Quantitative analysis of immobilized proteins and protein mixtures by amino acid analysis

Biomolecular surface engineering of materials often requires precise, versatile and efficient quantification of immobilized proteins at solid surfaces. Acidic hydrolysis of surface-bound proteins and subsequent HPLC analysis of fluorescence-derivatized amino acids were adapted and critically evaluated for that purpose. Contaminations and concentration-dependent amino acid retrieval during HPLC were found to influence the accuracy of the method. In addition to the choice of adequate conditions for hydrolysis, derivatization and chromatographic separation extensions of the data evaluation were suggested to improve the accuracy of the approach when applied to single protein systems: comparing the experimentally obtained amino acid ratio to the protein constitution enabled to identify the properly separated and detected amino acids. Those amino acids were selected for a more precise calculation of the amount of immobilized protein. To further increase the accuracy of the method, the retrieval of amino acids corresponding to protein amounts in the range between 0.5 and 4.0 microg was analyzed for a variety of proteins of interest to derive protein-specific correction factors. The evaluation of amino acid data was furthermore applied to quantify binary protein mixtures at similar settings. This method was proven useful to detect the composition of protein mixtures throughout a wide range of absolute and relative concentrations.     

Strategies for internal amino acid sequence analysis of proteins separated by polyacrylamide gel electrophoresis

An evaluation has been made of various strategies for obtaining internal amino acid sequence data from electrophoretically separated proteins. Electroblotting, in situ proteolysis and extraction, and direct electroelution are compared. Electroblotting of protein or peptides from gels resulted in poor yields (typically, 1-7%). However, higher yields (3-67%) were achieved by in situ enzymatic cleavage followed by acid extraction of the peptides from the gel. Peptides extracted from the gel were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), on short, small-bore columns (100 x 2.1 mm I.D.), to enable recovery of peptides in small volumes (ca. 50 microliters) suitable for microsequence analysis. Capillary zone electrophoresis under acidic conditions (pH 2.5) was used to assess peptide purity before sequence analysis. Cysteine residues were identified in unmodified proteins or peptides by a characteristic phenylthiohydantoin (PTH)-amino acid derivative during sequence analysis. This derivative does not co-chromatograph with any known PTH-amino acid. Direct electrophoretic elution of protein from gels yielded between 45-50% of applied protein. Proteins recovered from gels by electrophoretic elution required further purification by inverse-gradient RP-HPLC [R. J. Simpson, R. L. Moritz, E. C. Nice and B. Grego, Eur. J. Biochem., 165 (1987) 21] to remove sodium dodecylsulphate and acrylamide-related contaminants for sequence analysis.     

Site-specific incorporation of a redox-active amino acid into proteins

The redox-active amino acid 3,4-dihydroxy-l-phenylalanine (DHP), which can undergo two-electron oxidation to a quinone, has been incorporated selectively and efficiently into proteins in Escherichia coli in response to a TAG codon. We have demonstrated that DHP can be oxidized electrochemically within the protein. The ability to incorporate a redox-active amino acid site specifically into proteins should facilitate the study of electron transfer in proteins, as well as enable the engineering of redox proteins with novel properties.     

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.     

Molecular design of glycoprotein mimetics: glycoblotting by engineered proteins with an oxylamino-functionalized amino acid residue

The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O-(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation. Direct and chemoselective coupling between unmodified simple sugars and N-methylaminooxy group displayed on the engineered streptavidin allowed for the combinatorial synthesis of novel glycoprotein mimetics.     

Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix

Summary A method is described for obtaining peptide fragments for sequence analysis from microquantities of proteins separated by 1- or 2-dimensional polyacrylamide gel electrophoresis. After separation by electrophoresis, the proteins were stained with Coomassie Blue and excised. Proteolytic digestion with trypsin was performed directly in the polyacrylamide matrix. The resulting peptide fragments were eluted, separated by reversed phase HPLC, collected and sequenced in a gas phase sequencer. Excellent peptide recoveries allowed generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequences that can be used to generate oligonucleotide probes for molecular cloning, to design synthetic peptides for inducing antibodies, and to search sequence databases for related proteins.     

Recent advances in amino acid analysis by CE

This paper describes a number of articles that have been published on amino acid (AA) analysis using CE during the period from June 2005 to May 2007. This review article follows the format of the previous articles of Smith (Electrophoresis 1999, 20, 3078-3083), Prata et al.. (Electrophoresis 2001, 22, 4129-4138), and Poinsot et al.. (Electrophoresis 2003, 24, 4047-4062 and Electrophoresis 2006, 27, 176-194). Several new developments in AA analysis with CE are reported describing the use of laser-emitting diodes for LIF, MS, and chips. In addition, we describe articles concerning clinical studies and neuroclinical applications.     

Development of a derivatisation method for the analysis of aldehyde modified amino acid residues in proteins by Fourier transform mass spectrometry

A method was developed for the analysis of amino acids within bovine serum albumin (BSA) which had been modified by reaction with different enals. BSA was reacted with the aldehydes and the reaction products were stabilised by reaction with NaBH₄. The protein was then hydrolysed with 6N HCl and the hydrolysis products were analysed by liquid chromatography-mass spectrometry (LC-MS). The modified amino acids were derivatised with propylchloroformate. High resolution mass spectrometry carried out using an LTQ-Orbitrap instrument which was able to characterise a wide range of adducts. In addition double adducts were observed to be formed with 4-hydroxynonenal (HNE) and lysine or lysine + histidine. Qualitatively it was possible to consistently observe a pyridinium adduct formed between lysine and pentenal in human plasma from normal subjects.     

A compilation of amino acid analyses of proteins

The amino acid analyses of 183 proteins, as residues per 1000 residues, are given. In addition the carbohydrate content and the content of any noncommon amino acids are also given. The sources of all proteins are presented.     

A compilation of amino acid analyses of proteins

The amino acid analyses of 213 proteins, as residues per 1000 residues, are given. In addition, the carbohydrate content, the content of any noncommon amino acids, the sources of all proteins, and the necessary literature citations are given.     

Reversed-phase chromatography of phenylthiocarbamyl amino acid derivatives of physiological amino acids: an evaluation and a comparison with analysis by ion-exchange chromatography.

Reversed-phase chromatography of phenylthiocarbamyl (PTC) amino acid derivatives of physiological amino acids was evaluated and compared with the traditional method of ion exchange. The PTC amino acid derivatives were stable for at least 32 h at ambient temperature before injection. The relationship of detector response to concentration for the PTC derivatives was linear from 39 to 1250 pmol. With few exceptions, the within- and between-run precisions of plasma amino acid retention times were less than 0.2 and 0.3%, respectively; the within- and between-run precisions of their concentrations were less than 4.0 and 5.0%, respectively. Twenty-four plasma samples were quantitated by both reversed-phase and ion-exchange chromatography; fifteen of the twenty amino acids determined had correlation coefficients in the range 0.81-1.00. Nine non-standard amino acids and ten therapeutic drugs were added to plasma; D-glucosaminic acid and alpha-amino-beta-guanidinopropionic acid co-eluted with alpha-aminoadipic acid and threonine, respectively. Of the ten drugs added, only metronidazole and theophylline co-eluted with beta-alanine and histidine, respectively. The precision, stability, and sensitivity of the method render it ideal for the quantitation of plasma amino acids.     

A compilation of amino acid analyses of proteins

The amino acid analyses of 186 proteins are given as residues per 1000 residues. Additional information as carbohydrate composition, content of uncomon amino acids, and sources of all proteins are also presented.     

Fractional and amino acid compositions of micromycete proteins

A comparative study has been made of the fractional and amino acid compositions of the proteins of the biomasses ofTrichoderma harzianum, Fusarium moniliforme, and the hybrid culture FT-2. It has been established that the micromycete strains investigated are high-protein cultures containing from 36.0to 49.7% of protein. A study of the amino acid compositions of the micromycetes showed that the hybrid culture FT-2 possessed the largest amount of amino acids among the water-soluble proteins of the biomass (61.3 g/100g of biomass protein).Institute of Microbiology, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 41 71 29. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 428–431, May–June, 1997.     

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.     

Recent advances in amino acid analysis by capillary electrophoresis

Amino acids are studied extensively using capillary electrophoresis. In a previous article, we reviewed applications reported in the period 1999-early 2001 (Prata, C., Bonnafous, P., Fraysse, N., Treilhou, M., Poinsot, V., Couderc, F., Electrophoresis 2001, 22, 4129-4138). In this article we follow on with this review for the period end of 2001-beginning of 2003. We will report the developments of detection methods, separations of enantiomers, the new medical applications, and amino acids in food and plants. This review shows that CE is more and more important for the amino acid analysis.     

Recent advances in amino acid analysis by capillary electrophoresis

This paper describes the most important articles that have been published on amino acid analysis using CE during the period from June 2009 to May 2011 and follows the format of the previous articles of Smith (Electrophoresis 1999, 20, 3078-3083), Prata et al. (Electrophoresis 2001, 22, 4129-4138) and Poinsot et al. (Electrophoresis 2003, 24, 4047-4062; Electrophoresis 2006, 27, 176-194; Electrophoresis 2008, 29, 207-223; Electrophoresis 2010, 31, 105-121). We present new developments in amino acid analysis with CE, which are reported describing the use of lasers or light emitting diodes for fluorescence detection, conductimetry electrochemiluminescence detectors, mass spectrometry applications, and lab-on-a-chip applications using CE. In addition, we describe articles concerning clinical studies and neurochemical applications of these techniques.     

Recent advances in amino acid analysis by capillary electrophoresis

Amino acids are studied extensively using capillary electrophoresis. In this review we will report the different researchs which have been done in the literature since 1998. We will describe the developments of, detection methods, separations of enantiomers, the new medical applications, and amino acids in food and plants.     

Recent advances in amino acid analysis by capillary electrophoresis

This paper describes a number of articles that have been published on amino acid analysis using CE during the period from June 2003 to May 2005. This review article follows the previous ones of Smith (Electrophoresis 1999, 20, 3078-3083), Prata et al. (Electrophoresis 2001, 22, 4129-4138), and Poinsot et al. (Electrophoresis 2003, 24, 4047-4062). Several new developments in amino acid analysis with CE are reported concerning UV detection, LIF, MS, and NMR. In addition, we describe articles concerning clinical and pharmaceutical studies, neuroclinical applications, and agricultural and food analysis.