Cysteine-containing Peptides Research Articles

The role of copper in cysteine oxidation: study of intra- and inter-molecular reactions in mass spectrometry

Cysteine-containing peptide oxidation was studied both by using an inert platinum electrode and a sacrificial electrode (copper or zinc) generating metallic ions in electrospray ionization mass spectrometry (ESI-MS). Using peptides containing one, two and three cysteines, we have compared the different chemical and electrochemical oxidation pathways of cysteine (RS(-II)H) to cystine (RS(-I)S(-I)R) and to sulfenic, sulfinic and sulfonic acid (RS(0)OH, RS(II)O(2)H and RS(IV)O(3)H, respectively). In the absence of copper ions, intra-molecular reactions were the most abundant, whereas inter-molecular reactions were found to be enhanced by the presence of copper ions. These cations favor the formation of 2 : 1 (peptide : copper) complexes compared to 1 : 1 complexes, thus enhancing the formation of inter-molecular bridges. This study highlights the importance of the position of cysteine inside a peptide during disulfide bridge formation.

Identification of novel alternative splice isoforms of circulating proteins in a mouse model of human pancreatic cancer.

To assess the potential of tumor-associated, alternatively spliced gene products as a source of biomarkers in biological fluids, we have analyzed a large data set of mass spectra derived from the plasma proteome of a mouse model of human pancreatic ductal adenocarcinoma. MS/MS spectra were interrogated for novel splice isoforms using a nonredundant database containing an exhaustive three-frame translation of Ensembl transcripts and gene models from ECgene. This integrated analysis identified 420 distinct splice isoforms, of which 92 did not match any previously annotated mouse protein sequence. We chose seven of those novel variants for validation by reverse transcription-PCR. The results were concordant with the proteomic analysis. All seven novel peptides were successfully amplified in pancreas specimens from both wild-type and mutant mice. Isotopic labeling of cysteine-containing peptides from tumor-bearing mice and wild-type controls enabled relative quantification of the proteins. Differential expression between tumor-bearing and control mice was notable for peptides from novel variants of muscle pyruvate kinase, malate dehydrogenase 1, glyceraldehyde-3-phosphate dehydrogenase, proteoglycan 4, minichromosome maintenance, complex component 9, high mobility group box 2, and hepatocyte growth factor activator. Our results show that, in a mouse model for human pancreatic cancer, novel and differentially expressed alternative splice isoforms are detectable in plasma and may be a source of candidate biomarkers.

Assignment of Disulfide-Linked Peptides Using Automatic a1 Ion Recognition

We present a novel approach for the assignment of peptides containing disulfide linkages. Dimethyl labeling is introduced to generate labeled peptides which exhibit enhanced a1 ion signals during MS/MS fragmentation. For disulfide-linked peptides, multiple a1 ions can be observed due to multiple N-termini. This distinct feature allows sieving out the disulfide-linked peptides; meanwhile, the N-terminal amino acids can be identified. With such information, the number of possible peptide combinations involved in a disulfide bond dramatically narrows down. Furthermore, we developed a computational algorithm to perform target a1 ion screening followed by molecular weight matching of cysteine-containing peptides with specific amino acids at the N-termini. Once the protein sequence and the peak list from a LC-MS/MS survey scan of labeled peptides are imported, the identities of disulfide-linked peptides can be readily obtained. The presented approach is simple and straightforward, offering a valuable tool for protein structural characterization.

Prospective Type 1 and Type 2 Disulfides of Keap1 Protein

Experiments were carried out to detect cysteine residues on human Keap1 protein that may be sensors of oxidative stress that gives rise to changes in the GSH/GSSG redox couple. Human Keap1 protein, at a final concentration of 6 microM, was incubated for two hours in aqueous buffer containing 0.010 M GSH, pH 8, in an argon atmosphere. Subsequently, excess iodoacetamide and trypsin were added to generate a peptide map effected by LCMS analysis. Peptides containing all 27 carboxamidomethylated cysteines were identified. Replacement of GSH by 0.010 M GSSG yielded a map in which 13 of the original carboxamidomethylated peptides were unperturbed, while other caboxamidomethylated cysteine-containing peptides were undetected, and a number of new cysteine-containing peptide peaks were observed. By mass analysis, and in some cases, by isolation, reduction, carboxamidomethylation, and reanalysis, these were identified as S-glutathionylated (Type 1) or Cys-Cys (Type 2) disulfides. Such peptides derived from the N-terminal, dimerization, central linker, Kelch repeat and C-terminal domains of Keap1. Experiments were carried out in which Keap1 was incubated similarly but in the presence of various GSH/GSSG ratios between 100 and 1 ([GSH + GSSG] = 0.010 M), with subsequent caraboxamidomethylation and trypsinolysis to determine differences in sensitivities of the different cysteines to the type 1 and type 2 modifications. Cysteines most sensitive to S-glutathionylation include Cys77, Cys297, Cys319, Cys368, and Cys434, while cysteine disulfides most readily formed are Cys23-Cys38 and Cys257-Cys297. The most reducing conditions at which these modifications are at GSH/GSSG = 10, which computes to an oxidation potential of E h = -268.5 mV, a physiologically relevant value. Under somewhat more oxidizing, but still physiologically relevant, conditions, GSH/GSSG = 1 ( E h = -231.1 mV), a Cys319-Cys319 disulfide is detected far from the dimerization domain of the Keap1 homodimer. The potential impact on protein structure of the glutathionylation of Cys434 and Cys368, the two modified residues in the Kelch repeat domain, was analyzed by docking and energy minimizations of glutathione residues attached to the Kelch repeat domain, whose coordinates are known. The energy minimizations indicated marked alterations in structure with a substantial constriction of Neh2 binding domain of the Keap1 Kelch repeat domain. This alteration appears to be enforced by an extended hydrogen-bonding network between residues on the glutathione moiety attached to Cys434 and amino acid side chains that have been shown to be essential for repression of Nrf2 by Keap1. The modifications of Keap1 detected in the present study are discussed in the context of previous work of others who have examined the sensitivity of cysteines on Keap1 to electrophile assault.

Mixed Mode Thiol−Acrylate Photopolymerizations for the Synthesis of PEG−Peptide Hydrogels

Thiol−acrylate mixed mode photopolymerizations provide an easy, robust, cost-efficient, and cytocompatible reaction scheme for the incorporation of cysteine-containing peptide sequences into PEG hydrogels. With an acrylate:cysteine ratio of 4:1, ∼95% of the peptide is incorporated after 10 min of reaction with ∼5 mW/cm2 of 365 nm light. Specifically, thiol monomers, with thiol-presenting peptide groups in the form of CGGGGG, CRGGGG, CWGGGG, CDGGGG, CSGGGG, and CGGGGC, copolymerized with PEG4600 diacrylate were characterized. The chain transfer constant of acrylates to thiols was determined to range from 1.5 to 2, depending on the amino acid sequence. Complete conversion of the functional groups occurred after ∼40 s for all systems, excluding the tryptophan-containing peptide sequence which had a retarded polymerization, taking ∼60 s. The cross-linking densities of the final gels ranged from 1.5 × 10−2 to 2.0 × 10−2 mol/L when 10 mM of peptide was reacted with 40 mM of acrylate groups. Efficient incorporat...

Formation of Some Cysteine-Containing Peptide Monolayers on Au Electrodes and Their Applications for Metal Ion Sensing and Electrocatalytic Reactions

The use of biological molecules that already exist in nature offers advantages over using artificially made molecules in developing biosensors or electrocatalysts. Since those molecules have already been best adopted by nature to carry out specific functions, a lot of practical benefit could be acquired by utilizing or mimicking them rather than synthesizing complex molecules. In this regard, amino acids and peptides are favorite systems in metal ion sensing in a sense that they can act as very effective and often specific ligands for a variety of metal ions. While amine and carboxyl groups of amino acids participate in complexation, an amide nitrogen atom is also involved in complexation in the case of peptides. This often forms a chelate and a peptide-metal ion binding becomes significantly stronger as a result. Detailed accounts have been treated by Sigel and Martin and other authors. Despite the fact that amino acids and peptides readily form complexes with metal ions, electrochemical study of those complexes is scarce. It was not until recently when a first report by Yang et al. appeared for the electrochemical metal ion detection. They synthesized a tripeptide system, GlyGly-His, on top of an alkanethiol self-assembled Au electrode to detect copper ions. Since then, many papers regarding various electrochemical aspects of a peptide-metal binding have been published. Here we present our preliminary results of metal ion sensing and electrocatalytic reactions using cysteine-containing simple peptide monolayers constructed on a Au surface. Cysteine was chemisorbed on the Au surface via Au-S bonding and other amino acids were formed by the usual peptide synthesis method. This way two types of peptide monolayers were prepared: With a Au/ GSH-His system (GSH: glutathione, γ-L-glutamyl-L-cysteinyl-glycine), copper ions of nano molar concentration were detected by accumulating Cu on the peptide monolayer. With a Au/Cys-Cys-Cys/M(M = Cu, Fe) system, electrocatalytic oxidation of ascorbic acid (AA) and reduction of hydrogen peroxide were performed. Figure 1 shows cyclic voltammograms of a Au/GSH-His/ Cu system after immersing a Au/GSH-His electrode in a Cu-containing solution and transferring into the Cu-free electrolyte. Fairly stable voltammograms were obtained. Without copper ion, no faradaic current was observed. Redox peaks at +0.33 and +0.010 V are assigned to the 1-e− redox reactions of a Cu/Cu couple as indicated in other studies. With only GSH layer on Au, current slightly decreased upon multi potential cycling. This means copper ions form a more stable complex with histidine in which imidazole moiety of histidine is involved in complexation. Full copper ion accumulation was complete after ca. 10 min. Treating the Au/GSH-His/Cu electrode with EDTA solution, copper ions were completely removed from the modified

Aromatic Amino Acids Providing Characteristic Motifs in the Raman and SERS Spectroscopy of Peptides

Raman and surface-enhanced Raman spectroscopies (SERS) are potentially important tools in the characterization of biomolecules such as proteins and DNA. In this work, SERS spectra of three cysteine-containing aromatic peptides: tryptophan-cysteine, tyrosine-cysteine, and phenylalanine-cysteine, bound to Au nanoshell substrates, were obtained, and compared to their respective normal Raman spectra. While the linewidths of the SERS peaks are significantly broadened (up to 70%), no significant spectral shifts (<6 cm (-1)) of the major Stokes modes were observed between the two modalities. We show that the Raman and SERS spectra of penetratin, a cell-penetrating peptide oligomer, can be comprised quite reliably from the spectra of its constituent aromatic amino acids except in the backbone regions where the spectral intensities are critically dependent on the length and conformations of the probed molecules. From this study we conclude that, together with protein backbone groups, aromatic amino acid residues provide the overwhelmingly dominant features in the Raman and SERS spectra of peptides and proteins when present. It follows that the Raman modes of these three small constructed peptides may likely apply to the assignment of Raman and SERS features in the spectra of other peptides and proteins.

In-Gel S-Pyridylethylation of Gel-Resolved Proteins: Whole Gel Method

INTRODUCTIONThis protocol describes a method for performing reduction and S-alkylation of Coomassie-blue-stained proteins within an intact gel. Reduction is performed with dithiothreitol, and alkylation with 4-vinylpyridine. (Treatment of free cysteines with 4-vinylpyridine yields the S-β-(4-pyridylethyl) cysteinyl derivative.) S-β-(4-pyridylethyl) cysteine-containing peptides can be readily identified during RP-HPLC by their characteristic absorbance at 254 nm and during electrospray ionization tandem mass spectrometry by the appearance of a characteristic pyridylethyl fragment ion of 10(6) Da. The position of cysteine residues in a polypeptide sequence can be determined either by mass spectrometry or as phenylthiohydantoin S-β-(4-pyridylethyl) cysteine during Edman degradation.

In-Gel S-Pyridylethylation of Gel-Resolved Proteins: Individual Spot Method

INTRODUCTIONThis protocol describes a method for performing reduction and S-alkylation of Coomassie-blue-stained proteins within individually excised, stained protein spots or bands. Reduction is performed with dithiothreitol, and alkylation with 4-vinylpyridine. (Treatment of free cysteines with 4-vinylpyridine yields the S-β-(4-pyridylethyl) cysteinyl derivative.) S-β-(4-pyridylethyl) cysteine-containing peptides can be readily identified during RP-HPLC by their characteristic absorbance at 254 nm and during electrospray ionization tandem mass spectrometry by the appearance of a characteristic pyridylethyl fragment ion of 10(6) Da. The position of cysteine residues in a polypeptide sequence can be determined either by mass spectrometry or as phenylthiohydantoin S-β-(4-pyridylethyl) cysteine during Edman degradation.

Systematic comparison of two novel, thiol-reactive prosthetic groups for 18F labeling of peptides and proteins with the acylation agent succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

A systematic comparison of 4-[18F]fluorobenzaldehyde-O-(2-{2-[2-(pyrrol-2,5-dione-1-yl)ethoxy]-ethoxy}-ethyl)oxime ([18F]FBOM) and 4-[18F]fluorobenzaldehyde-O-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexyl]oxime ([18F]FBAM) as prosthetic groups for the mild and efficient 18F labeling of cysteine-containing peptides and proteins with the amine-group reactive acylation agent, succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), is described. All three prosthetic groups were prepared in a remotely controlled synthesis module. Synthesis of [18F]FBOM and [18F]FBAM was accomplished via oxime formation through reaction of appropriate aminooxy-functionalized labeling precursors with 4-[18F]fluorobenzaldehyde. The obtained radiochemical yields were 19% ([18F]FBOM) and 29% ([18F]FBAM), respectively. Radiolabeling involving [18F]FBAM and [18F]FBOM was exemplified by the reaction with cysteine-containing tripeptide glutathione (GSH), a cysteine-containing dimeric neurotensin derivative, and human native low-density lipoprotein (nLDL) as model compounds. Radiolabeling with the acylation agent [18F]SFB was carried out using a dimeric neurotensin derivative and nLDL. Both thiol-group reactive prosthetic groups show significantly better labeling efficiencies for the peptides in comparison with the acylation agent [18F]SFB. The obtained results demonstrate that [18F]FBOM is especially suited for the labeling of hydrophilic cysteine-containing peptides, whereas [18F]FBAM shows superior labeling performance for higher molecular weight compounds as exemplified for nLDL apolipoprotein constituents. However, the acylation agent [18F]SFB is the preferred prosthetic group for labeling nLDL under physiological conditions.

Quantitative Analysis of Snake Venoms Using Soluble Polymer-based Isotope Labeling

We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms. The SoPIL reagent selectively captures and isolates cysteine-containing peptides, and the subsequent tagged peptides are released and analyzed using nanoflow liquid chromatography-tandem mass spectrometry. The SoPIL strategy was used to quantify venom proteins from two pairs of venomous snakes: Crotalus scutulatus scutulatus type A, C. scutulatus scutulatus type B, Crotalus oreganus helleri, and Bothrops colombiensis. The hemorrhagic, hemolytic, clotting ability, and fibrinogenolytic activities of crude venoms were measured and correlated with difference in protein abundance determined by the SoPIL analysis. The SoPIL approach could provide an efficient and widely applicable tool for quantitative proteomics.

The origin and control of ex vivo oxidative peptide modifications prior to mass spectrometry analysis

The origin and control of ex vivo sample handling related oxidative modifications of methionine-, S-alkyl cysteine-, and tryptophan-containing peptides obtained from typical "in-solution" or "in-gel" proteolytic digestion strategies, have been examined by capillary HPLC and MS/MS. The origin of increased oxidation levels were found to be predominantly associated with the extensive ex vivo sample handling steps required for gel electrophoresis and/or in-gel proteolytic digestion of proteins prior to analysis by MS. Conditions for deliberately controlling the oxidation state (both oxidation and reduction) of these peptides, as well as for those containing cysteine, have been evaluated using a series of model synthetic peptides and standard tryptic protein digests. Essentially complete oxidation of methionine- and S-alkyl cysteine-containing peptides was achieved by reaction with 30% hydrogen peroxide/5% acetic acid at room temperature for 30 min. Under these conditions, cysteine was also converted to cysteic acid, while only limited oxidation of tryptophan to oxindolylalanine, and methionine and S-alkyl cysteine sulfoxides to their respective sulfones, were observed. Efficient reduction of methionine- and S-alkyl cysteine sulfoxide-containing peptides was achieved by reaction in 1 M dimethylsulfide/10 M hydrochloric acid at room temperature for 10 and 45 min, respectively. None of the reduction conditions evaluated were found to result in the reduction of oxindolylalanine, cysteic acid, or methionine sulfone.

MALDI In‐Source Photooxidation Reactions for Online Peptide Tagging

A TiO2 photoelectrode array has been designed to achieve efficient photoelectrochemical redox reactions with electron donors or acceptors during the laser desorption ionization process. By adding hydroquinone to a peptide mixture, the photoelectrochemically generated benzoquinone reacts with the cysteine residues to selectively form tagged cysteine-containing peptides, thus demonstrating that in-source oxidation reactions can be efficiently performed.

Remembering Bruce Merrifield

Although I am unable to contribute a scientific paper to this special issue, I cannot refrain from joining in the tribute that is rendered to this great scientist and personal friend. Creativity, persistence, modesty, kindness, courage, those are some of the words that come to mind with the souvenir of Bruce Merrifield. It certainly took a stroke of creative genius to think of using a terminal amino acid anchored to a solid support as starting point for the stepwise assembly of a polypeptide chain according to a predetermined sequence. To convert this brilliant idea into a dependable, fully automated machine, overcoming all the difficulties and obstacles that beset the enterprise, required an enormous amount of dogged persistence. Bruce had both. Success, when it came, changed nothing to Bruce's unassuming modesty. Even the Nobel Prize did not turn his head. It simply strengthened his commitment to basic, disinterested science and his feeling of social responsibility. It in no way altered his kindness and helpfulness, in spite of the many demands that were made on him. I can attest to this. When I wanted a difficult-to-synthesize, cysteine-containing peptide to test some crazy idea of mine, it took only a few weeks for Bruce to supply the material. Perhaps the most enduring souvenir left by Bruce is his unwavering fortitude in the face of the ordeal that plagued him during much of his life and finally got the better of his resistance. His gentle smile never left him. Neither did his devotion to his dear Libby, to whom our thoughts go today. Bruce Merrifield will be remembered with great affection and admiration by all his friends and colleagues.

Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.

Identification of the Protein Targets of the Reactive Metabolite of Teucrin A in Vivo in the Rat

Covalent modification of proteins is associated with the toxicity of many electrophiles, and the identification of relevant in vivo protein targets is a desirable but challenging goal. Here, we describe a strategy for the enrichment of adducted proteins utilizing single-chain fragment variable (ScFv) antibodies selected using phage-display technology. Teucrin A is a furan-containing diterpenoid found in the herb germander that is primarily responsible for the herb's hepatotoxicity in rodents and humans following metabolic activation by cytochrome P450 enzymes. Conjugates of the 1,4-enedial derivative of teucrin A, its presumed toxic metabolite, with lysine- and cysteine-containing peptides were synthesized and used to select ScFvs from a rodent phage-displayed library, which recognized the terpenoid moiety of the teucrin-derived adducts. Immunoaffinity isolation of adducted proteins from rat liver homogenates following administration of a toxic dose of teucrin A afforded a family of proteins that were identified by liquid chromatography/tandem mass spectrometry. Of the 46 proteins identified in this study, most were of mitochondrial and endoplasmic reticulum origin. Several cytosolic proteins were found, as well as four peroxisomal and two secreted proteins. Using Ingenuity Pathway Analysis software, two significant networks involving the target genes were identified that had major functions in gene expression, small molecule biochemistry, and cellular function and maintenance. These included proteins involved in lipid, amino acid, and drug metabolism. This study illustrates the utility of chemically synthesized biological conjugates of reactive intermediates and the potential of the phage display technology for the generation of affinity reagents for the isolation of adducted proteins.

Engineered Sarafotoxins as Tissue Inhibitor of Metalloproteinases-like Matrix Metalloproteinase Inhibitors

The sarafotoxins and endothelins are approximately 25-residue peptides that spontaneously fold into a defined tertiary structure with specific pairing of four cysteines into two disulfide bonds. Their structures show an interesting topological similarity to the core of the metalloproteinase interaction sites of the tissue inhibitors of metalloproteinases. Previous work indicates that sarafotoxins and endothelins can be engineered to eliminate or greatly reduce their vasopressive action and that their structural framework can withstand multiple sequence changes. When sarafotoxin 6b, which possesses modest matrix metalloproteinase inhibitory activity, was C-terminally truncated to remove its toxic vasopressive activity, the metalloproteinase inhibitory activity was essentially abolished. However, further changes, based on the sequences of peptides selected from libraries of sarafotoxin variants or suggested by analogy with tissue inhibitors of metalloproteinases, progressively enhanced the matrix metalloproteinase inhibitory activity. Peptide variants with multiple substitutions folded correctly and formed native disulfide bonds. Improvements in matrix metalloproteinase affinity have generated a peptide with micromolar K(i) values for matrix metalloproteinase-1 and -9 that are selective inhibitors of different metalloproteinases. Characterization of its solution structure indicates a close similarity to sarafotoxin but with a more extended C-terminal helix. The effects of N-acetylation and other changes, as well as docking studies, support the hypothesis that the engineered sarafotoxins bind to matrix metalloproteinases in a manner analogous to the tissue inhibitors of metalloproteinases.

Diagonal chromatographic selection of cysteinyl peptides modified with benzoquinones

The derivatization of cysteine-containing peptides with benzoquinone compounds is rapid, quantitative and specific in acidic media. The conversion of cysteines into hydrophobic benzoquinone-adducted residues in peptides is used here to alter the chromatographic properties of cysteinyl peptides during liquid chromatography separation. The benzoquinone derivatization is shown to allow the accurate selection of cysteine-containing peptides of bovine serum albumin tryptic digest by diagonal reversed-phase chromatography, which consists of one primary and a series of secondary identical liquid chromatographic separations, before and after a cysteinyl-targeted modification of the peptides by benzoquinone compounds.

FASTDXL: A Generalized Screen to Trap Disulfide-Stabilized Complexes for Use in Structural Studies

Structural studies of macromolecular complexes have produced extraordinary insights into a wide variety of biological processes. Unfortunately, as structural biologists pursue larger and more challenging assemblies, weakly stable and/or nonspecific interactions can become significant roadblocks to structure determination. We have developed a rapid and effective pool-based screen, termed FASTDXL (focused array screening technique for disulfide X-linking), to produce and identify disulfide-stabilized protein-nucleic acid assemblies. A significant strength of FASTDXL is that it can take advantage of prior structural knowledge about molecular interactions, but does not necessarily rely upon it. A detailed application of the approach to the difficult problem of trapping a bacterial primase-ssDNA complex is described, validating the method as a route toward obtaining diffracting crystals suitable for structure determination.

Isotope-Coded Affinity Tagging of Proteins

INTRODUCTIONQuantitative proteomics has traditionally been performed using 2D gel electrophoresis, where quantitation is accomplished by recreating differences in the staining patterns of proteins derived from two states of cell populations or tissues from a similar biological system. More recently, mass spectrometry (MS) methods based on stable isotope quantitation have been developed that show significant potential for differential expression proteomic studies. One such in vitro method, described in this protocol, involves the use of isotope-coded affinity tags (ICATs) with three functional moieties: a cysteine reactive moiety, a linker with either eight hydrogens (the light form of the reagent) or eight deuteriums (the heavy form of the reagent, having an isotope code or mass tag of 8 Da), and a biotin moiety (the affinity tag). Using this technique, the cysteine side chains in complex mixtures of proteins from two different states of a cell population (e.g., normal vs. disease) are reduced and alkylated using the light form of the reagent (d0-labeled tag) in one cell state and the heavy form of the reagent (d8-labeled tag) for proteins in the second cell state. The two mixtures (d0 and d8 labeled) are then combined and subjected to proteolytic digestion (typically, with trypsin and/or Lys-C). Generated cysteine-containing peptides are affinity-purified using an avidin column, resulting in a "simplified" mixture of peptides that contains ~10-fold fewer peptides than the original mixture. These peptides are analyzed by MS, and quantitation information based on the relative abundance of the d0 and d8 isotopes is obtained. The identification of proteins is obtained from the peptide molecular mass and MS/MS-derived amino acid sequence.