NORMAL GENOMIC

• Yamaguchi H, Miyazaki M, Maeda H
• Proteolysis approach without chemical modification for a simple and rapidanalysis of disulfide bonds using thermostable protease-immobilizedmicroreactors.
• Proteomics. 2010; 10: 2942-9
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• Disulfide bonds in proteins are important not only for the conformationalstability of the protein but also for the regulation ofoxidation-reduction in signal transduction. The conventional method forthe assignment of disulfide bond by chemical cleavage and/or proteolysisis a time-consuming multi-step procedure. In this study, we report asimple and rapid analysis of disulfide bond from protein digests that wereprepared by the thermostable protease-immobilized microreactors. Thefeasibility and performance of this approach were evaluated by digestinglysozyme and BSA at several temperatures. The proteins which stabilizetheir conformations by disulfide bonds were thermally denatured duringproteolysis and were efficiently digested by the immobilized protease butnot by free protease. The digests were directly analyzed by ESI-TOF MSwithout any purification or concentration step. All four disulfide bondson lysozyme and 10 of 17 on BSA were assigned from the digests by thetrypsin-immobilized microreactor at 50 degrees C. The procedure forproteolysis and the assignment were achieved within 2 h without anyreduction and alkylation procedure. From the present results, theproteolysis approach by the thermostable protease-immobilized microreactorprovides a strategy for the high-throughput analysis of disulfide bond inproteomics.

• Kim HI, Beauchamp JL
• Mapping disulfide bonds in insulin with the Route 66 Method: selectivecleavage of S-C bonds using alkali and alkaline earth metal enolatecomplexes.
• J Am Soc Mass Spectrom. 2009; 20: 157-66
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• Simple and fast identification of disulfide linkages in insulin isdemonstrated with a peptic digest using the Route 66 method. This isaccomplished by collisional activation of singly and doubly chargedcationic Na(+) and Ca(2+) complexes generated using electrosprayionization mass spectrometry (ESI-MS). Collisional activation of doublycharged metal complexes of peptides with intermolecular disulfide linkagesyields two sets of singly charged paired products separated by 66 massunits resulting from selective SC bond cleavages. Highly selectiveelimination of 66 mass units, which corresponds to the molecular weight ofhydrogen disulfide (H(2)S(2)), is observed from singly charged metalcomplexes of peptides with disulfide linkages. The mechanism proposed forthese processes is initiated by formation of a metal-stabilized enolate atCys, followed by cleavage of the S-C bond. Further activation of theproducts yields sequence information that facilitates locating theposition of the disulfide linkages in the peptic digest fragments. Forexample, the doubly charged Ca(2+) complex of the peptic digest productGIVEQCCASVCSL/FVNQHLCGSHL yields paired products separated by 66 massunits resulting from selective SC bond cleavages at an intermoleculardisulfide linkage under low-energy collision-induced dissociation. Furtheractivation of the product comprising the A chain reveals the presence of asecond disulfide bridge, an intramolecular linkage. Experimental andtheoretical studies of the disulfide linked model peptides providemechanistic details for the selective cleavage of the S-C bond.

• Zhang M, Kaltashov IA
• Mapping of protein disulfide bonds using negative ion fragmentation with abroadband precursor selection.
• Anal Chem. 2006; 78: 4820-9
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• Fast mapping of disulfide bonds in proteins containing multiple cysteineresidues is often required in order to assess the integrity of thetertiary structure of proteins prone to degradation and misfolding or todetect distinct intermediate states generated in the course of oxidativefolding. A new method of rapid detection and identification ofdisulfide-linked peptides in complex proteolytic mixtures utilizes thetendency of collision-activated peptide ions to lose preferentially sidechains of select amino acids in the negative ion mode. Cleavages ofcysteine side chains result in efficient dissociation of disulfide bondsand produce characteristic signatures in the fragment ion mass spectra.While cleavages of other side chains result in insignificant loss of massand full retention of the peptide ion charge, dissociation of externaldisulfide bonds results in physical separation of two peptides and,therefore, significant changes of both mass and charge of fragment ionsrelative to the precursor ion. This feature allows the fragment ionsgenerated by dissociation of external disulfide bonds to be easilydetected and identified even if multiple precursor ions are activatedsimultaneously. Such broadband selection of precursor ions for consecutiveactivation is achieved by lowering the dc/rf amplitude ratio in the firstquadrupole filter of a hybrid quadrupole time-of-flight mass spectrometer.The feasibility of the new method is demonstrated by partial mapping ofdisulfide bridges within a 37-kDa protein containing 16 cysteine residuesand complete disulfide mapping within a lysozyme (14.5 kDa) containing 8cysteine residues. In addition to detecting peptide pairs connected by asingle external disulfide, the new method is also shown to be capable ofidentifying peptides containing both external and internal disulfidebonds. The two major factors determining the efficiency of disulfidemapping using the new methodology are the effectiveness of proteolysis andthe ability of the resulting proteolytic fragments to form multiplycharged negative ions.

• Flensburg J, Tangen A, Prieto M, Hellman U, Wadensten H
• Chemically-assisted fragmentation combined with multi-dimensional liquidchromatography and matrix-assisted laser desorption/ionization post sourcedecay, matrix-assisted laser desorption/ionization tandem time-of flightor matrix-assisted laser desorption/ionization tandem mass spectrometryfor improved sequencing of tryptic peptides.
• Eur J Mass Spectrom (Chichester, Eng). 2005; 11: 169-79
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• Derivatization of tryptic peptides using an Ettan CAF matrix-assistedlaser desorption/ionization (MALDI) sequencing kit in combination withMALDI-post source decay (PSD) is a fast, accurate and convenient way toobtain de novo or confirmative peptide sequencing data. CAF (chemicallyassisted fragmentation) is based on solid-phase derivatization using a newclass of water stable sulfonation agents, which strongly improves PSDanalysis and simplifies the interpretation of acquired spectra. Thederivatization is performed on solid supports, ZipTip(microC18, limitingthe maximum peptide amount to 5 microg. By performing the derivatizationin solution enabled the labeling of tryptic peptides derived from 100microg of protein. To increase the number of peptides that could besequenced, derivatized peptides were purified using multidimensionalliquid chromatography (MDLC) prior to MALDI sequencing. Following thefirst dimension strong cation exchange (SCX) chromatography step, modifiedpeptides were separated using reversed-phase chromatography (RPC). Duringthe SCX clean up step, positively charged peptides are retained on thecolumn while properly CAF-derivatized peptides (uncharged) are not. Amoderately complex tryptic digest, prepared from six different proteins ofequimolar amounts, was CAF-derivatized and purified by MDLC. Fractionsfrom the second dimension nano RPC step were automatically sampled andon-line dispensed to MALDI sample plates and analyzed using MALDI massspectrometry fragmentation techniques. All proteins in the derivatizedprotein mixture digest were readily identified using MALDI-PSD or MALDItandem mass spectrometry (MS/MS). More than 40 peptides were unambiguouslysequenced, representing a seven-fold increase in the number of sequencedpeptides in comparison to when the CAF-derivatized protein mix digest wasanalyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion,MDLC purification of CAF-derivatized peptides significantly increases thesuccess rate for de novo and confirmative sequencing using various MALDIfragmentation techniques. This new approach is not only applicable tosingle protein digests but also to more complex digests and could, thus,be an alternative to electrospray ionization MS/MS for peptide sequencing.

• Zhang W, Marzilli LA, Rouse JC, Czupryn MJ
• Complete disulfide bond assignment of a recombinant immunoglobulin G4monoclonal antibody.
• Anal Biochem. 2002; 311: 1-9
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• Recombinant monoclonal antibodies (mAbs) are an emerging therapeutic area.However, there are few reports on disulfide bond assignment of recombinantmAbs. This work describes the complete disulfide bond assignment of arecombinant immunoglobulin G4 (IgG4) mAb. N-ethylmaleimide (NEM) was usedto mask free sulfhydryl groups present in the mAb. Digestion of the mAbwith endoproteinase Lys-C without disulfide scrambling was achieved bydenaturing the mAb in the presence of NEM in guanidine hydrochloride(GuHCl). The Lys-C digest was subsequently reduced with dithiothreitol(DTT). Native and reduced Lys-C digests were mass analyzed by on-linereversed-phase-high-performance liquid chromatography mass spectrometry(RP-HPLC/MS). Disulfide-containing peptides were sequenced by off-linenanoelectrospray quadrupole time-of-flight mass spectrometry (nanoESI-QTOFMS) and N-terminal Edman sequencing for verifying connectivities. Therecombinant IgG4 mAb was found to contain the expected disulfide linkageswith the proposed method. The NEM alkylating reagent was critical inminimizing disulfide scrambling during the denaturation and digestion ofthe mAb. This integrated approach, combining MS and N-terminal Edmansequencing, was capable of assigning the disulfide pattern of the IgG4 mAbrapidly and completely, and should be applicable for disulfide bondassignment and structural analysis of other mAbs and large proteins withmultiple disulfide bonds.

• Stensballe A, Andersen S, Jensen ON
• Characterization of phosphoproteins from electrophoretic gels by nanoscaleFe(III) affinity chromatography with off-line mass spectrometry analysis.
• Proteomics. 2001; 1: 207-22
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• Detailed characterization of phosphoproteins as well as otherpost-translationally modified proteins is required to fully understandprotein function and regulatory events in cells and organisms. Here wepresent a mass spectrometry (MS) based experimental strategy for theidentification and mapping of phosphorylation site(s) using onlylow-picomole amounts of phosphoprotein starting material. Miniaturizedsample preparation methods for MS facilitated localization ofphosphorylation sites in phosphoproteins isolated by polyacrylamide gelelectrophoresis. Custom made, nanoscale immobilized Fe(III) affinitychromatography (Fe(III)-IMAC) columns were employed for enrichment ofphosphorylated peptides from crude peptide mixtures prior to off-lineanalysis by matrix-assisted laser desorption/ionization (MALDI) MS ornanoelectrospray tandem mass spectrometry (MS/MS). An optimized andsensitive procedure for alkaline phosphatase treatment of peptide mixtureswas implemented, which in combination with nano-scale Fe(III)-IMAC andMALDI-MS allowed unambiguous identification of phosphopeptides byobservation of 80 Da mass shifts. Nanoelectrospray MS/MS was used forphosphopeptide sequencing for exact determination of phosphorylationsites. The advantages and limitations of the experimental strategy wasdemonstrated by enrichment, identification and sequencing ofphosphopeptides from the model proteins ovalbumin and bovine beta-caseinisolated by gel electrophoresis. Furthermore, an autophosphorylation siteat Ser-3 in recombinant human casein kinase-2 beta subunit was determined.The potential of miniaturized Fe(III)-IMAC and MALDI-MS forcharacterization of in vivo phosphorylated proteins was demonstrated byidentification of tryptic phosphopeptides derived from the human p47/phoxphosphoprotein isolated by two-dimensional gel electrophoresis.

• Merewether LA, Le J, Jones MD, Lee R, Shimamoto G, Lu HS
• Development of disulfide peptide mapping and determination of disulfidestructure of recombinant human osteoprotegerin chimera produced inEscherichia coli.
• Arch Biochem Biophys. 2000; 375: 101-10
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• Recombinant human osteoprotegerin chimera is a 90-kDa protein containing ahuman IgG Fc domain fused to human osteoprotegerin. The molecule is adimer linked by two intermolecular disulfide bonds and contains elevenintramolecular disulfide bonds per monomer. A cysteine-rich region inosteoprotegerin contains nine disulfide bridges homologous to thecysteine-rich signature structure of the tumor necrosis factorreceptor/nerve growth factor receptor superfamily. In this report, we havedeveloped peptide mapping procedures suitable to generatedisulfide-containing peptides for disulfide structure assignment of thefusion molecule. The methods employed included proteolytic digestion usingendoproteinases Glu-C and Lys-C in combination followed by LC-MS analyses.Disulfide linkages of peptide fragments containing a single disulfide bondwere assigned by sequence analysis via detection of(phenylthiohydantoinyl) cystine and/or by MS analysis. Disulfide bonds ofa large, core fragment containing three peptide sequences linked by fourdisulfides were assigned after generation of smaller disulfide-linkedpeptides by a secondary thermolysin digestion. Disulfide structures ofpeptide fragments containing two disulfide bonds were assigned usingmatrix-assisted laser desorption ionization mass spectrometry withpostsource decay. Both the inter- and intramolecular disulfide linkages ofthe chimeric dimer were confirmed.

• McMullen BA, Fujikawa K, Davie EW, Hedner U, Ezban M
• Locations of disulfide bonds and free cysteines in the heavy and lightchains of recombinant human factor VIII (antihemophilic factor A).
• Protein Sci. 1995; 4: 740-6
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• The locations of disulfide bonds and free cysteines in the heavy and lightchains of recombinant human factor VIII were determined by sequenceanalysis of fragments produced by chemical and enzymatic digestions. TheA1 and A2 domains of the heavy chain and the A3 domain of the light chaincontain one free cysteine and two disulfide bonds, whereas the C1 and C2domains of the light chain have one disulfide bond and no free cysteine.The positions of these disulfide bonds are conserved in factor V andceruloplasmin except that the second disulfide bond in the A3 domain ismissing in both factor V and ceruloplasmin. The positions of the threefree cysteines of factor VIII are the same as three of the four cysteinespresent in ceruloplasmin. However, the positions of the free cysteines infactor VIII and ceruloplasmin are not conserved in factor V.

• Tam JP, Lin YZ, Liu W, Wang DX, Ke XH, Zhang JW
• Mapping the receptor-recognition site of human transforming growthfactor-alpha.
• Int J Pept Protein Res. 1991; 38: 204-11
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• The receptor-recognition site human transforming growth factor-alpha (TGFalpha), a 50-residue tricyclic peptide with three disulfide bonds, wasmapped by a set of 46 peptide analogs consisting of linear, monocyclic,bicyclic, and tricyclic structures representing individual and overlappingsubdomains of human TGF alpha. Linear overlapping fragments ranging from 7to 18 residues and spanning the entire length of TGF alpha as well asmonocyclic analogs with one disulfide linkage were found to be inactive inboth receptor-binding and mitogenic assays. Bicyclic analogs with twodisulfide linkage and representing either the amino or carboxyl two-thirdsof TGF alpha showed low activity at 0.1-0.9 mM concentrations. Tricyclicanalogs containing all three disulfide linkages but lacking either theamino or carboxyl terminal heptapeptide was, respectively, 3% and 0.1% asactive as TGF alpha. These results show that determinants for the receptorbinding cannot be represented by a short continuous fragment or a singlesubdomain, but are located on a discontinuous surface on a foldedstructure with disulfide restraints. Furthermore, these results whencombined with our previous results which shows that the middle subdomain(second disulfide loop) is not involved in the receptor binding suggestthat the receptor-binding residues are constituted of three fragmentslocated at the first and third subdomains as well as the external carboxylpeptide.

• Maekawa K, Tsunasawa S, Dibo G, Sakiyama F
• Primary structure of nuclease P1 from Penicillium citrinum.
• Eur J Biochem. 1991; 200: 651-61
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• The primary structure of nuclease P1, which cleaves both RNA andsingle-stranded DNA, from Penicillium citrinum was elucidated. Thecomplete amino acid sequence consisting of 270 residues was determined byanalysis of peptides obtained by digestion with Achromobacter protease Iof the reduced and S-aminoethylated protein and by digestion withStaphylococcus aureus V8 protease of the reduced and S-carboxymethylatedprotein. Four half-cystine residues were assigned to Cys72-Cys217 andCys80-Cys85. N-Glycosylated asparagine residues were identified atpositions 92, 138, 184 and 197. Fast-atom-bombardment and laser-ionizationMS were successfully used to confirm the determined amino acid sequencesof peptides and to estimate the molecular mass of this glycoprotein havingheterogenous sugar moieties, respectively. Comparison of the amino acidsequence of nuclease P1 with other nucleases revealed that the protein hasa high degree of sequence identity (50%) with nuclease S1 from Aspergillusoryzae. The His-Phe-Xaa-Asp-Ala sequence (positions 60-64) is similar tothe sequence (His-Phe-Asp-Ala) involving the active-site His119 of bovinepancreatic RNase A, and the Pro-Leu-His sequence (positions 124-126) isidentical with the sequence involving the active-site His134 of porcinepancreatic DNase I.

• Wang N, Southan C, DeWolf WE Jr, Wells TN, Kruse LI, Leatherbarrow RJ
• Bovine dopamine beta-hydroxylase, primary structure determined by cDNAcloning and amino acid sequencing.
• Biochemistry. 1990; 29: 6466-74
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• A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has beenisolated from bovine adrenal glands. The clone hybridizes to twooligonucleotide probes, one based on a previously reported active sitepeptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] andthe other based on the human DBH sequence [Lamouroux, A., et al. (1987)EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame thatcodes for the soluble form of bovine DBH, with the exception of the firstsix amino acids. Direct confirmation of 93% of the cDNA-derived sequencewas obtained from cleavage peptides by protein sequencing and massspectrometry. Differences were found between these two sequences at onlytwo positions. Of the four potential N-linked carbohydrate attachmentsites, two, Asn-170 and Asn-552, were shown to be partially and fullyglycosylated, respectively. Within the 69% of the protein sequenceconfirmed by mass spectrometry, no other covalent modifications weredetected.