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.     

Development of structural analysis of sulfated N-glycans by multidimensional high performance liquid chromatography mapping methods

Although the biological importance of sulfated oligosaccharides has been widely recognized, there are only a few reports that describe detailed structures of sulfated N-glycans. This is largely due to the lack of a convenient method to identify structures of sulfated glycans found in low incidence. Here we develop multidimensional high performance liquid chromatography (HPLC) mapping methods for rapid and convenient identification of sulfated N-glycans. By using adequate quantities of sulfated N-glycans derived from LS12 cells, which are transfected with sulfotransferase cDNA, 40 different sulfated glycans have been successfully mapped. Furthermore, we have applied the HPLC data to identification of isomeric products resulting from an enzymatic reaction of N-acetylglucosamine 6-O-sulfotransferase-1 in vitro and revealed that this enzyme preferentially catalyzes sulfation of the GlcNAcbeta1-->2Manalpha1-->3Man branch in a biantennary acceptor.     

Recent advances in peptide separation by multidimensional liquid chromatography for proteome analysis

Shotgun proteomics dominates the field of proteomics. The foundations of the strategy consist of multiple rounds of peptide separation where chromatography provides the bedrock. Initially, the scene was relatively simple with the majority of strategies based on some types of ion exchange and reversed phase chromatography. The thirst to achieve comprehensivity, when it comes to proteome coverage and the global characterization of post translational modifications, has led to the introduction of several new separations. In this review, we attempt to provide a historical perspective to separations in proteomics as well as indicate the principles of their operation and rationales for their implementation. Furthermore, we provide a guide on what are the possibilities for combining different separations in order to increase peak capacity and proteome coverage. We aim to show how separations enrich the world of proteomics and how further developments may impact the field.     

Serially coupled microcolumn reversed phase liquid chromatography for shotgun proteomic analysis

Microcolumn RPLC (μRPLC) is one of the optimum separation modes for shotgun proteomic analysis. To identify as many proteins as possible by MS/MS, the improvement on separation efficiency and peak capacity of μRPLC is indispensable. Although the increase in column length is one of the effective solutions, the preparation of a long microcolumn is rather difficult due to the high backpressure generated during the packing procedure. In our recent work, through connecting microcolumns of 5, 10, and 15 cm length via unions with minimal dead volume, long microcolumns with length up to 30 cm were obtained, with which 318 proteins were identified from proteins extracted from Escherichia coli by μRPLC‐ESI MS/MS, and similar distributions of M_w and pI were found with single and various coupled microcolumns. Furthermore, by using MS/MS with improved sensitivity, with such a serially coupled 30 cm long microcolumn, 1692 proteins were identified within 7 h from rat brain tissue, with false positive rate (FPR) <1%. All these results demonstrated that serially couple microcolumns might be of great promising to improve the separation capacity of μRPLC in shotgun proteomic analysis.     

Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography-tandem mass spectrometry

Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale two-dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S-carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC-MS/MS system in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G-protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell-cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery.     

An accurate proteomic quantification method: Fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry

A facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using MS for quantification, the FLAQ method is a chromatography-based quantification in combination with MS for identification. Multidimensional liquid chromatography (MDLC) with laser-induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. Several requirements should be met for fluorescent labeling in MS identification: Labeling completeness, minimum side-reactions, simple MS spectra, and no extra tandem MS fragmentations for structure elucidations. A fluorescence dye, 5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALDI MS identification. Quantitative labeling was achieved with optimization of reacting conditions. A synthesized peptide and model proteins, BSA (35 cysteines), OVA (five cysteines), were used for verifying the completeness of labeling. Proteins were separated through MDLC and quantified based on fluorescent intensities, followed by MS identification. High accuracy (RSD% < 1.58) and wide linearity of quantification (1-10(5) ) were achieved by LIF detection. The limit of quantitation for the model protein was as low as 0.34 amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ.     

Novel strategy of high-abundance protein depletion using multidimensional liquid chromatography

In this study, for the first time, a comprehensive two-dimensional (2D) liquid-phase separation system, coupling strong cation exchange chromatography (SCX) to reversed-phase high performance liquid chromatography (RPLC), instead of specificity depletion method, was developed at the intact protein level for depletion of high-abundance proteins from rat liver. Proteins were prefractionated by SCX in the first dimensional separation, followed by RPLC with high resolution separation. UV absorption intensity was used to differentiate high-abundance proteins. The proteins with the absorbance intensity above 0.1 AU were defined as high abundance proteins and depleted. After removal of high-abundance proteins; other proteins were pooled, digested, and subsequently separated by capillary liquid chromatography coupled with MALDI-TOF/TOF mass spectrometry analysis. The high efficiency of the strategy was demonstrated by analyzing the soluble protein extracted from rat liver tissue. In total, 77 high-abundance proteins were depleted in one experiment flow. The ratio of depleted content of high-abundance proteins to that of total proteins was about 34.5%. In total, 1530 proteins were identified using the depletion strategy. Quantitative estimation of high-abundance proteins through liquid chromatography combined with UV absorption spectra was achieved. On the basis of the reproducible experimental results, a rapid and high-throughput depletion protocol was put forward. Along with depletion of the most (79.1%) high-abundance proteins and the separation of digested peptides, the total separation time could be less than 30 h. This strategy has no bias for depleting high-abundance proteins and enhances the number of identified proteins; therefore, it can be widely used in the global proteins analysis.     

Enantiomeric determination of pantoprazole in human plasma by multidimensional high-performance liquid chromatography

Multidimensional HPLC is a powerful tool for the analysis of samples of a high degree of complexity. This work reports the use of multidimensional HPLC by coupling a RAM column with a chiral polysaccharide column to the analysis of Pantoprazole in human plasma by direct injection. The enantiomers from the plasma samples were separated with high resolution on a tris(3,5-dimethoxyphenylcarbamate) of amylose phase after clean-up by a RAM BSA octyl column. Water was used as solvent for the first 5 min in a flow-rate of 1.0 ml/min for the elution of the plasmatic proteins and then acetonitrile-water (35:65 v/v) for the transfer and analysis of pantoprazole enantiomers, which were detected by UV at 285 nm. Analysis time was 28 min with no time spent on sample preparation. A good linear relationship was obtained in the concentration range of 0.20 to 1.5 microg/ml for each enantiomer. Inter and intra-day precision and accuracy were determined by one low (0.24 microg/ml), one medium (0.70 microg/ml) and one high (1.3 microg/ml) plasma concentration and gave a C.V. varying from 1.80 to 8.43% and accuracy from 86 to 92%. Recoveries of pantoprazole enantiomers were in the range of 93.7-101.2%. The validated method was applied to the analysis of the plasma samples obtained from ten Brazilian volunteers who received an 80 mg oral dose of racemic pantoprazole and was able to quantify the enantiomers of pantoprazole in all clinical samples analyzed.     

On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated.     

Size-exclusion chromatography in multidimensional separation schemes for proteome analysis

Size-exclusion chromatography (SEC) is a separation technique with a relatively low resolving power, compared to those usually utilized in proteomics. Therefore, it is often overlooked in experimental protocols, when the main goal is resolving complex biological mixtures. In this report, we introduce innovative multidimensional schemes for proteomics analysis, in which SEC plays a practical role. Liquid isoelectric focusing (IEF) was combined with SEC, and experimental results were compared to those obtained by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), well-established techniques relying upon similar criteria for separation.Additional experiments were performed to evaluate the practical contribution of SEC in multidimensional chromatographic separations. Specifically, we evaluated the combination of SEC and ion exchange chromatography in an analytical scheme for the mass spectrometric analysis of protein-extracts obtained from bacterial cultures grown in stable isotope enriched media. Experimental conditions and practical considerations are discussed.     

High-performance liquid chromatography of fatty acid isopropylidene hydrazides and its application in lipid analysis

Fatty acid isopropylidene hydrazides, prepared by stepwise treatment of acyl lipids with hydrazine and acetone, were analyzed by high-performance liquid chromatography on a reversed-phase column. These derivatives could be easily eluted with 15% water in methanol and monitored by measuring absorbance at 229 nm with a uv detector. Their elution behavior, in general, was similar to that of methyl esters and some commonly used ultraviolet-absorbing derivatives of fatty acids. The new method has been used for fatty acid analysis of some oils.     

A multidimensional chromatography technology for in-depth phosphoproteome analysis

Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation. Its application to the yeast Saccharomyces cerevisiae after DNA damage led to the identification of 8764 unique phosphopeptides from 2278 phosphoproteins using tandem MS. Analysis of two low abundance proteins, Rad9 and Mrc1, revealed that approximately 50% of their phosphorylation was identified via this global phosphorylation analysis. Thus, this technology is suited for in-depth phosphoproteome studies.     

Linear multidimensional liquid chromatography in the preparative scale purification of calmodulin from brain extract

Rapid preparative scale purification of calmodulin from crude bovine brain extract is achieved in a single chromatographic run by physically coupling two different liquid chromatography columns which employ different separation mechanisms. In this case columns packed with newly commercialized 40-microns silica-based hydrophobic interaction and 5-microns micron silica-based weak anion-exchange chromatography media were used. The only sample preparation required for conducting this purification procedure is the addition of salt to the crude brain supernatant to promote the initial binding of calmodulin to the hydrophobic interaction chromatography media. Chromatography carried out on such linear arrangements of columns has been referred to as linear multidimensional liquid chromatography.     

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.     

Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli

A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.     

A 2-D liquid-phase chromatography for proteomic analysis in plant tissues

Two-dimensional liquid chromatography based on a high-performance chromatofocusing in the first dimension followed by high-resolution reversed-phase chromatography in the second dimension can be used as a complementary approach to protein separation with two-dimensional gel electrophoresis. In this work, Arabidopsis thaliana proteins obtained from different tissue extracts were resolved by using a new automated system, ProteomeLab PF 2D commercialized by Beckman Coulter (Fullerton, CA, USA). In particular, protein patterns obtained after two different extraction procedures (MgSO4 and urea buffer) were compared. Reproducibility of the protein patterns was also confirmed in different injections of the same sample and in the comparative analyses of some proteins by MALDI-TOF/MS. Computer analysis of the chromatograms revealed that with this two-dimensional liquid phase technique, hundreds of "virtual bands" can be identified and compared in crude plant protein lysates.     

Improved proteomic discovery by sample pre-fractionation using dual-column ion-exchange high performance liquid chromatography

Clinically relevant biomarkers are urgently needed for improving patient diagnosis, risk stratification, prognosis and therapeutic treatments. There is a particularly compelling motivation for identifying protein-based indicators of early-stage disease for more effective interventions. Despite recent progress, the proteomic discovery process remains a daunting challenge due to the sheer heterogeneity and skewed protein abundances in biofluids. Even the most advanced mass spectrometry systems exhibit limiting overall dynamic ranges and sensitivities relative to the needs of modern biomedical applications. To this end, we report the development of a robust, rapid, and reproducible high performance ion-exchange liquid chromatography pre-fractionation method that allows for improved proteomic detection coverage of complex biological specimens using basic tandem mass spectrometry screening procedures. This form of sample simplification prior to global proteomic profiling, which we refer to collectively as 'fractionomics', increases the number and diversity of proteins that can be confidently identified in tissue and cell lysates as compared to the straight analysis of unfractionated crude extracts.     

Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags

The effectiveness of proteome-wide protein identification and quantitative expression profiling is dependent on the ability of the analytical methodologies employed to routinely obtain information on low-abundance proteins, as these are frequently of great biological importance. Two-dimensional gel electrophoresis, the traditional method for proteome analysis, has proven to be biased toward highly expressed proteins. Recently, two-dimensional chromatography of the complex peptide mixtures generated by the digestion of unseparated protein samples has been introduced for the identification of their components, and isotope-coded affinity tags (ICAT) have been introduced to allow for accurate quantification of the components of protein mixtures by mass spectrometry. Here, we demonstrate that the combination of isotope coded affinity protein tags and multidimensional chromatography/mass spectrometry of tryptic peptide mixtures is capable of detecting and quantifying proteins of low abundance in complex samples.     

Chlorophyll analysis by high-performance liquid chromatography

The separation and determination of chlorophylls by high-performance liquid chromatography (HPLC) is described. Chlorophylls and their derivatives were separated by reversed-phase HPLC based on hydrophobic interaction between solute and support, using an octadecyl silica column and elution with 100% methanol. Separated pigments were detected fluorometrically with a sensitivity in the picomole range: the fluorescence response was linear over a wide pigment concentration range. Resolution of five chlorophylls a and four protochlorophyll species esterified with different alcohols was achieved within 22 min in a single experiment. This method can be used for the determination of chlorophyll b, bacteriochlorophyll a esters and products synthesized from chlorophyll, but not for nonesterified pigments, i.e., chlorophyllide, protochlorophyllide and chlorophyll c. The chromatographic mobility of chlorophyll a esterified with different alcohols increases with increasing number of carbon atoms in the esterifying alcohols. The plots obtained from the logarithm of the capacity factor (k′) of these pigments versus the numbers of carbon atoms of the alcohol molecule gave a straight line, thus permitting the estimation of the chain length of unknown pigment esterifying alcohols. This HPLC separation technique did not cause the formation of artifacts. The deviation of the individual retention time for each pigment is less than ±0.5%, thus making this method suitable for the rapid identification and quantification of unknown pigments.