Use of multidimensional lectin affinity chromatography in differential glycoproteomics

This paper reports studies comparing the relative degree of sialylation among human serum glycoproteins carrying complex biantennary N-linked, hybrid, and high-mannose oligosaccharides. Comparisons were made by coupling lectin affinity selection with stable isotope coding of peptides from tryptic digests of serum. After proteolysis, samples were split and differentially acetylated with stable isotope coding agents according to either origin or the separation method by which they would be fractionated. A lectin column prepared from Sambucus nigra agglutinin (SNA) was used to select and compare the concentration of sialic acid containing glycopeptides. The relative standard deviation in quantification using this method was 4%. Using this method the concentration of sialic acid containing glycoproteins from a normal individual were compared to those in a pooled serum sample from a large number of normal individuals. It was found that sialylation varied less than 2-fold in all but four or five glycoproteins. Further studies were done on the degree of sialylation within glycoproteins. Samples labeled with the light isoform of the coding agent were applied to a set of serial lectin columns consisting of a concanavalin A (Con A) column coupled to an SNA column for selecting sialic acid appended to glycopeptides with complex biantennary N-linked, hybrid, and high-mannose glycans. In contrast, samples labeled with the heavy isoform of the coding agent were applied to a Con A lectin column alone to select glycopeptides containing complex biantennary N-linked, hybrid, and high-mannose glycans, without regard to sialylation. Glycopeptides thus selected were mixed, deglycosylated by PNGase F, and fractionated by reversed-phase chromatography (RPC). The RPC fractions were then analyzed by ESI-MS. The relative standard deviation of the method was 4%. All glycopeptides identified contained sialic acid except one. Peptides in which the relative abundance of isotopic isoforms was equal were considered to indicate that the protein parent was fully sialylated at that specific glycosylation site.     

Comparison of inlet geometry in microfluidic cell affinity chromatography

Cell separation based on microfluidic affinity chromatography is a widely used methodology in cell analysis research when rapid separations with high purity are needed. Several successful examples have been reported with high separation efficiency and purity; however, cell capture at the inlet area and inlet design have not been extensively described or studied. The most common inlets-used to connect the microfluidic chip to pumps, tubing, etc.-are vertical (top-loading) inlets and parallel (in-line) inlets. In this work, we investigated the cell capture behavior near the affinity chip inlet area and compared the different performances of vertical inlet devices and parallel inlet devices. Vertical inlet devices showed significant cell capture capability near the inlet area, which led to the formation of cell blockages as the separation progressed. Cell density near the inlet area was much higher than that in the remaining channel, whereas for parallel inlet chips cell density at the inlet area was similar to that in the rest of the channel. In this paper, we discuss the effects of inlet type on chip fabrication, nonspecific binding, cell capture efficiency, and separation purity. We also discuss the possibility of using vertical inlets in negative-selection separations. Our findings show that inlet design is critical and must be considered when fabricating cell affinity microfluidic devices.     

Negative enrichment of target cells by microfluidic affinity chromatography

A three-dimensional microfluidic channel was developed for high-purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of nontarget cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multistep processes in microfluidic systems. In previous work (Li, P.; Tian, Y.; Pappas, D. Anal. Chem.2011, 83, 774-781), we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel system showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity and ease of fabrication and use is suitable for cell separations when subsequent analysis of target cells is required.     

Immobilized-metal affinity and hydroxyapatite chromatography of genetically engineered subtilisin

High-performance immobilized-metal affinity and hydroxyapatite chromatography were employed to investigate the engineered subtilisin S1 binding site microenvironment. Although these methods are classified as affinity techniques, unlike traditional affinity columns, both are capable of probing the entire surface of a molecule. The metal chelate study employed gradient elution to assemble retention maps for a wide range of mobile-phase pH. Resolution of single substitution variants was achieved at the optimum mobile-phase pH. A total of four metals were applied separately to the metal chelate column to investigate ligand specificity with respect to protein retention. Hydroxyapatite chromatography, albeit an established technique, has only recently been developed as a high-performance chromatographic method. Gradient elution separations were performed to determine selectivity. Immobilized-metal affinity chromatography was found to be the more effective method for the separation of site-specific variants.     

Development of sulfhydryl-reactive silica for protein immobilization in high-performance affinity chromatography

Two techniques were developed for the immobilization of proteins and other ligands to silica through sulfhydryl groups. These methods made use of maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method). The resulting supports were tested for use in high-performance affinity chromatography by employing human serum albumin (HSA) as a model protein. Studies with normal and iodoacetamide-modified HSA indicated that these methods had a high selectivity for sulfhydryl groups on this protein, which accounted for the coupling of 77-81% of this protein to maleimide- or iodoacetyl-activated silica. These supports were also evaluated in terms of their total protein content, binding capacity, specific activity, nonspecific binding, stability, and chiral selectivity for several test solutes. HSA columns prepared using maleimide-activated silica gave the best overall results for these properties when compared to HSA that had been immobilized to silica through the Schiff base method (i.e., an amine-based coupling technique). A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods. These features make these supports attractive in the development of protein columns for such applications as the study of biological interactions and chiral separations.     

Immobilized gallium(III) affinity chromatography of phosphopeptides.

A novel procedure for micropurification of phosphorylated peptides, as a front end to mass spectrometric analysis, is described. As a result of a systematic study, we propose the use of an immobilized metal affinity chromatography (IMAC) in a microtip (Erdjument-Bromage, H.; et al. J. Chromatogr., A 1998, 826, 167-181) format, more specifically in combination with Ga(III) ions. Manual Ga-(III) IMAC is easy to perform; phosphopeptides are retrieved in a near-quantitative and highly selective manner, to yield a concentrated sample for direct analysis by matrix-assisted laser desorption/ionization time-of-flight and nanoelectrospray ionization mass spectrometry. Ga-(III) ions offer distinct advantages over the use of other metals, such as Fe(III) and Al(III), in terms of both selectivity and versatility, including facile base elution. Selectivity is best illustrated by effective enrichment of phosphopeptides that were present in a molar ratio of approximately 2% on a background of unphosphorylated protein, a situation very typical perhaps for protein phosphorylation states in the cell. The system was also used to retrieve and tentatively identify five previously uncharacterized phosphopeptides from a tryptic digest of human beta 4 integrin, isolated from cell extracts by immunoprecipitation.     

Fluorous affinity chromatography for enrichment and determination of perfluoroalkyl substances

The adsorption behavior of four perfluoroalkyl acids, including the environmentally relevant perfluorooctanoic acid, has been investigated on a straight-chain perfluorohexyl adsorbent. The aim of this study was to probe the potential of perfluorinated materials for the analysis and enrichment of perfluoroalkyl analytes. Water/acetonitrile mixtures, to which formic acid had been added (generally, 0.1%), were employed as mobile phases. For all perfluorinated acids, a U-shaped retention profile was observed by changing the amount of acetonitrile in the mobile phase. This behavior has been correlated to the excess adsorption of the organic component on the adsorbent surface. The concept of perfluoromethylene selectivity has been defined as the ability of a chromatographic system to discriminate between molecules that differ by a single perfluoromethylene group. The contribution to the Gibbs free energy of phase transfer for the passage of a perfluoroalkyl carbon from the mobile to the stationary phase has been evaluated. This information, in addition to the traditional van't Hoff analysis, has also been used to estimate the analogous contribution for the transfer of a carboxylic unit. Finally, insights into the retention mechanisms of perfluoroalkyl acids on straight-chain perfluorohexyl sorbents are discussed.     

Selection of ligands for affinity chromatography using quartz crystal biosensor

This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (k(ass) and k(diss)) and equilibrium dissociation constants (K(D)) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10(-5) M, which is within the range of 10(-4)-10(-8) M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins.     

Analysis of glycopeptides using lectin affinity chromatography with MALDI-TOF mass spectrometry

Glycopeptides prepared from 1 nmol of a mixture of glycoproteins, transferrin, and ribonuclease B by lysylendopeptidase digestion were isolated by lectin and cellulose column chromatographies, and then they were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry which enables the performance of MS ( n ) analysis. The lectin affinity preparation of glycopeptides with Sambucus nigra agglutinin and concanavalin A provides the glycan structure outlines for the sialyl linkage and the core structure of N-glycans. Such structural estimation was confirmed by MALDI-TOF MS and MALDI-QIT-TOF MS/MS. Amino acid sequences and location of glycosylation sites were determined by MALDI-QIT-TOF MS/MS/MS. Taken together, the combination of lectin column chromatography, MALDI-TOF MS, and MALDI-QIT-TOF MS ( n ) provides an easy way for the structural estimation of glycans and the rapid analysis of glycoproteomics.     

Analytical potential of protein A for affinity chromatography of polyclonal and monoclonal antibodies

Protein A based rapid affinity chromatography for quantitation of various immunoglobulins of class G (IgG) is described. Three-minute analysis using either citrate or phosphate buffers and detection with 220- or 280-nm ultraviolet absorption was found to be optimum for quantitation of IgG from 0.25 to 250 micrograms of IgG on-column with a percent relative standard deviation (% RSD) of 2-3% RSD. The method has a detection limit estimated to be 100 ng of IgG on-column. It has been used to analyze a variety of IgG-containing samples from such diverse sources as hybridoma selection, media cultivation, and purification studies. Gradient elution studies and the relationship of IgG elution to IgG isoelectric point (pI) are also described.     

Examination of glycan profiles from IgG-depleted human immunoglobulins facilitated by microscale affinity chromatography

Among the most important proteins involved in disease and healing processes are the immunoglobulins (Igs). Although many of the Igs have been studied through proteomics, aside from IgG, immunoglobulin carbohydrates have not been extensively characterized in different states of health. It seems valuable to develop techniques that permit an understanding of changes in the structures and abundances of Ig glycans in the context of disease onset and progression. We have devised a strategy for characterization of the glycans for the Ig classes other than IgG (i.e., A, D, E, and M) that contain kappa light chains that requires only a few microliters of biological material. First, we designed a microcolumn containing recombinant Protein L that was immobilized on macroporous silica particles. A similarly designed Protein G microcolumn was utilized to first perform an online depletion of the IgG from the sample, human blood serum, and thereby facilitate enrichment of the other Igs. Even though only 3 μL of serum was used in these analyses, we were able to recover a significantly enriched fraction of non-IgG immunoglobulins. The enrichment properties of the Protein L column were characterized using a highly sensitive label-free quantitative proteomics LC-MS/MS approach, and the glycomic profiles of enriched immunoglobulins were measured by MALDI-TOF MS. As a proof of principle, a comparative study was conducted using blood serum from a small group of lung cancer patients and a group of age-matched cancer-free individuals to demonstrate that the method is suitable for investigation of glycosylation changes in disease. The results were in agreement with a glycomic investigation of whole blood serum from a much larger lung cancer cohort.     

High-performance affinity monolith chromatography: development and evaluation of human serum albumin columns

Several immobilization methods were explored for the preparation of high-performance affinity monolithic columns containing human serum albumin (HSA). These monoliths were based on a copolymer of glycidyl methacrylate and ethylene dimethacrylate. In one method, the epoxy groups of this copolymer were used directly for the immobilization of HSA through its amine residues (i.e., the epoxy method); in other approaches, these epoxy groups were converted to diols for later use in the carbonyldiimidazole, disuccinimidyl carbonate, and Schiff base methods. Each HSA monolith was evaluated in terms of its total protein content and its retention of several model compounds, including (R/S)-warfarin and D/L-tryptophan. The greatest amount of immobilized HSA was obtained by the Schiff base method, whereas the epoxy method gave the lowest protein content. The Schiff base method also gave the best resolution in chiral separations of (R/S)-warfarin and D/L-tryptophan. All of the immobilization methods gave similar relative activities for HSA in its binding to (R)- and (S)-warfarin, but some differences were noted in the activity of the immobilized HSA for D- and L-tryptophan. The efficiency of these monoliths was found to be greater than that of silica-based HSA columns for (R/S)-warfarin (i.e., analytes with high retention), but little or no difference was seen for D- and L-tryptophan (analytes with weak retention).     

Global kinase screening. Applications of frontal affinity chromatography coupled to mass spectrometry in drug discovery

Utilizing frontal affinity chromatography with mass spectrometry detection (FAC-MS), we have identified novel applications in the discovery of small-molecule hits to protein targets that are difficult if not impossible to accomplish using traditional assays. We demonstrate for the first time an ability to distinguish between competitive ligands for the ATP and substrate sites of protein kinase C independently in the same experiment and show that ATP competitive ligands using a functionally inactive receptor tyrosine kinase can be identified. This ability of FAC-MS to simultaneously monitor binding at the ATP and substrate binding sites, as well as measure ligand binding to both active and inactive kinases, suggests that FAC-MS can be used as a "global kinase binding assay".     

Direct MALDI-MS/MS of phosphopeptides affinity-bound to immobilized metal ion affinity chromatography beads

Immobilized metal ion affinity chromatography (IMAC) is a useful method to selectively isolate and enrich phosphopeptides from a peptide mixture. Mass spectrometry is a very suitable method for exact molecular weight determination of IMAC-isolated phosphopeptides, due to its inherent high sensitivity. Even exact molecular weight determination, however, is not sufficient for identification of the phosphorylation site if more than one potential phosphorylation site is present on a peptide. The previous method of choice for sequencing the affinity-bound peptides was electrospray tandem mass spectrometry (ESI-MS/MS). This method required elution and salt removal prior to MS analysis of the peptides, which can lead to sample loss. Using a matrix-assisted laser desorption/ionization (MALDI) source coupled to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer with true MS/MS capabilities, direct sequencing of IMAC-enriched peptides has been performed on IMAC beads applied directly to the MALDI target. The utility of this new method has been demonstrated on a protein with unknown phosphorylation sites, where direct MALDI-MS/MS of the tryptic peptides bound to the IMAC beads resulted in the identification of two novel phosphopeptides. Using this technique, the phosphorylation site determination is unambiguous, even with a peptide containing four potentially phosphorylated residues. Direct analysis of phosphorylated peptides on IMAC beads does not adversely affect the high-mass accuracy of an orthogonal injection QqTOF mass spectrometer, making it a suitable technique for phosphoproteomics.     

Adsorption property and affinity chromatography of polystyrene derivative sorbent towards cow's milk xanthine oxidase

On the basis of the biospecific molecular recognition between complementary chemical groups of xanthine oxidase (XO) and their ligands particularly sulphated glycoaminoglycans and heparin. Poly (styrene chlorosulfonyl) particles modified by sulfonate sodium groups was synthesized and its adsorption property towards cow's milk XO was established. The adsorption of XO onto this functional polymer was performed in batch at 4 °C and at pH 6.0 during 30 min. of incubation. The adsorbed XO content at the interface allows establishing the chemisorption isotherm curve. The affinity association estimated from this adsorption isotherm according to the Langmuir equation was found to be significantly high in the magnitude of 1.25 × 10⁶ M^? 1. Affinity chromatography on column using this functional polymer as a stationary phase confirms its high ability to adsorb XO at low ionic strength. In fact, the xanthine oxidase of the crude extract is strongly adsorbed onto the sorbent and is eluted at high ionic strength with out any significant loss of its biological activity. The purified enzyme possesses a protein flavin ratio (PFR) of 6.05 with a specific activity of 1.78 UI/mg. On the other hand, the electrophoresis of XO fraction showed a single band with a molecular weight of about 150 kDa. Thus, the synthesized beads functionalized by sulfonate group could be used efficiently and advantageously in the purification of XO instead of other conventional chromatographic methods which need several steps.     

A smart microfluidic affinity chromatography matrix composed of poly(N-isopropylacrylamide)-coated beads

The efficient upstream processing of complex biological or environmental samples for subsequent biochemical analysis remains a challenge in many analytical systems. New microfluidic platforms that provide multidiagnostic capabilities on single chips face a similar challenge in getting specific analytes purified or contaminants removed in different fluid streams. Here, stimuli-responsive polymers have been used to construct "smart" beads that can be reversibly immobilized on microfluidic channel walls to capture and release targets. The 100-nm latex beads were surface-modified with the temperature-sensitive polymer poly(N-isopropylacrylamide) (PNIPAAm). At room temperature, a suspension of these beads flows through a microfluidic channel constructed of poly(ethylene terephthalate). However, when the temperature in the channel is raised above the lower critical solution temperature (LCST) of PNIPAAm, the beads aggregate and adhere to the walls of the channel. The adhered beads are stable for long durations on the channel walls (demonstrated up to 70 min) in the presence of flow. The beads were further modified with the affinity moiety biotin, which tightly binds streptavidin. The dual-modified beads were adhered to the channel walls and functioned as a chromatographic affinity separation matrix, capable of binding streptavidin that was flowed through the microfluidic channel. Upon the reverse thermal stimulation to below the PNIPAAm LCST, the beads and captured streptavidin were observed to quickly dissolve and elute from the channel walls. This temperature-responsive affinity chromatography matrix can thus be flowed into a column and aggregated via temperature change, followed by the controlled release of affinity-captured targets back into the microfluidic flow stream.