Inhibition of protein carbamylation in urea solution using ammonium-containing buffers

Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times, and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium-containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium-containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium-containing buffers was developed to facilitate its application in proteomic research.     

Peptide mapping of heterogeneous protein samples.

A simple two-dimensional electrophoretic method for peptide mapping of heterogeneous protein samples is presented. The reduced and denatured proteins of the mixture are separated in a first dimension by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. After completion of the electrophoresis, the whole gel lane is equilibrated in stacking gel buffer and is transferred at right angles onto a second slab gel. A protease solution is overlayed on the gel lane and a partial proteolysis of the proteins to be analyzed is performed during the stacking phase of the second electrophoresis. The second electrophoresis resolves the characteristic pattern of peptides of each individual protein as a series of spots located below the original position of the undigested protein. The peptide maps of the following samples are presented as examples: protein P23 and P23* of bacteriophage T4, membranes of Dictyostelium discoideum, membranes of human erythrocytes, and 35S-labeled proteins of D. discoideum synthesized in vivo or in a cell-free wheat germ extract. In complex samples, up to 20 individual proteins can be analyzed at once and a protein comprising only 1% of the total sample generates a clearly identifiable peptide pattern. Good reproducibility of the patterns obtained allows the comparison of samples of different origins.     

Microwave-assisted protein preparation and enzymatic digestion in proteomics

The combinations of gel electrophoresis or LC and mass spectrometry are two popular approaches for large scale protein identification. However, the throughput of both approaches is limited by the speed of the protein digestion process. Present research into fast protein enzymatic digestion has been focused mainly on known proteins, and it is unclear whether these results can be extrapolated to complex protein mixtures. In this study microwave technology was used to develop a fast protein preparation and enzymatic digestion method for protein mixtures. The protein mixtures in solution or in gel were prepared and digested by microwave-assisted protein enzymatic digestion, which rapidly produces peptide fragments. The peptide fragments were further analyzed by capillary LC and ESI-ion trap-MS or MALDI-TOF-MS. The technique was optimized using bovine serum albumin and then applied to human urinary proteins and yeast lysate. The method enabled preparation and digestion of protein mixtures in solution (human urinary proteins) or in gel (yeast lysate) in 6 or 25 min, respectively. Equivalent (in-solution) or better (in-gel) digestion efficiency was obtained using microwave-assisted protein enzymatic digestion compared with the standard overnight digestion method. This new application of microwave technology to protein mixture preparation and enzymatic digestion will hasten the application of proteomic techniques to biological and clinical research.     

Isolation of N-terminal protein sequence tags from cyanogen bromide cleaved proteins as a novel approach to investigate hydrophobic proteins

A novel method for the isolation of protein sequence tags to identify proteins in a complex mixture of hydrophobic proteins is described. The PST (Protein Sequence Tag) technology deals with the isolation and MS/MS based identification of one N-terminal peptide from each polypeptide fragment generated by cyanogen bromide cleavage of a mixture of proteins. PST sampling takes place after sub-cellular fractionation of a complex protein mixture to give enrichment of mitochondrial proteins. The method presented here combines effective sample preparation with a novel peptide isolation protocol involving chemical and enzymatic cleavage of proteins coupled to chemical labeling and selective capture procedures. The overall process has been very successful for the analysis of complex mixtures of hydrophobic proteins, particularly membrane proteins. This method substantially reduces the complexity of a protein digest by "sampling" the peptides present in the digest. The sampled digest is amenable to analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods of "sampling" protein digests have great value' if they can provide sufficient information to identify substantially all of the proteins in the sample while reducing the complexity of the sample to maximize the efficient usage of LC-MS/MS capacity. The validity of the process is demonstrated for mitochondrial samples from S. cerevisiae. The proteins identified by the PST technology are compared to the proteins identified by the conventional technology 2-D gel electrophoresis as a control.     

Trichoplusia ni granulosis virus granulin: a phenol-soluble, phosphorylated protein.

Trichoplusia ni granulosis virus granulin consists of one major polypeptide component with an estimated molecular weight of 28,000. The protein is phenol soluble, phosphorylated, and acidic. A protease activated by alkaline conditions is also associated with solubilized granulin preparations. If not properly inactivated, the protease will introduce extensive artifact into the protein giving rise to ambiguous and incorrect results as analyzed by SDS-polyacrylamide gel electrophoresis and peptide mapping. Procedures are documented for enzyme inactivation and the preparation of granulin in highly purified form for characterization.     

Overproduction and purification of Lon protease fromEscherichia coli using a maltose-binding protein fusion system

Lon protease, which plays a major role in degradation of abnormal proteins inEscherichia coli, was overproduced and efficiently purified using the maltose-binding protein (MBP) fusion vector. The MBP-Lon fusion protein was expressed in a soluble form inE. coli and purified to homogeneity by amylose resin in a single step. Lon protease was split from MBP by cleaving a fusion point between MBP and Lon with factor Xa and purified by amylose resin and subsequent gel filtration. In this simple method, Lon protease was purified to homogeneity. Purified MBP-Lon fusion protein and Lon protease showed similar breakdown activities with a peptide (succinyl-l-phenylalanyl-l-leucyl-phenylalanyl--d-methoxynaphthylamide) and protein (-casein) in the presence of ATP. Therefore, the gene-fusion approach described in this study is useful for the production of functional Lon protease. MBP-Lon fusion protein, which both binds to the amylose resin and has ATP-dependent protease activity, should be especially valuable for its application in the degradation of abnormal proteins by immobilized enzymes.     

A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA

Amyloid fibrils from a patient with diffuse amyloid disease are dissociated in 6 m guanidine hydrochloride and fractionated by gel chromatography. Two major components are separated on Sepharose 6B. Both proteins are characterized by chromatography, immunodiffusion, discontinuous gel electrophoresis, amino acid tryptic peptide mapping and amino acid sequence analysis. The smaller of the two components is typical of the known protein AA by size (8400 daltons), amino acid composition and a 30-residue N-terminal sequence. The larger of the components (25,000 daltons) undergoes electrophoresis as a single band and appears unaffected by thiol reduction. It differs from protein AA in amino acid content and by its tryptic peptide map, although it contains an N-terminal amino acid sequence identical to protein AA when carried to 20 residues. Treatment of this larger component by mild acid hydrolysis results in the release of the 8400-dalton protein AA. Fractionation after guanidine hydrochloride treatment of this particular amyloid fibril preparation is compared to the fractionation of a typical secondary amyloid preparation that contains only protein AA as the major component. The origin and relationship of the 8,400- and 25,000-dalton protein components is discussed.     

Characterization of the actin polymerization-inhibiting protein from chicken gizzard smooth muscle

An actin polymerization-inhibiting protein, occurring in crude preparations of vinculin from chicken gizzard, has been found to be heterogeneous. The molecular masses of the polymerization-inhibiting peptides have been reported to range from 20 kDa to 80 kDa [Schr?er, E. & Wegner, A (1985) Eur. J. Biochem. 153, 515-520]. In this paper, a 21-kDa peptide was isolated from the bulk of the other peptides by gel chromatography. The 21-kDa peptide was identified as a polymerization-inhibiting peptide by its ability to retard nucleated actin polymerization and to bind polymeric actin when it was blotted onto nitrocellulose. Antiserum raised to the 21-kDa peptide was found to react with almost all peptides of the blotted heterogeneous polymerization-inhibiting protein. The same peptides which reacted with antiserum cosedimented with polymeric actin. The major peptides of the blotted polymerization-inhibiting protein bound polymeric actin. The largest peptide which reacted with antiserum and cosedimented with polymeric actin had a molecular mass of 85 kDa. The results suggest that the preparation of polymerization-inhibiting protein contains mainly polymerization-inhibiting peptides and only some contaminants, and that all the polymerization-inhibiting peptides are proteolytic fragments stemming from a common precursor.     

Gel‐aided sample preparation (GASP)—A simplified method for gel‐assisted proteomic sample generation from protein extracts and intact cells

We describe a “gel‐assisted” proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel‐aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy‐to‐use on a wide range of sample types, and accessible to nonspecialists.     

Sequence homology analysis of a heterogenous protein population by chemical and enzymic digestion using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel system

A procedure for examining possible sequence homology between two or more proteins in a heterogenous protein mixture using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis system is described. Three different chemical reagents (cyanogen bromide, hydroxylamine, and acetic acid) and three enzymes (α-chymotrypsin, trypsin, and Staphylococcus aureus protease) have been used as the cleavage reagents for the peptide mapping studies. Potential application of this technique in conjunction with radioactive labeling and immunological studies was also demonstrated.     

Purification of a multicatalytic protease complex from developing winged bean seeds by indirect immunoaffinity chromatography

Many protease inhibitors have been characterized from leguminous seeds but very little is known about seed proteases which are supposedly regulated by these inhibitors. We have developed an indirect immunoaffinity chromatography system for the purification of cognate proteases from the same source, based on preferential high salt elution of the enzyme from a ternary complex of the protease, the inhibitor, and the anti-inhibitor IgG. Using anti-winged bean chymotrypsin inhibitor (WbCI) IgG as an affinity ligand, a multicatalytic protease complex has been purified from developing winged bean (Psophocarpus tetragonolobus) seeds. The purified preparation resolves into two large proteolytically active components when subjected to gel permeation chromatography under nondenaturing conditions, while SDS/PAGE analysis shows the presence of approximately 15 polypeptide chains in the 20- to 115-kDa range. The preparation cleaves known synthetic peptide substrates of trypsin, chymotrypsin, and V8 protease and it is only partially inhibited by a number of class-specific protease inhibitors. Western blot analysis shows the presence of WbCI in the purified preparation even after its extensive removal by the IgG-Sepharose column. The versatility of the indirect immunoaffinity chromatography system is attested by its extension to the soybean seeds.     

Purification and characterization of an acidic amino acid-specific endopeptidase of Bacillus subtilis obtained from a commercial preparation (Protease Type XVI, Sigma)

An acidic amino acid-specific endopeptidase was purified from Protease Type XVI (Sigma), a commercial product from culture filtrate of Bacillus subtilis, by a series of column chromatographies on CM-Toyopearl (Fractogel) and Mono-S, guided by activity assay using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase HPLC. The molecular weight of the protease was estimated to be 18,000 by gel filtration on TSK gel G3000SWXL column using 6 M guanidine hydrochloride as an eluent, and 17,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the protease was 7.7. Studies on the substrate specificity with peptide p-nitroanilides and natural peptides revealed that the protease hydrolyzes the peptide bonds on the carboxyl-terminal side of acidic amino acids, especially of glutamic acid. The protease was completely inactivated by DFP, indicating the serine protease nature of the protease. The activity of the protease was also inhibited by EDTA and GEDTA, and reactivated by Ca2+. The protease contained 1.3 +/- 0.2 mol/mol protein of Ca2+. These results suggest that Ca2+ plays a vital role in the protease activity.     

Mitochondrial phosphate transport. Large scale isolation and characterization of the phosphate transport protein from beef heart mitochondria

The phosphate transport protein from beef heart mitochondria has been purified on a large scale by hydroxylapatite chromatography in the presence of sodium dodecyl sulfate and urea. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain), the pure phosphate transport protein preparation consists of two protein bands (alpha and beta, ratio 1:1) with similar mobilities (34 kDa) which display identical peptide maps if fragmented with either CNBr or HCl/dimethyl sulfoxide/HBr. The complete amino acid composition of phosphate transport protein is presented. Quantitative determination of N-terminal amino acids underlines the purity of the preparation and shows for alpha and beta the identical amino-terminals H2N-Ala-Val-Glu-Glu-Glx-Tyr-. Qualitative digestion shows that carboxypeptidase A is able to release at least three amino acids from the C termini of the alpha as well as the beta band of phosphate transport protein. The nature of these two protein bands is discussed. The sum of phosphate transport protein (alpha + beta) per total mitochondrial protein amounts to 2.3% or 1.4 nmol of phosphate transport protein (34 kDa) per nmol of cytochrome b.     

Challenging protein purification from anammox bacteria

The anaerobic ammonium oxidation (anammox) is a fascinating microbial pathway contributing to the global biogeochemical nitrogen cycle. The anammox pathway of nitrogen conversion can only be elucidated after the responsible proteins have been purified and characterised. The anammox bacteria have a complex cell envelope consisting of protein and lipopolysaccharide and they grow in dense cell aggregates. Preparing cell extract and purifying proteins from the cell aggregates is hampered by the extracellular polymeric material and by gel formation. It was demonstrated that protein-protein (i.e. disulfide formation) as well as protein-polysaccharide interaction caused this gel formation in extracts. Cell extract gelled upon freezing/thawing and boiling. Additionally, proteins aggregated on various chromatography media upon concentration and during desalting. The polysaccharides clogged the matrix of chromatographic materials and the pores of ultrafiltration membranes. The precipitation of proteins and polysaccharides caused very low resolution and streaking on SDS- and two-dimensional polyacrylamide gels. The present work describes the potential causes for gel formation in anammox cell extracts. Optimized protocols for sample preparation for polyacrylamide gel electrophoresis and ion exchange chromatography are presented. High-resolution gel electrophoresis of the cell extract was achieved after clarification from polymeric substances with denaturating phenol extraction and the purification of a 10 kDa cytochrome c is presented as an example.     

lon gene product of Escherichia coli is a heat-shock protein

The product of the pleiotropic gene lon is a protein with protease activity and has been tentatively identified as protein H94.0 on the reference two-dimensional gel of Escherichia coli proteins. Purified Lon protease migrated with the prominent cellular protein H94.0 in E. coli K-12 strains. Peptide map patterns of Lon protease and H94.0 were identical. A mutant form of the protease had altered mobility during gel electrophoresis. An E. coli B/r strain that is known to be defective in Lon function contained no detectable H94.0 protein under normal growth conditions. Upon a shift to 42 degrees C, however, the Lon protease was induced to high levels in K-12 strains and a small amount of protein became detectable at the H94.0 location in strain B/r. Heat induction of Lon protease was dependent on the normal allele of the regulatory gene, htpR, establishing lon as a member of the high-temperature-production regulon of E. coli.     

Large-scale electrophoresis for protein purification: exploiting isoelectricity

Preparative electrophoresis methods (including isoelectric focusing in immobilized pH gradients) in gel phases are characterized by low loadings barely a few mg protein per ml matrix), low recoveries (rarely exceeding 70%), and heavy contamination from neurotoxic gel materials (the unreacted gel monomers and ungrafted oligomers). These drawbacks can be minimized by a version of isoelectric focusing in which the need for protein of interest to pass the gel is eliminated: only the contaminants traverse the gel. This is achieved by circulating a liquid sample between two gels held at controlled pHs. The method can provide: (1) high rate of sample processing (up to 1 g h⁻¹); (2) high purification (in general to charge homogeneity); and (3) high recoveries (>95%). A large-scale membrane apparatus has been built, with a cross- sectional diameter of 9 cm. Large Pt electrode disks provide even current flow. In this electrolyser, 10 g of Eglin C (produced by recombinant DNA technology) have been purified to homogeneity in around 10 h from 1 l of a partially enriched preparation.     

Effect of protein backbone folding on the stability of protein-ligand complexes

The role played by the degree of folding of protein backbones in explaining the binding energetics of protein-ligand interactions has been studied. We analyzed the protein/peptide interactions in the RNase-S system in which amino acids at two positions of the peptide S have been mutated. The global degree of folding of the protein S correlates in a significant way with the free energy and enthalpy of the protein-peptide interactions. A much better correlation is found with the local contribution to the degree of folding of one amino acid residue: Thr36. This residue is shown to have a destabilizing interaction with Lys41, which interacts directly with peptide S. Another system, consisting of the interactions of small organic molecules with HIV-1 protease was also studied. In this case, the global change in the degree of folding of the protease backbone does not explain the binding energetics of protein-ligand interactions. However, a significant correlation is observed between the free energy of binding and the contribution of two amino acid residues in the HVI-1 protease: Gly49 and Ile66. In general, it was observed that the changes in the degree of folding are not restricted to the binding site of the protein chain but are distributed along the whole protein backbone. This study provides a basis for further consideration of the degree of folding as a parameter for empirical structural parametrizations of the binding energetics of protein folding and binding.     

Profiling the alkaline membrane proteome of Caulobacter crescentus with two-dimensional electrophoresis and mass spectrometry

Attempts at protein profiling in the alkaline pH region using isoelectric focusing have often proved difficult, greatly limiting the scope of proteome analysis. We investigated several parameters using custom pH 8-11 immobilized pH gradients to separate a Caulobacter crescentus membrane preparation. These included sample application, quenching endoosomotic flow and gel matrix composition. Among these factors, the sample application position was the predominant parameter to affect two-dimensional gel quality. Separated proteins were silver stained and profiled using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The use of a prototype MALDI-Q-Tof mass spectrometer assisted identification of several proteins by providing highly informative peptide fragmentation data from the sample digests. Thirty-two unique alkaline proteins were identified in this study, which complements our previously described C. crescentus membrane proteome. Our experiments point towards new options for proteomic researchers aiming to both extend the scope of analysis, and simplify methods of identifying proteins with high confidence.     

An optimized protein in-gel digest method for reliable proteome characterization by MALDI-TOF-MS analysis

Human lung epithelial cells (A549) were used as a model to develop a reliable proteome characterization method by peptide mass fingerprinting (PMF). Lung cell lysate proteins and protein standards were separated by 2D-gel electrophoresis, stained with Coomassie blue, gel plugs were subjected to commonly adapted as well as optimized in-gel digestion/sample preparation methods. Samples were analyzed by MALDI-TOF-MS. Optimization parameters included, use of NH(4)OAc in destaining and in-gel digestion buffers, detergent/salt removal prior to in-gel digestion, use of solvents of varying polarities (0%, 30%, 60% ACN containing 0.1% TFA) to improve peptide recoveries, matrix composition (alpha-cyano-4-hydroxycinamic acid-organic solvent combinations) and on-target salt removal. This led to enhanced mass spectral information and a sensitivity gain in the order of 6-10 fold compared to that of common procedures, yielding reliable, unambiguous protein identification with femtomol protein sensitivity by Autoflex MALDI-TOF-MS. Triplicate analyses by two analysts revealed consistent, wide range m/z values including in < 1200Da region by relieving matrix-exerted signal suppression, requiring one trial to obtain a unique protein identification with superior PMF results for the optimized method. Analyses of ten A549 proteins in replicates using the optimized method yielded fast, reliable characterization, suggesting the potential application of this method in high-throughput protein identification by PMF.     

Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil cathepsin G from normal donors

Human neutrophil cathepsin G from normal donors has been purified 82-fold using an isolation procedure which included sequential sodium chloride extraction, Aprotonin-Sepharose affinity chromatography, CM-cellulose ion-exchange chromatography, and AcA44 gel filtration chromatography. The inclusion of this last purification step was crucial for separating inactive lower molecular weight species from the active forms of neutrophil cathepsin G and resulted in a higher specific activity of the final preparation. SDS polyacrylamide gradient gel electrophoresis of the purified reduced protein demonstrated three discrete polypeptides of Mr 31,000, 30,000, and 29,500. Peptide analysis of tryptic digests indicated that these three polypeptides are structurally related to each other and represent microheterogeneity of the purified protein. The cathepsin G peptide maps were distinctly different from the peptide maps of neutrophil elastase. The apparent isoelectric points of these forms as determined by two-dimensional electrophoresis was approximately 8.0. Utilizing microsequencing techniques, the first 25 residues of normal neutrophil cathepsin G have been determined and shown to be identical (except for residue 11) with the sequence of 21 residues of cathepsin G isolated from leukemic myeloid cells. A high degree of homology was found when the amino-terminal regions of neutrophil cathepsin G, rat mast cell protease II (65%) and two human serine proteinases, factor D (52%) and neutrophil elastase (48%), were compared. A precipitating monospecific antiserum to cathepsin G was produced by repeated immunizations of guinea pigs. This antiserum has been used in immunoblotting experiments to demonstrate that the intracellular form(s) of this enzyme is the same approximate Mr as the purified enzyme, and to develop a solid-phase radioimmunoassay for measuring neutrophil cathepsin G in the range 5-50 ng/ml.