Analysis of relative isotopologue abundances for quantitative profiling of complex protein mixtures labelled with the acrylamide/D3-acrylamide alkylation tag system

The new method of analysis of relative isotopologue abundances (ARIA) applied here is based on the evaluation of total isotope patterns of tryptic protein fragments measured by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) to calculate the mixing ratios of composites consisting of stable isotope labelled and isotopically natural (unlabelled) proteins, as described in an accompanying paper in this issue. Recently, Sechi (Rapid Commun. Mass Spectrom. 2002; 16: 1416-1424) and Gehanne et al. (Rapid Commun. Mass Spectrom. 2002; 16: 1692-1698) introduced the use of differential quantitative mass analysis by MALDI-TOFMS using mixtures of standard proteins alkylated prior to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with either acrylamide (AA) or deuterium-labelled [2,3,3'-D(3)]-acrylamide (D3AA). In the present study we validate the AA/D3AA system, firstly by measuring the yield of proteins alkylated with AA, and secondly by using differential radioactive labels ((125)I and (131)I) to quantitatively establish that non-comigration in 2D-PAGE is negligible. ARIA is then applied to quantitatively estimate the relative proportions of peptides labelled with AA or D3AA in the validated system, using typical silver-stained 2D-PAGE protein spots from 2D gels loaded with 150 microg of total liver protein. The precision and limitations of ARIA quantification of peptides differentially alkylated with isotopomeric reagents are discussed.     

Synthesis, characterisation and application of enantiomeric isotopomers of Evans’ oxazolidinones

The synthesis of a series of enantiomerically pure deuterium-labelled isotopomeric Evans’ oxazolidinones is discussed.     

Isotopomeric diols by “one-pot” Ru-catalyzed homogeneous hydrogenation of dicarboxylic acids

The “one-pot” homogeneous hydrogenation of γ-butyrolactone and succinic or fumaric acid to 1,4-butandiol, have been successfully realized in the presence of the catalytic system [Ru(acac)₃]/triphos] [triphos:MeC(CH₂PPh₂)₃]. The influence of some reaction parameters on the regioselectivity and the rate of the reaction were investigated. The study was then extended to the “one-pot” synthesis of isotopomeric 1,4-butandiols by deuteration of the appropriate substrates in a deuterated solvent. 1,4-butandiol-d₈, which was fully characterized, was obtained with 96% yield and 100% isotopomeric selectivity. A mechanism was proposed to rationalize the role of catalyst, solvent and deuterium distribution.     

Quantitative variability in the 252Cf plasma desorption mass spectra of triglycerides and waxes

A series of mixtures of solid triglycerides and waxes are examined using ²⁵²Cf plasma desorption mass spectrometry. The relative abundances of diagnostic ions are found to vary with sample preparation, handling, and composition. It is proposed that the less efficiently packing, shorter chain, compounds are forced to the analysis site, i.e. the surface. This takes place at varying rates that reflect the bulk properties of the mixture as revealed by its phase diagram and thermal history. Mixtures that remain liquids during analysis show ion abundances correctly reflecting their composition. Mixtures of isotopomeric glycerides also show normal ion abundances.     

High affinity capture surface for matrix-assisted laser desorption/ionisation compatible protein microarrays

A surface for the capture of biotin-tagged proteins on matrix-assisted laser desorption/ionisation (MALDI) targets has been investigated. Binding of a poly-L-lysine poly(ethylene glycol)-biotin polymer to glass and gold surfaces has been demonstrated using dual wavelength interferometry. Biotinylated proteins were captured onto this surface using tetrameric neutravidin as a multivalent bridging molecule. Biotin tagging of proteins was achieved by chemical biotinylation or by expressing a protein with a biotinylation consensus sequence in E. coli. The specificity of the surface for biotin-tagged proteins allowed the purification of biotin-tagged glutathione-S-transferase from a bacterial lysate directly onto a MALDI target. Subsequently, the protein was digested on the MALDI target and a protein fingerprint analysis confirmed its presence directly, but no E. coli proteins were detected. Therefore, we conclude that this surface is highly specific for the capture of biotin-labelled proteins and has low non-specific binding properties for non-biotinylated proteins. Furthermore, protein-protein interactions using biotinylated lectins were investigated, and the selective capture of the glycoprotein fetuin with wheat germ agglutinin was demonstrated. Also, immobilised Arachis hypogea agglutinin recognised a minor asialo component of this glycoprotein on the array. The high affinity immobilisation of proteins onto this surface allowed effective desalting procedures to be used which improved the desorption of high molecular weight proteins. Another aspect of this surface is that a highly ordered coupling of the analyte can be achieved which eliminates the search for the sweet spot and allows the creation of densely packed protein microarrays for use in mass spectrometry.     

Experimental study of albumin and lysozyme adsorption onto acrylic acid (AA) and 2-hydroxyethyl methacrylate (HEMA) surfaces

Many commercial soft contact lenses are based on poly-2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AA) hydrogels. The adsorption of proteins, albumin and lysozyme, on such contact lens surfaces may cause problems in their applications. In this work the adsorption of proteins, albumin and lysozyme, on hydrogel surfaces, AA and HEMA, was investigated as a function of concentration of protein. Also the effects of pH and ionic strength of protein solution on the adsorption of protein were examined. The obtained results indicated that the degree of adsorption of protein increased with the concentration of protein, and the adsorption of albumin on HEMA surface at the studied pHs (6.2-8.6) was higher than AA surface, whereas the adsorption of lysozyme on AA surface at the same pHs was higher than HEMA. The change in ionic strength of protein solution affected the proteins adsorption on both AA and HEMA surfaces. Also, the amount of sodium ions deposited on the AA surface was much higher than HEMA surface. This effect can be related to the negative surface charge of AA and its higher tendency for adsorption of sodium ions compared to the HEMA surface.     

Monitoring protein distributions based on patterns generated by protein adsorption behavior in a microfluidic channel

We report a unique monitoring technique of protein distributions based on distinctive patterns generated by protein adsorption behavior on a solid surface in a microfluidic channel. Bare gold and COOH-modified self-assembled monolayer (SAM) sensing surfaces were pre-adsorbed with one of four different proteins: lysozyme, albumin, transferrin, or IgG. Each surface provides a thermodynamically governed platform for immobilizing proteins and generates analyte-specific response patterns. Each surface has its own thermodynamic energy governing pre-adsorbed protein behaviors, so that sample proteins react with the pre-adsorbed ones to different extents depending on their sizes, isoelectric points (pI), and characteristics of the sensing surfaces. Modified surfaces were mounted and monitored in real time using surface plasmon resonance (SPR). Buffer-prepared sample matrices (α1-antitrypsin, haptoglobin, C-reactive protein (CRP), and IgM) characterized protein response patterns. Each surface generated distinctive patterns based on individual SPR angle shifts. We classified each sample with 95% accuracy using linear discriminant analysis (LDA). Our method also discriminated between different concentrations of CRP in the cocktail sample, detecting concentrations as low as 1 nM with 91.7% accuracy. This technique may be integrated with a microfluidic lab-on-a-chip system and monitor the distribution of a specific group of proteins in human serum.     

A comparison of the adsorption of saliva proteins and some typical proteins onto the surface of hydroxyapatite

Adsorption of protein from saliva on hydroxyapatite was compared with adsorption of several typical proteins with different electric charges, i.e. lysozyme, human serum albumin, β-lactoglobulin and ovalbumin. Adsorbed amounts of these proteins were determined and electrophoretic mobilities of protein-covered hydroxyapatite particles were measured, at different values for the adsorbed mass and, therefore, at various degrees of surface coverage. Also, adsorption kinetics were investigated by streaming potential measurements of a hydroxyapatite surface in contact with a protein solution, allowing monitoring of changes in the zeta-potential of the protein-covered hydroxyapatite surface in real time. The adsorbed amounts show that, as compared to most of the other proteins, the saliva proteins have remarkably low adsorption affinity. The measured values for the electrophoretic mobilities indicate that the positively charged proteins in the saliva mixture preferentially adsorb onto the negatively charged hydroxyapatite surface; this is most pronounced at low protein concentration in solution (i.e. at low coverage of the surface by the protein). Preferential uptake of the positively charged saliva proteins during the initial stages of the adsorption process is also concluded from the results of the kinetics experiments. Preferential adsorption of positive proteins is somewhat suppressed by the presence of Ca²⁺ ions in the medium. The results suggest that an acquired pellicle on a tooth in an oral environment contains a significant fraction of positively charged proteins. The positively charged proteins in the pellicle reduce the zeta-potential at the tooth surface to low values; consequently, electrostatic forces are expected to play only a minor role in the interaction with other components (e.g. bacterial cells).     

Synthesis and reactivity of dimethyl platinum(IV) hydrides in water

New hydrophilic ligands of the di(2-pyridyl)methanesulfonate family, L = dpms and Me-dpms, enable the synthesis of methyl platinum(IV) hydrides, LPtMe2H, the study of very fast CH reductive coupling, and reductive elimination of these complexes in water. In dichloromethane solutions, 13CH4 reacts with (Me-dpms)PtMe2H to produce isotopomeric complexes.     

Spatially addressable chemoselective C-terminal ligation of an intein fusion protein from a complex mixture to a hydrazine-terminated surface

Protein immobilization on surfaces is useful in many areas of research, including biological characterization, antibody purification, and clinical diagnostics. A critical limitation in the development of protein microarrays and heterogeneous protein-based assays is the enormous amount of work and associated costs in the purification of proteins prior to their immobilization onto a surface. Methods to address this problem would simplify the development of interfacial diagnostics that use a protein as the recognition element. Herein, we describe an approach for the facile, site-specific immobilization of proteins on a surface without any preprocessing or sample purification steps that ligates an intein fusion protein at its C-terminus by reaction with a hydrazine group presented by a surface. Furthermore, we demonstrate that this methodology can directly immobilize a protein directly from cell lysate onto a protein-resistant surface. This methodology is also compatible with soft lithography and inkjet printing so that one or more proteins can be patterned on a surface without the need for purification.     

Use of isotopomers for cluster size and geometry determination

Formulas are presented for the determination of the number of isotopomeric structures possible for a cluster of any geometry and size. Linear clusters are shown to be a special case of the general formulas. Extension to multi-isotope, single element clusters and multi-isotope, multi-element clusters is also made. Examples of each class are given.     

SURFACE PROPERTIES OF SOME LOW MOLECULAR WEIGHT PROTEINS

Surface properties of four proteins having molecular weights less than 5,000 are reported at air/water and alumina/water interface at pH 7.0. Reversibility in the adsorption of these proteins at the alumina/water interface is tested. The adsorption on alumina/water interface has been found to be controlled by electrostatic interaction. Positive adsorption was obtained when protein and alumina surface had opposite charges and negative adsorption was obtained when both protein and surface had same charges. Of the four proteins reversibility in adsorption was observed with the one having the lowest molecular weight of 3100. The adsorption behavior apparently had no correlation with their surface hydrophobic!ty. Time dependent changes in air/water interfacial tension was observed for all the four proteins indicating time dependent loosening of compact protein structure and surface unfolding.     

Functionalized self-assembled monolayers on ultraflat gold as platforms for single molecule force spectroscopy and imaging

Single molecule force spectroscopy is a valuable tool for studying unfolding and nanomechanical properties of proteins. The common practice is to stretch proteins from a surface that was dosed to give a reasonable hit rate and to analyze the curves that exhibit the expected characteristics of a single polymer. Whether the surface-bound proteins are indeed single and isolated remains unclear, and the undesirable protein/surface interactions that obscure informative features of the force curves are implicitly assumed to be absent. In this study, mixed self-assembled monolayers (SAMs) consisting of N-hydroxysuccinimide (NHS) and oligoethylene glycol (OEG) terminated thiols on an ultraflat gold surface were used to covalently immobilize proteins via lysine residues. By the optimization of attachment sites via lysine-NHS linkages amidst a protein-resistant layer of the OEG SAM, it was possible to isolate single proteins for study in a controlled fashion. The single protein distribution on the surface is clearly demonstrated by atomic force microscopy (AFM) imaging. The OEG also significantly reduces nonspecific tip-surface interactions between the cantilever and surface. Stretching covalently attached single proteins produces high-quality and reproducible force-extension curves. This experimental strategy is an attractive platform with which to study protein structure, interactions, and nanomechanical properties of single proteins.     

Specific labeling of cell surface proteins with chemically diverse compounds

The specific and covalent labeling of fusion proteins with synthetic molecules opens up new ways to study protein function in the living cell. Here we present a novel method that allows for the specific and exclusive extracellular labeling of proteins on the surfaces of live cells with a large variety of synthetic molecules including fluorophores, protein ligands, or quantum dots. The approach is based on the specific labeling of fusion proteins of acyl carrier protein with synthetic molecules through post-translational modification catalyzed by phosphopantetheine transferase. The specificity and versatility of the labeling should allow it to become an important tool for studying and manipulating cell surface proteins and for complementing existing approaches in cell surface engineering.     

Influence of surface modification on protein retention in ion-exchange chromatography: Evaluation using different retention models

A large number of different stationary phases for ion-exchange chromatography (IEC) from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (α-lactalbumin, β-lactoglobulin A, bovine serum albumin and alcohol dehydrogenase) were determined for three anion-exchange adsorbents based on synthetic copolymer beads with differences in the functional group chemistry. Fractogel EMD DEAE and Fractoprep DEAE consist of functional groups bound to the surface via “tentacles”, ToyopearlDEAE by a short linker. Three models which describe chromatographic retention were used to analyse the characteristic parameters of the protein/stationary-phase interactions. The number of electrostatic interaction between the stationary phase and the model proteins, the protein specific surface charge densities and the interacting surface of the proteins with the adsorptive layer of the chromatographic media depend on the surface modification as well as on the molecular mass of the model proteins. In general, protein retention of the model proteins on the weak anion exchangers was found to be greater if the stationary phase carries tentacles and protein mass is above 60 kDa.     

Protein-protein binding-sites prediction by protein surface structure conservation

A new algorithm to predict protein-protein binding sites using conservation of both protein surface structure and physical-chemical properties in structurally similar proteins is developed. Binding-site residues in proteins are known to be more conserved than the rest of the surface, and finding local surface similarities by comparing a protein to its structural neighbors can potentially reveal the location of binding sites on this protein. This approach, which has previously been used to predict binding sites for small ligands, is now extended to predict protein-protein binding sites. Examples of binding-site predictions for a set of proteins, which have previously been studied for sequence conservation in protein-protein interfaces, are given. The predicted binding sites and the actual binding sites are in good agreement. Our algorithm for finding conserved surface structures in a set of similar proteins is a useful tool for the prediction of protein-protein binding sites.     

Surface recognition of a protein using designed transition metal complexes.

Each protein has a unique pattern of histidine residues on the surface. This paper describes the design, synthesis, and binding studies of transition metal complexes to target the surface histidine pattern of carbonic anhydrase (bovine erythrocyte). When the pattern of cupric ions on a complex matches the surface pattern of histidines of the protein, strong and selective binding can be achieved in aqueous buffer (pH = 7.0). The described method of protein recognition is applicable to proteins of known structures. With rapidly increasing number of solved protein structures, the method has wide applicability in purification, targeting, and sensing of proteins.     

Molecular packing of lysozyme,fibrinogen, and bovine serum albumin on hydrophilic and hydrophobic surfaces studied by infrared-visible sum frequency generation and fluorescence microscopy

Infrared-visible sum frequency generation (SFG) vibrational spectroscopy, in combination with fluorescence microscopy, was employed to investigate the surface structure of lysozyme, fibrinogen, and bovine serum albumin (BSA) adsorbed on hydrophilic silica and hydrophobic polystyrene as a function of protein concentration. Fluorescence microscopy shows that the relative amounts of protein adsorbed on hydrophilic and hydrophobic surfaces increase in proportion with the concentration of protein solutions. For a given bulk protein concentration, a larger amount of protein is adsorbed on hydrophobic polystyrene surfaces compared to hydrophilic silica surfaces. While lysozyme molecules adsorbed on silica surfaces yield relatively similar SFG spectra, regardless of the surface concentration, SFG spectra of fibrinogen and BSA adsorbed on silica surfaces exhibit concentration-dependent signal intensities and peak shapes. Quantitative SFG data analysis reveals that methyl groups in lysozyme adsorbed on hydrophilic surfaces show a concentration-independent orientation. However, methyl groups in BSA and fibrinogen become less tilted with respect to the surface normal with increasing protein concentration at the surface. On hydrophobic polystyrene surfaces, all proteins yield similar SFG spectra, which are different from those on hydrophilic surfaces. Although more protein molecules are present on hydrophobic surfaces, lower SFG signal intensity is observed, indicating that methyl groups in adsorbed proteins are more randomly oriented as compared to those on hydrophilic surfaces. SFG data also shows that the orientation and ordering of phenyl rings in the polystyrene surface is affected by protein adsorption, depending on the amount and type of proteins.     

Multicolor imaging of cell surface proteins

We report on a method for the multicolor imaging of cell surface proteins which is based on the labeling of carrier protein (CP) fusion proteins with different fluorophores. In one application, different generations of a cell surface protein can be sequentially labeled to discriminate between old and newly made copies. In another application, fusions to different CPs can be selectively labeled with different fluorophores in one sample. Both applications open up new ways for studying the properties of cell surface proteins of living cells.     

Direct correlation of the crystal structure of proteins with the maximum positive and negative charge states of gaseous protein ions produced by electrospray ionization

Electrospray mass spectrometric studies in native folded forms of several proteins in aqueous solution have been performed in the positive and negative ion modes. The mass spectra of the proteins show peaks corresponding to multiple charge states of the gaseous protein ions. The results have been analyzed using the known crystal structures of these proteins. Crystal structure analysis shows that among the surface exposed residues some are involved in hydrogen-bonding or salt-bridge interactions while some are free. The maximum positive charge state of the gaseous protein ions was directly related to the number of free surface exposed basic groups whereas the maximum negative charge state was related to the number of free surface exposed acidic groups of the proteins. The surface exposed basic groups, which are involved in hydrogen bonding, have lower propensity to contribute to the positive charge of the protein. Similarly, the surface exposed acidic groups involved in salt bridges have lower propensity to contribute to the negative charge of the protein. Analysis of the crystal structure to determine the maximum charge state of protein in the electrospray mass spectrum was also used to interpret the reported mass spectra of several proteins. The results show that both the positive and the negative ion mass spectra of the proteins could be interpreted by simple consideration of the crystal structure of the folded proteins. Moreover, unfolding of the protein was shown to increase the positive charge-state because of the availability of larger number of free basic groups at the surface of the unfolded protein.