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PubMed1990

Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase.

In these studies we demonstrate that insulin stimulates both tyrosine and serine phosphorylation of the insulin receptor after its partial purification on wheat germ-agarose, and after affinity purification on insulin-agarose. Analysis of the serine phosphate incorporated into partially purified or highly purified insulin receptor suggests that an insulin-sensitive serine kinase (IRSK) copurifies with the insulin receptor. Following trypsin digestion, reversed-phase high pressure liquid chromatography (HPLC) analysis of the phosphorylated, affinity-purified insulin receptor preparation reveals phosphopeptide profiles similar to those of trypsin-digested receptors immunoprecipitated from 32P-labeled fibroblasts overexpressing the human insulin receptor. The major insulin-stimulated HPLC phosphopeptide peak from insulin receptors labeled in intact cells contains a hydrophilic phosphoserine-containing peptide which rapidly elutes from a C18 column. HPLC and two-dimensional separation indicate that the same phosphopeptide is obtained when affinity-purified insulin receptors are phosphorylated by IRSK. The serine containing tryptic peptide within the cytoplasmic domain of the human insulin receptor predicted to elute most rapidly upon HPLC had the sequence SSHCQR corresponding to residues 1293-1298. A synthetic peptide containing this sequence is phosphorylated by the insulin receptor/IRSK preparation. After alkylation and trypsin digestion, the synthetic phosphopeptide comigrates with the alkylated, tryptic phosphopeptide derived from insulin receptor phosphorylated in vitro by IRSK. We propose that serine 1293 or 1294 of the human insulin receptor is a major site(s) phosphorylated on the insulin receptor in intact cells and is phosphorylated by IRSK. Furthermore, insulin added directly to affinity-purified insulin receptor/IRSK preparations stimulates the phosphorylation of synthetic peptides corresponding to this receptor phosphorylation site and another containing threonine 1336. Kemptide phosphorylation is not stimulated by insulin under these conditions. No phosphorylation of peptide substrates for Ca2+/calmodulin-dependent protein kinase, protein kinase C, casein kinase II, or cGMP-dependent protein kinase by IRSK is detected. These data indicate that IRSK exhibits specificity for the insulin receptor and may be activated by the insulin receptor tyrosine kinase in an insulin-dependent manner.

Amino Acids↗

PubMed2025

Genome-Wide Analysis of DtxR and HrrA Regulons Reveals Novel Targets and a High Level of Interconnectivity Between Iron and Heme Regulatory Networks in Corynebacterium glutamicum.

Iron is vital for most organisms, serving as a cofactor in enzymes, regulatory proteins, and respiratory cytochromes. In Corynebacterium glutamicum , iron and heme homeostasis are tightly interconnected and controlled by the global regulators DtxR and HrrA. While DtxR senses intracellular Fe2+, HrrSA is activated by heme. This study provides the first genome-wide analysis of DtxR and HrrA binding dynamics under varying iron and heme conditions using chromatin affinity purification and sequencing (ChAP-Seq). We revealed 25 novel DtxR targets and 210 previously unrecognized HrrA targets. Among these, metH, encoding homocysteine methyltransferase, and xerC, encoding a tyrosine recombinase, were bound by DtxR exclusively under heme conditions, underscoring condition-dependent variation. Activation of metH by DtxR links iron metabolism to methionine synthesis, potentially relevant for the mitigation of oxidative stress. Beyond novel targets, 16 shared targets between DtxR and HrrA, some with overlapping operator sequences, highlight their interconnected regulons. Strikingly, we demonstrate the significance of weak ChAP-Seq peaks that are often disregarded in global approaches, but feature an impact of the regulator on differential gene expression. These findings emphasize the importance of genome-wide profiling under different conditions to uncover novel targets and shed light on the complexity and dynamic nature of bacterial regulatory networks.

Corynebacterium glutamicum↗

PubMed1993

Phosphorylcholine-binding proteins from the seminal fluids of different species share antigenic determinants with the major proteins of bovine seminal plasma.

The major proteins of bovine seminal plasma, BSP-A1, BSP-A2, BSP-A3, and BSP-30kDa (collectively named BSP proteins) bind to phospholipids containing the phosphorylcholine moiety. An affinity purification method using a p-aminophenyl phosphorylcholine-Agarose (PPC-Agarose) affinity matrix was developed for their purification. In this study, we investigated the distribution of BSP-like analogues in seminal fluid of the human, porcine, hamster, mouse, and rat using this affinity matrix. Alcohol precipitates of the seminal plasma/seminal vesicle secretions (SP/SVS) were further delipidated using isopropyl ether:n-butanol (60:40). The protein preparations obtained were solubilized in a minimal volume of buffer A (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.02% NaN3), dialyzed against the same buffer, and applied to a PPC-Agarose column connected to a FPLC system. The unbound material was washed out and the adsorbed proteins eluted with buffer A containing 10 mM phosphorylcholine (PrC) and 10 M urea. The fractions were separated by SDS-PAGE, stained or transferred onto a nitrocellulose membrane, and probed with rabbit polyclonal anti-BSP antibodies. Anti-BSP cross-reacting proteins were detected in the seminal fluids of all the species investigated. Moreover, many of these proteins bound to the affinity matrix. The BSP proteins and their immunoreacting analogues appear to be ubiquitous in mammals and may possibly be involved in a common function such as in the modification of the lipid content of the sperm plasma membrane.

Animals↗

PubMed1990

A dual-affinity gene fusion system to express small recombinant proteins in a soluble form: expression and characterization of protein A deletion mutants.

A novel gene fusion system to express and purify small recombinant proteins in Escherichia coli has been constructed. The concept allows for affinity purification of soluble gene products by sequential albumin- and Zn2(+)-affinity chromatography. The dual-affinity system is well suited for expression of unstable proteins as only full-length protein is obtained after purification and proteins gain proteolytic stability in the fusion protein. Here we show that the dual-affinity approach can be used for the expression of various unstable derivatives of a single IgG-binding domain based on staphylococcal protein A. Analysis of the proteolytic stabilities and the IgG-binding properties of the different mutant proteins suggest that the model for the structure of an IgG-binding domain must be re-evaluated.

Base Sequence↗

PubMed2000

Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct.

Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the C-terminal (scFv-His6) or N-terminal (His6-scFv) of the coding sequence. Solid-phase radioimmunoassay for scFv-His6 showed only 20-25% binding whereas both His6-scFv and scFv (no His-tag) showed 60-65% binding. Surface plasmon resonance studies by BIAcore revealed the binding affinity constant (KA) for His6-scFv and scFv as 1.19 x 10(6) M(-1) and 3.27 x 10(6) M(-1), respectively. No K(A) value could be calculated for scFv-His6 due to poor binding kinetics (kon and koff). Comparative homology modeling for scFv and scFv-His6 showed that the C-terminal position of the His-tag partially covered the antigen-binding site of the protein. The study demonstrates that the C-terminal position of His-tag on the CC49 scFv adversely affects the binding properties of the construct. The results emphasize the importance of functional characterization of recombinant proteins expressed with purification tags.

Animals↗

PubMed2002

Development and characterization of Ni-NTA-bearing microspheres.

BACKGROUND: For ease of purification, proteins are often expressed with a short affinity sequence of five or six adjacent histidine residues (His-tag). This His-tag binds to the metal of metal chelator complexes such as Ni(2+)-nitrilotriacetic acid (Ni-NTA) or -iminodiacetic acid (Ni-IDA). Chromatography resins bearing covalently attached metal chelator complexes are used widely for the easy affinity purification of His-tagged proteins or peptides. Because Ni-NTA microspheres were not commercially available at the beginning of our studies, we prepared and characterized such microspheres to immobilize His-tagged proteins and study their interactions. Our microspheres are of three types: (a) metal chelator complexes bound covalently to polystyrene microspheres, (b) metal chelator complexes bound covalently to silica microspheres, and (c) lipid-linked metal chelator complexes adsorbed to silica microspheres forming self-assembled bilayer membranes where the metal chelators have lateral mobility. METHODS: The microspheres bearing covalently attached Ni-chelator were synthesized by reacting a primary amine-bearing Ni-NTA ligand with carboxy-functionalized microspheres and then loading with Ni(2+). Microspheres with laterally mobile metal chelator were made by incubating glass microspheres with liposomes containing phosphatidylcholine (PC) and the metal chelating lipid 1,2-dioleoyl-sn-glycero-3-[(N (5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]. Binding of a His-tagged enhanced green fluorescent protein (EGFP) was used to characterize these microspheres by flow cytometry for their specificity, sensitivity, capacity and stability. RESULTS: While all micospheres specifically bind His-tagged proteins, the conditions to achieve this are different for the polystyrene- and silica-based spheres. All three types of microspheres bind His-EGFP with saturation occurring at 30-50 nM and an apparent avidity (concentration of half-maximal binding) of approximately 1 to 2 x 10(-8) M at pH 7.4. Binding of His-EGFP is inhibited by imidazole or ethylene-diaminetetraacetic acid (EDTA). Polystyrene Ni-NTA microspheres showed significant nonspecific binding as measured by binding in the presence of imidazole or EDTA or by binding of fluorescent proteins lacking a His-tag. This nonspecific binding of proteins to and aggregation of polystyrene spheres could only be prevented by the inclusion of low concentrations of Tween 20, but not by including bovine serum albumin (BSA), polyethylene glycols, or polyvinylpyrrolidones as blocking agents. In contrast, silica-based microspheres with covalently attached Ni-NTA or silica microspheres bearing adsorbed bilayers that contain Ni-NTA-lipid showed little nonspecific binding in the presence of BSA. Our results on the stability of immobilization indicate that washing destabilizes the binding of His-tagged proteins to Ni-NTA microspheres. This binding consists of two interactions of different affinities. We also demonstrate that limited multiplexed analysis with differently sized silica microspheres bearing the Ni-NTA-lipid is feasible. CONCLUSIONS: The microspheres described are well suited to selectively immobilize His-tagged proteins to analyze their interactions by flow cytometry. The affinity and kinetic stability of the interaction of His-tagged proteins with Ni-NTA are insufficient to use Ni-NTA microspheres in multiplexed analysis formats where different His-tagged proteins are bound to distinct microspheres. Improvements towards this end (improved chelators and/or improved affinity tags) are critical for extending the use of this method. We are currently working on novel chelators to strengthen the stability of immobilization of His-tagged proteins to surfaces. Such improvements would greatly enhance the analysis of interactions of immobilized His-tagged proteins and could make the development of microsphere-based arrays with His-tagged protein/antibody possible.

Binding Sites↗

PubMed1992

Autoantigenic epitopes of the B polypeptide of Sm small nuclear RNP particles. Identification of regions accessible only within the U1 small nuclear RNP.

OBJECTIVE: To analyze the autoantigenic epitopes of the Sm B polypeptide of the U1 small nuclear RNP (snRNP) using complementary DNA (cDNA) clones. METHODS: Expression of Sm B fusion proteins in lambda phage vectors, immunoblots, immunoprecipitations, and affinity purification of antibodies. RESULTS: Immunoblots using antibodies affinity-purified from B fusion proteins demonstrated that there were cross-reactive epitopes between the B'/B and A polypeptides of the U1 snRNP. Immunoprecipitation assays suggested that there were at least 3 different autoantigenic epitopes on the B polypeptide that could be classified into 2 general groups based upon autoantibody reactivity. The first group of autoantibodies, which bound 2 separate autoantigenic epitopes (BU1-1, BU1-2), participated in immunoprecipitation of the U1 snRNP alone. The second group, which bound the third type of autoantigenic epitope (BSm-1), immunoprecipitated all the abundant Sm snRNPs. CONCLUSION: There is at least 1 region on the B proteins that is accessible to antibodies only within the structure of the U1 snRNP, as well as a region that is accessible on all Sm snRNPs. These data support the notion that the native U1 RNP, perhaps containing B proteins in a different conformation than those found on other Sm snRNPs, may drive the humoral immune response in systemic lupus erythematosus.

Amino Acid Sequence↗

PubMed1992

Design of novel affinity adsorbents for the purification of trypsin-like proteases.

A number of ligands for the selective purification by affinity chromatography of the trypsin-like protease, porcine pancreatic kallikrein, were designed de novo by computer-aided molecular design. The ligands were designed to mimic the side-chains of a number of arginyl dipeptides and included a benzamidine moiety substituted on a triazine ring. The ligands displayed inhibitory activities against pancreatic kallikrein which mirrored the specificity constants of the dipeptides they were designed to mimic. The ligand with the highest affinity for the enzyme, an analogue of a Phe-Arg dipeptide, when immobilized to Sepharose CL-4B via a hexamethylene spacer arm, purified pancreatic kallikrein 110-fold in one step from a crude pancreatic acetone extract.

Benzamidines↗

PubMed2005

Dual-affinity avidin molecules.

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.

Animals↗

PubMed2006

Molecular organization of peroxisomal enzymes: protein-protein interactions in the membrane and in the matrix.

The beta-oxidation of fatty acids in peroxisomes produces hydrogen peroxide (H2O2), a toxic metabolite, as a bi-product. Fatty acids beta-oxidation activity is deficient in X-linked adrenoleukodystrophy (X-ALD) because of mutation in ALD-gene resulting in loss of very long chain acyl-CoA synthetase (VLCS) activity. It is also affected in disease with catalase negative peroxisomes as a result of inactivation by H2O2. Therefore, the following studies were undertaken to delineate the molecular interactions between both the ALD-gene product (adrenoleukodystrophy protein, ALDP) and VLCS as well as H2O2 degrading enzyme catalase and proteins of peroxisomal beta-oxidation. Studies using a yeast two hybrid system and surface plasmon resonance techniques indicate that ALDP, a peroxisomal membrane protein, physically interacts with VLCS. Loss of these interactions in X-ALD cells may result in a deficiency in VLCS activity. The yeast two-hybrid system studies also indicated that catalase physically interacts with L-bifunctional enzyme (L-BFE). Interactions between catalase and L-BFE were further supported by affinity purification, using a catalase-linked resin. The affinity bound 74-kDa protein, was identified as L-BFE by Western blot with specific antibodies and by proteomic analysis. Additional support for their interaction comes from immunoprecipitation of L-BFE with antibodies against catalase as a catalase- L-BFE complex. siRNA for L-BFE decreased the specific activity and protein levels of catalase without changing its subcellular distribution. These observations indicate that L-BFE might help in oligomerization and possibly in the localization of catalase at the site of H2O2 production in the peroxisomal beta-oxidation pathway.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1996

An inducible expression system of histidine-tagged proteins in Streptomyces lividans for one-step purification by Ni2+ affinity chromatography.

An expression and purification cassette containing the aminoglycoside phosphotransferase gene (aph) as selective marker has been constructed in the Escherichia coli vector pULHis2. DNA fragments inserted in the cassette can be easily subcloned in pIJ699 to give vectors for overexpression of genes in Streptomyces and purification of proteins by a one-step procedure. The expression system uses the thiostrepton-inducible promoter tipA for expression and a six histidine coding nucleotide sequence that is fused in frame to the foreign gene inserted in the polylinker. The pULHis2-derived expression vector has been used satisfactorily to express and to purify the P7 and P8 proteins of Nocardia lactamdurans which carry out the methoxylation of cephalosporin C to 7-methoxycephalosporin C.

Bacterial Proteins↗

PubMed1993

Efficient parvovirus B19 DNA purification and molecular cloning.

A simple and efficient method of purification and molecular cloning of Parvovirus B19 DNA directly from small quantities of viremic sera was developed. Purified virions were lysed in annealing conditions, then viral DNA purification in double strand (ds) DNA form was achieved using an affinity DNA binding matrix. Affinity purification yielded a consistently high recovery of viral DNA. Using affinity purified ds viral DNA, we efficiently and stably cloned the complete coding internal unique sequence of B19 DNA. In our cloning strategy AatII and BamHI restriction endonuclease sites were exploited. This permitted cleavage of the 5.0 kbp AatII fragment in two AatII-BamHI fragments which could be efficiently cloned in a directional way in pUC18 plasmid vector. The availability of the two cloned AatII-BamHI fragments thus allowed the construction of a full length clone in a single ligation reaction.

Cloning, Molecular↗

PubMed2001

Study on the scale-up of human IgG3 purification using protein A affinity chromatography.

The purification of human IgG3 subclass out of IgG (Immunoglobulin-G) was studied using protein A-Sepharose affinity chromatography. The effect of operational parameters such as flow rate, ionic strength, pH and size of sample was investigated, and the process was scaled-up 10-fold. The use of 0.5 m NaCl in the loading buffer had a dramatic effect in the purity of IgG3 recovered in the flowthrough fraction (values in the order of 97% were consistently obtained). This was attributed to a more effective binding of IgG subclasses 1, 2 and 4 to protein A (well known classical mechanism based in Fc fragment) and in some extent to a decrease in the binding of subclass 3 to protein A by the alternative mechanism based in the Fab fragment. The increase in residence time also increased in a relevant way the purity of IgG3. This is attributed to an increased effectiveness of the mechanisms mentioned above. The recovery yields in the IgG3 rich fraction were in the range 21-32% and are possibly a consequence of binding to protein A by the alternative mechanism and also due to deactivation during processing.

Chromatography, Affinity↗

PubMed1977

Synthesis of a disulfide affinity adsorbent for purification of estrogen receptor.

Synthesis of an estrogen affinity adsorbent containing a disulfide linkage between the steroid and stationary matrix permitted facile purification of high affinity estrogen binding proteins. Following affinity chromatography of either antibody directed against estrone 17-carboxymethyloxime - bovine serum albumin or immature calf uterine cytoplasmic estrogen receptor proteins, the specifically bound mercaptoethanol. Crude antibody and uterine cytosol was 10(-3) to 10(-2)M cystamine (S-S) to block SH-containing proteins, in order to protect the adsorbent against protein-mediated S-S in equilibrium SH exchange. Cystamine was found to markedly stabilize crude cytosol receptor protein by 200-300% compared with preparations obtained under ordinary conditions. Disulfide affinity adsorbents are versatile in that they can be used either under conventional conditions of specific protein recovery, or with 2-mercaptoethanol which removes the ligand and bound protein from the stationary matrix quantitatively.

Animals↗

PubMed1998

Photocleavable aminotag phosphoramidites for 5'-termini DNA/RNA labeling.

We report the design and evaluation of two non-nucleosidic photocleavable aminotag phosphor-amidites. These reagents introduce a photocleavable amino group on the 5'-terminal phosphate of synthetic oligonucleotides. The 5' photocleavable amino group enables introduction of a variety of amine-reactive markers onto synthetic oligonucleotides as well as immobilization on activated solid supports. The photocleavable bond on the 5'-phosphate can then be selectively cleaved by near-UV illumination, thereby enabling release of the marker or detachment of the oligonucleotide from a solid support. The preparation of photocleavable conjugates with biotin, digoxigenin and tetramethylrhodamine are described. In the case of biotin, a conjugate was used in a high sensitivity hybridization assay as a photocleavable probe for a complementary sequence immobilized on beads. It is also demonstrated that the 5'-PC-amino group can be used as an affinity tag for photocleavage-mediated affinity purification and phosphorylation of synthetic oligonucleotides in conjunction with activated supports. Such 5'-PC-amino labeled oligonucleotides should be useful in a variety of applications in molecular biology including multiple non-radioactive probing of DNA/RNA blots, affinity isolation and purification of nucleic acids binding proteins, diagnostic assays requiring release of the probe-target complex or specific marker, cassette mutagenesis and PCR. They will also enable the spatially-addressable photorelease of the probe-target complexes or marker molecules for diagnostic purposes.

Amides↗

PubMed1985

A comparative study of choline acetyltransferase from normal and Alzheimer brain.

A comparative study was made of the enzyme choline acetyltransferase (ChAT) from normal and Alzheimer (senile dementia of the Alzheimer type) brain. The number of molecular weight and charge forms of the enzyme were determined in the caudate region of both brains. Efficient purification of active ChAT was achieved using immuno-affinity purification. It was shown that the purified enzyme was identical in both cases, exhibiting a single charge (apparent pI approximately 8.2) and a single molecular weight (mol. wt. = 68,000). The idea of a selective loss of one particular isoform to explain the reduced levels of ChAT observed in Alzheimer's disease can be ruled out.

Alzheimer Disease↗

PubMed2006

Single-step purification of recombinant anthrax lethal factor from periplasm of Escherichia coli.

Lethal toxin (LT) that composed by protective antigen and lethal factor (LF) is the major virulence factor of Bacillus anthracis. The treatments of LT in animals could reproduce most manifestations of B. anthracis infections that greatly improves our knowledge in LT-mediated pathogenesis and facilitates anthrax-related researches without having to directly contact the hazardous bacterium B. anthracis. The recombinant protein of LF (rLF), however, still lacks a simple purification method. Herein, we developed single-step nickel affinity purification of rLF with yield up to 3mg/l. By fusion to the leader sequence of outer membrane protein OmpA, rLF could easily be purified from the periplasm of Escherichia coli. To investigate whether the rLT is functional in our system, both wild type rLF and the catalytic mutant rLF that contains a single amino acid substitution at zinc-binding site (LF(E687A)), were subjected to macrophage cytotoxicity analysis. Our data showed that the rLT is fully functional, while the LF(E687A) fail to induce cell death of tested macrophage cells. These findings suggested that the purification protocol herein is a user-friendly method that allows researchers to obtain the functional rLF by single-step purification.

Animals↗

PubMed2006

Method to site-specifically identify and quantitate carbonyl end products of protein oxidation using oxidation-dependent element coded affinity tags (O-ECAT) and nanoliquid chromatography Fourier transform mass spectrometry.

Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. This presents a significant advancement over current methods, combining strengths of current methods and adding the abilities to multiplex, quantitate, and probe more modified amino acids. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1,4,7,10-tetraazacyclododecane-N,N',N' ',N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. O-ECAT can be loaded with a variety of metals, which yields the ability to generate mass pairs and multiplex multiple samples. The O-ECAT moiety also serves as a handle for identification, quantitation, and affinity purification. After proteolysis, the AOD-tagged peptides are affinity purified and analyzed by nanoLC-FTICR-MS (nanoliquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry), which provides high specificity in extracting coeluting AOD mass pairs with a unique mass difference and allows relative quantitation based on isotopic ratios. Using this methodology, we have quantified and mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation both at the protein and amino acid levels. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic, and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure, validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities toward oxidation, independent of amino acid residue. This novel methodology not only has the ability to identify and quantitate oxidized proteins but also yields site-specific quantitation on a variety of individual amino acids. We expect to extend this methodology to study disease-related oxidation.

Affinity Labels↗