since there is not one unique way that leads to the correct model and proper validation. We discuss many possibilities for feature selection, classification and biomarker discovery. For validation we advice a combination of cross-validation and permutation testing, a validation strategy supported in the literature.
Keywords: Statistical validation; Permutation test; Classification; Biomarker discovery; Double cross-validation; Feature selection; Curse of dimensionality; Multivariate data analysis; Proteomics;

Advanced polymers for molecular recognition and sensing at the interface by Marcella Chiari; Marina Cretich; Francesco Damin; Gabriele Di Carlo; Claudio Oldani (89-103).
In the few last years, the need of reliable, fast and inexpensive methods for selective analysis of specific substances in complex mixtures has grown exponentially. In particular, the detection of biomolecules, such as oligonucleotides, proteins, peptides and carbohydrates is of outstanding importance in gene expression, drug design and medicine studies. To these purposes, molecular recognition on microarray-configured devices is one of the most promising tools. This technology uses a number of different substrates such as glass, silicon, alumina or gold-coated slides. The use of polymers is a very effective way to tailor surface properties introducing functional groups able to bind biomolecules and prevent denaturation and non-specific binding. Furthermore, advanced polymers, thanks to their particular physico-chemical properties, can be used to improve selectivity and sensitivity during assays. This review will provide very recent examples of polymer-mediated molecular recognition between guest molecules in solution and host molecules located at the solid phase.
Keywords: Polymers; Molecular recognition; Surfaces; Coatings; Poly(acrylic acid); Poly(dimethylacrylamide); Conjugated polymers;

Recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices by Wei-Hua Huang; Feng Ai; Zong-Li Wang; Jie-Ke Cheng (104-122).
Cells are the fundamental unit of life, and studies on cell contribute to reveal the mystery of life. However, since variability exists between individual cells even in the same kind of cells, increased emphasis has been put on the analysis of individual cells for getting better understanding on the organism functions. During the past two decades, various techniques have been developed for single-cell analysis. Capillary electrophoresis is an excellent technique for identifying and quantifying the contents of single cells. The microfluidic devices afford a versatile platform for single-cell analysis owing to their unique characteristics. This article provides a review on recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices; focus areas to be covered include sampling techniques, detection methods and main applications in capillary electrophoresis, and cell culture, cell manipulation, chemical cytometry and cellular physiology on microfluidic devices.
Keywords: Single-cell analysis; Capillary electrophoresis; Microfluidic devices; Chemical cytometry; Cellular physiology;

Recent development of multi-dimensional chromatography strategies in proteome research by Jia Tang; Mingxia Gao; Chunhui Deng; Xiangming Zhang (123-132).
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.
Keywords: Multi-dimensional liquid chromatography; Proteomic analysis; Bottom-up and top-down approaches;

Many recombinant proteins (rPRTs) have a high bioactivity and some of them may eventually be classified as drugs beneficial to human health, recombinant human protein drugs (rPDs). rPDs are a high-technology product with all the associated economic benefits, therefore the liquid chromatography (LC) of rPRT is different from that of proteins isolated in laboratory scale for purely research purposes. The design of a purification scheme for an rPRT depends on the intended function of the purified rPRT, as a pure sample for research in small scale, or as a product for industrial production. This review paper mainly deals with the latter instance, producing rPD at a large scale. Pharmaceutical economics is considered not only for each step of purification, but also the whole production process. This strategy restricts the content of this review paper to the factors affecting the optimization source, the character of rPRT in up-stream technology and the purification of the rPRT in down-stream production. In the latter instance, the purification step is required to be as efficient as possible and LC is the core of the refined purification method, which is either a single LC method or combination of LC methods, sometimes, it may be a combination of LC and other non-LC separation methods comprising an optimized purification technology. Here some typical examples of rPRT purification at the large scale, recent developments, such as protein folding liquid chromatography, short column chromatography, and new packing material and column techniques are introduced.
Keywords: Liquid chromatography; Biotechnology; Recombinant protein; Protein drug; Purification; Protein folding; Large scale; Packings; Column techniques; Pharmaceutical economics;

The analysis by capillary electrophoresis of less commonly analyzed biofluids is reviewed. The sample matrices considered include airway surface fluid, sputum, synovial fluid, amniotic fluid, saliva, cerebrospinal fluid, aqueous humor, vitreous humor, and sweat. Many of the techniques used in the analysis of abundant and commonly tested biofluids such as plasma or urine can be applied to these other matrices, e.g. sample extraction prior to analysis. However, for some of these alternative biofluids the available sample amounts are only in the nanoliter or low microliter range, which places constraints on the sample preparation options which are available. For such samples, direct sample injection may be necessary, possibly coupled with on-capillary concentration or derivatization approaches. Particular attention is paid in this review to analyses where the sample is directly injected onto the separation capillary or where minimal sample preparation is performed.
Keywords: Airway surface fluid; Sputum; Synovial fluid; Amniotic fluid; Saliva; Cerebrospinal fluid; Aqueous humor; Vitreous humor; Sweat; Capillary electrophoresis; Micellar electrokinetic capillary chromatography; Direct sample injection;