Homogeneous fluorescent derivatization of large proteins

A method of homogeneously derivatizing large proteins for highly sensitive analysis is described. Homogeneity of the derivative was realized by tagging all the free amino groups of proteins. With this method, alpha-chymotrypsinogen A, ovalbumin and bovine serum albumin were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Prior to the derivatization, all the proteins were reduced and alkylated. After reacting the resulting unfolded proteins with excessive amounts of AQC, the samples were analyzed with matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to determine the derivatization degree. The results indicated that all three proteins had been, or had almost been, fully derivatized. HPLC and CE were used for characterizing these protein derivatives. Under the optimized fluorescence detection conditions, the detectability of the tagged proteins was 2400-6200 times better than that detected at UV 280 nm, 170-300 times better than detected at UV 214 nm, and 150-420 times better than measured with their native fluorescence.     

Capillary column high-performance liquid chromatographic-electrospray ionization triple-stage quadrupole mass spectrometric analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis application to cerebellar protein mapping

A method is presented for the structural characterization of proteins separated by two-dimensional poly-acrylamide gel electrophoresis (2D-PAGE). The method includes separation of a protein mixture by 2D-PAGE, recovery of proteins from the gel spots revealed by copper staining and analysis of the proteins by triple-stage quadrupole mass spectrometry using an electrospray ionization interface (ESI-TSQMS). Prior to the mass spectrometric analysis, the extracted proteins were passed through a small reversed-phase column (10 × 4.0 mm I.D.) to remove salts and gel-derived contaminants and then introduced into the mass spectrometer through a reversed-phase capillary column with 0.25 mm I.D. Application of the method to the analysis of rat cerebellar proteins suggests that the molecular mass could be accurately determined with sub-picomole amounts of protein samples derived from one or two 2D gels. The method was also useful for peptide mapping and determination of amino acid sequences of proteins micro-prepared from the 2D gel. Because 2D-PAGE has an excellent resolving power in protein separation and because capillary LC-ESI-TSQMS provides structural information with very small amounts of samples, the combined system of 2D-PAGE and capillary LC-ESI-TSQMS described here should allow wide applications to molecular studies of genes and proteins, such as identifications of protein spots on 2D gels, confirmation of gene/protein sequences and analysis of post-translational modification of proteins present naturally in tissue/cell extracts or expressed by recombinant DNA techniques.     

Structural analysis of recombinant proteins prepared by semi-dry electroblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Proteins that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were electroblotted onto polyvinylidene difluoride membranes in procedures to prepare homogeneous recombinant proteins for direct N-terminal sequence analysis. A semi-dry blotting procedure was employed to immobilize protein bands on the membranes for subsequent sequence analysis. This method has been used routinely to evaluate the quality of recombinant proteins, which are present in crude cell extracts produced by different expression systems or under different expression conditions. N-Terminal processing, amino acid misincorporation, as well as the inefficient secretion of recombinant proteins can be detected by direct N-terminal sequence analysis of the purified electroblotted samples. Consequently, time-consuming chromatographic procedures can be eliminated. These procedures are also especially valuable for determining degradation sites of a purified recombinant protein, as well as evaluating multiple gene products expressed by isolated cluster genes.     

Separation and characterization of proteins from green and etiolated shoots of rice (Oryza sativa L.): towards a rice proteome

Proteins extracted from green and etiolated shoots of rice were separated by two-dimensional polyacrylamide gel electrophoresis and relative molecular weights and isoelectric points were determined. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 85 proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino acid sequences of 21 out of 85 proteins were determined in this manner. N-terminal regions of the remaining proteins could not be sequenced. The internal amino acid sequences of proteins were determined by sequence analysis of peptides obtained by the Cleveland peptide mapping method and compared with those of known plant and animal protein sequences to understand the nature of the proteins. Green shoots revealed the presence of photosynthetic proteins as expected; however, as etiolated shoots were not photosynthetic, only precursors of the photosynthetic proteins were identified. Interestingly, the presence of L-ascorbate peroxidase only in etiolated shoots suggests a cellular protectant function for this antioxidant enzyme in the etiolating shoots. Using this experimental approach, we could identify the major proteins involved in growth regulation in photosynthetic green shoots as well as in etiolating rice seedlings.     

Amino acid sequences of 60B8 antigens induced in HL-60 cells by 1,25-dihydroxyvitamin D3. The antigens are identical with macrophage-related protein-14 and -8

A differentiation antigen 60B8 appeared in human promyelocytic leukemia HL-60 cells which had been induced to differentiate into macrophage-like cells by treatment with 1,25-dihydroxyvitamin D3. The antigen was purified by immunoaffinity chromatography and separated into two proteins, 60B8-A and -B antigens, by reverse-phase high performance liquid chromatography (HPLC). Both proteins were digested with proteases, and the resulting peptides were subjected to amino acid sequence analysis after purification by reverse-phase HPLC. The amino acid sequences of 60B8-A and -B antigens were identical with those of the proteins MRP-14 and -8, respectively, which were recently predicted from the nucleotide sequences of their complementary deoxyribonucleic acid (cDNA) clones by Odink et al. (Nature (London), 330, 80 (1987)). Although they did not characterize the chemical properties of the two proteins, our results clearly indicate that macrophage-related protein (MRP)-14 and -8 are expressed without post-translational modification, except that the amino-terminus of MRP-14 is blocked, in differentiated HL-60 cells.     

Use of amino acid analysis for estimating the individual concentrations of proteins in mixtures

The concentrations of five individual proteins in a mixture were determined from one amino acid analysis of the mixture by solving for each protein using simultaneous equations. Dried casein and whey were separated into five individual protein components using reversed-phase HPLC. Individual proteins were collected and analyzed for amino acid composition. These data were used as standards. Mixtures of purified proteins were analyzed for total amino acid composition and the concentrations of individual proteins in the mixtures were determined by solving simultaneous equations based on the amino acid analysis composition of the standards.     

Use of phage display methods to identify heptapeptide sequences for use as affinity purification 'tags' with novel chelating ligands in immobilized metal ion affinity chromatography

This study describes the screening of a peptide phage display library for amino acid sequences that bind with different affinities to a novel class of chelating ligands complexed with Ni2+ ions. These chelating ligands are based on the 1,4,7-triazacyclononane (TACN) structure and have been chosen to allow enhanced efficiency in protein capture and decreased propensity for metal ion leakage in the immobilized metal ion affinity chromatographic (IMAC) purification of recombinant proteins. Utilising high stringency screening conditions, various peptide sequences containing multiple histidine, tryptophan, and/or tyrosine residues were identified amongst the different phage peptide sequences isolated. The structures, and particularly the conserved locations of these key amino acid residues within the selected heptapeptides, form a basis to design specific peptide tags for use with these novel TACN ligands as a new mode of IMAC purification of recombinant proteins.     

Optimizing high-performance liquid chromatography method for quantification of glucosamine using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization in rat plasma: application to a pharmacokinetic study

A sensitive and reliable HPLC method with fluorescence detection based on the precolumn derivatization of glucosamine with 6-aminoquinolyl-N-hydroxylsuccinimidyl carbamate (AQC) was established for the quantitative determination of glucosamine in rat plasma. The plasma protein was precipitated by acetonitrile, followed by vortex mixing and centrifugation. The supernatant was divided into the organic layer and aqueous layer by adding sodium chloride, and then the aqueous layer was derivatized with AQC in 0.2 M borate buffer of pH 8.8 before the HPLC analysis. An amino acid analysis column (3.9 x 150 mm, 4 microm) was applied, with 140 mM sodium acetate buffer (pH = 5.25) and acetonitrile as mobile phase at a flow rate of 1 mL/min. A linear correlation coefficient of 0.9987 was calculated within the range of 0.1-30 microg/mL of the standard curve for glucosamine. The limit of detection was 30 ng/mL. The intra- and inter-day precisions (as RSD) were less than 7.38 and 12.72%, respectively. The intra- and inter-day accuracy ranged from 91.8 to 110.0%. Extraction recoveries of glucosamine in plasma were more than 90%. The validated method was successfully applied for the quantitative determination of glucosamine in rat plasma and evaluation for pharmacokinetic study of glucosamine. It was also possible to be applied for the quantitative determination of other compounds containing amino group in biological samples.     

Advanced dress-up chiral columns: New removable chiral stationary phases for enantioseparation of chiral carboxylic acids

This paper describes the preparation of new dress-up columns featuring reproducibly removable and replaceable chiral stationary phases. After synthesizing perfluroalkylated quinine and quinidine derivatives as chiral stationary phase compounds (F-CSPs), we adsorbed them reversibly onto a fluorous LC column through pumping of their solutions. Using this dress-up chiral column and fluorophobic elution of aqueous ammonium formate/MeOH mixtures, we could enantioseparate four racemic N-acetyl amino acids, dichlorprop, and sixteen fluorescent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized amino acids. Dressing and undressing of the coated F-CSPs could be controlled by varying the fluorophilicity and fluorophobicity of the eluent. The relative standard deviations of the retention times, the retention factors, the number of theoretical plates, the enantioseparation factors, and the resolutions of each of four preparations of such dress-up columns were all less than or equal to 5.26% (from 20 repeated analyses); the reproducibilities from four different preparations were all less than or equal to 10.6%. These columns also facilitated highly sensitive and selective analyses of AQC-amino acids when detected using LC–MS/MS.     

Comparative analysis of amino acid composition in the active site of nirk gene encoding copper-containing nitrite reductase (CuNiR) in bacterial spp.

The nirk gene encoding the copper-containing nitrite reductase (CuNiR), a key catalytic enzyme in the environmental denitrification process that helps to produce nitric oxide from nitrite. The molecular mechanism of denitrification process is definitely complex and in this case a theoretical investigation has been conducted to know the sequence information and amino acid composition of the active site of CuNiR enzyme using various Bioinformatics tools. 10 Fasta formatted sequences were retrieved from the NCBI database and the domain and disordered regions identification and phylogenetic analyses were done on these sequences. The comparative modeling of protein was performed through Modeller 9v14 program and visualized by PyMOL tools. Validated protein models were deposited in the Protein Model Database (PMDB) (PMDB id: PM0080150 to PM0080159). Active sites of nirk encoding CuNiR enzyme were identified by Castp server. The PROCHECK showed significant scores for four protein models in the most favored regions of the Ramachandran plot. Active sites and cavities prediction exhibited that the amino acid, namely Glycine, Alanine, Histidine, Aspartic acid, Glutamic acid, Threonine, and Glutamine were common in four predicted protein models. The present in silico study anticipates that active site analyses result will pave the way for further research on the complex denitrification mechanism of the selected species in the experimental laboratory.     

Identification of mouse brain proteins after two-dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis

Two-dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time-consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low microgram range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two-dimensional electrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R-250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N-terminal sequence determination.     

Determination of amino acids by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and high performance liquid chromatography with ultraviolet detection

A precolumn derivatization method for the determination of amino acids using 6-aminoquinolyl-N-hyroxy-succinimidyl carbamate (AQC) followed by high-performance liquid chromatography is described. Ultraviolet detection was used for the assay of AQC derivatives of amino acids with the detection wavelength set at 248 nm. The reagent peak interference was minimized by optimizing the pH of the eluent and the gradient elution profile to improve the resolution between the reagent peak and amino acid derivatives. All nineteen amino acids were separated in 35 min with resolutions 1.6. The correlation coefficients of the calibration graphs for seventeen amino acids were fairly good (r 0.9999) at concentrations of 25–500 μM. The detection limits for all common amino acids including cystine and trytophan were at the range of 0.07–0.3 pmol. Good reproducibility and accuracy of the method were demonstrated by the determination of amino acids in three typical kinds of samples (protein, peptide and feed.) The average relative standard deviations for bovine serum albumin (BSA) and neuromedin were 0.86% and 1.36, respectively, and the average relative errors were 3.2% and 2.3%, respectively. The results of the analysis of feed hydrolysates agreed with those obtained by an ion-exchange method and the average recovery of the method for feed hydrolysates was 98%.     

Conformation Analysis of Ile-Ala-Val-Pro Peptide and Its Derivatives by Circular Dichroism

Ile-Ala-Val-Pro as a hypocholesterolemic peptide was isolated from soybean protein. We have synthesized four peptides, Ile-Ala-Val-Pro-Gly-Glu-Val-Ala, Leu-Ile-Ala-Val-Pro-Gly-Glu-Val-Ala, Ile-Ala-Val-Pro-Thr-Gly-Val-Ala, Leu-Ile-Ala-Val-Pro-Thr-Gly-Val-Ala, with a conserved Ile-Ala-Val-Pro amino acid sequence, for circular dichroism investigations. These four peptide sequences were also found in the amino acid sequence in soybean protein, which was defined from the genomic sequence. Additionally for a detailed analysis of conformation features of these peptides, the Ile-Ala-Val-Pro and Leu-Ile-Ala-Val-Pro were also synthesized. All peptides were prepared using standard fluorenylmethyloxycarbonyl methodology and the peptide yields ranged from 90 to 95% of the theoretical yields with purity after purification above 99%.     

溴化氰裂解结合电喷雾串联质谱测定电泳分离蛋白的C端序列

C端测序是蛋白质及多肽一级结构确认的重要组成部分,也是重组蛋白药物质量控制的重要依据。建立了溴化氰裂解结合电喷雾串联质谱测定蛋白质C端序列的方法,并应用于重组人肿瘤坏死因子受体和纽兰格林的C端测序。首先根据待测蛋白序列进行溴化氰理论裂解,如果C-端肽段理论分子量在500~5000U之间,则将待测样品进行SDS-PAGE分离,考马斯亮兰染色,然后进行胶内溴化氰裂解,最后应用基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)测定C-端肽段的分子量,电喷雾串联质谱对C端肽段进行测序。应用本方法分别测定了这两个蛋白质C端19个和11个氨基酸残基序列。研究结果表明:本方法灵敏、有效、实用性较强,可适用于部分重组蛋白药物的质量控制和蛋白质的结构确证,是对目前蛋白质C端测序方法的有效补充。     

Prediction of protein structural classes by a new measure of information discrepancy

Since it was observed that the structural class of a protein is related to its amino acid composition, various methods based on amino acid composition have been proposed to predict protein structural classes. Though those methods are effective to some degree, their predictive quality is confined because amino acid composition cannot sufficiently include the information of protein sequences. In this paper, a measure of information discrepancy is applied to the prediction of protein structural classes; different from the previous methods, this new approach is based on the comparisons of subsequence distributions; therefore, the effect of residue order on protein structure is taken into account. The predictive results of the new approach on the same data set are better than those of the previous methods. As to a data set of 1401 sequences with no more than 30% redundancy, the overall correctness rates of resubstitution test and Jackknife test are 99.4 and 75.02%, respectively, and to other data sets the similar results are also obtained. All tests demonstrate that the residue order along protein sequences plays an important role on recognition of protein structural classes, especially for alpha/beta proteins and alpha+beta proteins. In addition, the tests also show that the new method is simple and efficient.     

A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori

Background

A group of abundant proteins of ~30 kDa is synthesized in silkworm larval peripheral fat body (PPFB) tissues and transported into the open circulatory system (hemolymph) in a time-depended fashion to be eventually stored as granules in the pupal perivisceral fat body (PVFB) tissues for adult development during the non-feeding stage. These proteins have been shown to act anti-apoptotic besides being assigned roles in embryogenesis and defense. However, detailed protein structural information for individual PPFB and PVFB tissues during larval and pupal developmental stages is still missing. Gel electrophoresis and chromatography were used to separate the 30 kDa proteins from both PPFB and PVFB as well as hemolymph total proteomes. Mass spectrometry (MS) was employed to elucidate individual protein sequences. Furthermore, 30 kDa proteins were purified and biochemically characterized.

Results

One- and two-dimensional gel electrophoresis (1/2D-PAGE) was used to visualize the relative changes of abundance of the 30 kDa proteins in PPFB and PVFB as well as hemolymph from day 1 of V instar larval stage to day 6 of pupal stage. Their concentrations were markedly increased in hemolymph and PVFB up to the first two days of pupal development and these proteins were consumed during development of the adult insect. Typically, three protein bands were observed (~29, 30, 31 kDa) in 1D-PAGE, which were subjected to MS-based protein identification along with spots excised from 2D-gels run for those proteomes. Gas phase fragmentation was used to generate peptide sequence information, which was matched to the available nucleotide data pool of more than ten highly homologous insect 30 kDa lipoproteins. Phylogenetic and similarity analyses of those sequences were performed to assist in the assignment of experimentally identified peptides to known sequences. Lipoproteins LP1 to LP5 and L301/302 could be matched to peptides extracted from all bands suggesting the presence of full length and truncated or modified protein forms in all of them. The individual variants could not be easily separated by classical means of purification such as 2D-PAGE because of their high similarity. They even seemed to aggregate as was indicated by native gel electrophoresis. Multistep chromatographic procedures eventually allowed purification of an LP3-like protein. The protein responded to lipoprotein-specific staining.

Conclusions

In B. mori larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph. The concentration of these proteins changed progressively during development from their synthesis in PPFB, transport in hemolymph to storage in PVFB. While the 30 kDa proteins could be reproducibly separated in three bands electrophoretically, the exact nature of the individual protein forms present in those bands remained partially ambiguous. The amino acid sequences of all known 30 kDa proteins showed very high homology. High-resolution separation techniques will be necessary before MS and other structural analysis can shed more light on the complexity of the 30 kDa subproteome in B. mori. A first attempt to that end allowed isolation of a B. mori LP3-like protein, the complete structure, properties and function of which will now be elucidated in detail.     

Predicting protein folding rates using the concept of Chou's pseudo amino acid composition

One of the most important challenges in computational and molecular biology is to understand the relationship between amino acid sequences and the folding rates of proteins. Recent works suggest that topological parameters, amino acid properties, chain length and the composition index relate well with protein folding rates, however, sequence order information has seldom been considered as a property for predicting protein folding rates. In this study, amino acid sequence order was used to derive an effective method, based on an extended version of the pseudo-amino acid composition, for predicting protein folding rates without any explicit structural information. Using the jackknife cross validation test, the method was demonstrated on the largest dataset (99 proteins) reported. The method was found to provide a good correlation between the predicted and experimental folding rates. The correlation coefficient is 0.81 (with a highly significant level) and the standard error is 2.46. The reported algorithm was found to perform better than several representative sequence-based approaches using the same dataset. The results indicate that sequence order information is an important determinant of protein folding rates.     

A statistical method for predicting protein unfolding rates from amino acid sequence

The prediction of protein unfolding rates from amino acid sequences is one of the most important challenges in computational biology and chemistry. The analysis on the relationship between protein unfolding rates and physical-chemical, energetic, and conformational properties of amino acid residues provides valuable information to understand and predict the unfolding rates of two- and three-state proteins. We found that the classification of proteins into different structural classes shows an excellent correlation between amino acid properties and unfolding rates of two- and three-state proteins, indicating the importance of native-state topology in determining the protein unfolding rates. We have formulated three independent linear regression equations to different structural classes of proteins for predicting their unfolding rates from amino acid sequences and obtained an excellent agreement between predicted and experimentally observed unfolding rates of proteins; the correlation coefficients are 0.999, 0.990, and 0.992, respectively, for all-alpha, all-beta, and mixed-class proteins. Further, we have derived a general equation applicable to all structural classes of proteins, which can be used for predicting the unfolding rates for proteins of an unknown structural class. We observed a correlation of 0.987 and 0.930, respectively, for back-check and jack-knife tests. These accuracy levels are better than those of other methods in the literature.     

Combination of two-dimensional gel electrophoresis with microsequencing and amino acid composition analysis: improvement of speed and sensitivity in protein characterization

High-resolution two-dimensional polyacrylamide electrophoresis (2-DE) is commonly used as an analytical approach to resolve and detect most of the numerous protein species of an organism. However, the isolation of microgram amounts of protein in a 2-DE spot in a form suitable for microsequence analysis and amino acid composition analysis is a key step in the chemical characterization of these proteins. With the development of chemically inert membranes it is now possible to retain proteins present in low quantities from the polyacrylamide matrix with high yields. The immobilized proteins are suitable for direct sequence analysis and amino acid composition analysis. The combination of protein chemical and electrophoretic techniques makes it possible to obtain chemical information from subpicomole quantities of protein, resulting in the availability of a new set of biologically important proteins for structural analysis. This paper summarizes the methods and strategies for the chemical protein analysis of 2-DE spots in our laboratory.