Enzymatic Digestion of Proteins and HPLC Peptide Isolation in the Sub-Nanomole Range

Stone, Kathryn L.; LoPresti, Mary B.; Williams, Nancy; Crawford, Janet; DeAngelis, Raymond; Williams, Kenneth R.

doi:10.1016/b978-0-12-682001-0.50046-4

Cited by 40 publications

(16 citation statements)

References 5 publications

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“…Reactions were found to be essentially complete even at 0°C within the time required for reagent separation on spin columns, and it was not possible to obtain rapid kinetic measurements. Chemical Modification of IscU and IscS-Cysteine residues of IscS were alkylated in 0.1 M Tris buffer, pH 7, using N-ethylmaleimide (21), and cysteine residues of IscU were carboxymethylated in 0.2 M potassium phosphate buffer, pH 7, using iodoacetic acid (22). For each reaction, ϳ0.1 mM IscS or IscU was incubated with 1 mM labeling reagent for 1 h. Excess N-ethylmaleimide and iodoacetic acid were removed by repeated cycles of ultrafiltration and dilution with 50 mM Tris, pH 7.0.…”

mentioning

confidence: 99%

Transfer of Sulfur from IscS to IscU during Fe/S Cluster Assembly

Urbina

Silberg²,

Hoff

et al. 2001

Journal of Biological Chemistry

246

259

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mentioning

confidence: 99%

Transfer of Sulfur from IscS to IscU during Fe/S Cluster Assembly

Urbina

Silberg²,

Hoff

et al. 2001

Journal of Biological Chemistry

246

259

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“…Following proteolytic digestion, peptides were separated by narrow-bore reverse-phase HPLC on a Vydac 2.1 mm x 150 mm C18 column. The gradient employed was a modification of that described by Stone et al (11). Briefly, where buffer A was 0.06% trifluoroacetic acid/H20 and buffer B was 0.055% trifluoroacetic acid/acetonitrile, a gradient of 5% buffer B at 0 min, 33% buffer B at 63 min, 60%o buffer B at 95 min, and 80% buffer B at 105 min with a flow rate of 0.15 ml/min was used.…”

mentioning

confidence: 99%

Transmission-blocking antibodies recognize microfilarial chitinase in brugian lymphatic filariasis.

Fuhrman

Lane

Smith

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. The monoclonal antibody MF1, which mediates clearance of peripheral microfilaremia in a gerbil infection model, recognizes two stage-specific proteins, p70 and p75, in B. malayi microrflariae. cDNA coding for the MF1 antigen was sequenced, and the predicted protein sequence shows significant similarities to chitinases from bacteria and yeast. When microfilarial extracts and purified preparations of the MF1 antigen were tested for chitinase activity, strong bands of chitin-degrading activity comigrated in SDS/PAGE with p70 and p75 and showed a reductiondependent mobility shift characteristic of the MFl antigen. Thus, the MF1 antigen is microfrarial chitinase, which may function to degrade chitin-containing structures in the microfilaria or in its mosquito vector during parasite development and transmission.Lymphatic filariasis afflicts nearly 100 million people worldwide (1). Of the three species of lymphatic filariids that infect humans, Brugia malayi can most readily be maintained in the laboratory by passage between gerbils (another natural host) and a mosquito vector. Thus, B. malayi has been useful for studying pathogenesis and transmission of filariasis at the molecular level.Passive transfer of the monoclonal antibody MF1 was previously shown to mediate the transient clearance of first stage larvae, or microfilariae (mf), from the peripheral blood of gerbils infected with B. malayi (2). The MF1 antibody recognizes two stage-specific proteins (p70 and p75) in extracts of mf that appear only after the mf have matured for several days in the vertebrate host (3). The appearance of the MF1 antigen corresponds with the onset of the parasite's ability to infect the mosquito (3).It was thus of great interest to define more precisely the role of the MF1 antigen in filarial development and transmission. With the expectation that the proteins' sequences might clarify their function, we obtained partial amino acid sequences for p70 and p75 and then used this information to isolate and to sequence the corresponding cDNA.1 We report here that the predicted protein sequence of the MF1 antigen has several regions of significant similarity to bacterial and yeast chitinases. Furthermore, the native MF1 antigen is demonstrated to degrade chitin in vitro.MATERIALS AND METHODS Parasite Material. Gerbils (Meriones unguiculatus) were infected intraperitoneally with B. malayi either in our own facilities or at the Filariasis Repository Research Service, Athens, GA. mf were purified and proteins were extracted as described (3). Poly(A) mRNA was isolated from purified mf according to Cox and Smulian (4). Genomic DNA was made from adult B. malayi as described (5).Protein Purification and Amino Acid Sequencing. The MF1 antigen was partially purified from extracts of mf by DE-52 chromatography as described (6). N-terminal sequence analysis was performed on the combined p70 and p75 following elution with 0.5 M NaCI from DE-52, using an ABI model 4...

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“…'500µ1) and high SDS concentration ('5.3%, determined by the method of Waite and Wang [17]). p23 could not be recovered from the electroeluate by conventional RP-HPLC because of the known effect of SDS on column efficiency [18,19] and the high absorbance of Coomassie blue and other acrylamide-related contaminants interfering in the interpretation of RP-HPLC peptide maps [20]. As shown in Fig.…”

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confidence: 99%

Isolation of Low Picomole Levels of Peptides and Proteins for Microsequence Analysis

1991

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Developments in molecular genetics over the past decade have made it possible, in principle, to isolate and to clone essentially any gene in an organism. These gene-cloning techniques, combined with the ability to overexpress cloned genes now enable the large-scale production of recombinant protein for the purpose of conducting detailed protein structure-function studies, for vaccines and for replacement therapeutics.The importance of amino acid sequence information (albeit, partial sequence data) to many gene-cloning strategies is well known. Although current protein sequencing technologies now permit amino acid sequence information to be obtained at low picomole levels -levels at which many biologically important proteins can be isolated -it is now generally recognized that the major obstacle in obtaining such information is not the sensitivity of sequence analysis per se, but the ability to isolate and micromanipulate samples at these low levels [for a review, see refs. 1-3).Protein structural analysis is complicated by the need to interface high-resolution protein and peptide isolation techniques (eg., RP-HPLC, acrylamide gel electrophoresis, affinity chromatography) with current automated protein sequence analysers. Accordingly, we have developed a number of strategies for the high-yield purification of microgram quantities of peptide and protein for sequence analysis. The purpose of this article is to outline strategies for obtaining sequence data from proteins resolved by 2-DE. Two examples from our laboratory will be given -murine aB-crystallin [4] and a number of murine plasmacytoma proteins [5,6]. Strategies for obtaining sequence data on RP-HPLC and affinity chromatography-derived proteins are reported elsewhere [5,7,8].

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Enzymatic Digestion of Proteins and HPLC Peptide Isolation in the Sub-Nanomole Range

Cited by 40 publications

References 5 publications

Transfer of Sulfur from IscS to IscU during Fe/S Cluster Assembly

Transfer of Sulfur from IscS to IscU during Fe/S Cluster Assembly

Transmission-blocking antibodies recognize microfilarial chitinase in brugian lymphatic filariasis.

Isolation of Low Picomole Levels of Peptides and Proteins for Microsequence Analysis

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