Abstract
The amino acid sequence of the thioredoxin isolated from rabbit bone marrow was determined chiefly by high performance tandem mass spectrometry and fast atom bombardment mass spectrometry combined with manual Edman degradation. The sequences of peptides generated by digestion with trypsin alone or in combination with Staphylococcus aureus protease V8 or thermolysin were determined from their collision-induced dissociation mass spectra. Alignment of these sequences and additional sequence information were obtained from the collision-induced dissociation mass spectra of peptides obtained from digestion of the intact protein with S. aureus protease V8 and alpha-chymotrypsin. The resulting sequence of 104 residues is as follows: Val-Lys-Gln-Ile-Glu-Ser-Lys-Ser-Ala-Phe-Gln- Glu-Val-Leu-Asp-Ser-Ala-Gly-Asp-Lys-Leu-Val-Val- Val-Asp-Phe-Ser-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys- Met-Ile-Lys-Pro-Phe-Phe-His-Ala-Leu-Ser-Glu-Lys- Phe-Asn-Asn-Val-Val-Phe-Ile-Glu-Val-Asp-Val-Asp- Asp-Cys-Lys-Asp-Ile-Ala-Ala-Glu-Cys-Glu-Val-Lys- Cys-Met-Pro-Thr-Phe-Gln-Phe-Phe-Lys-Lys- Gly-Gln-Lys-Val-Gly-Glu-Phe-Ser-Gly-Ala-Asn-Lys- Glu-Lys-Leu-Glu-Ala-Thr-Ile-Asn-Glu-Leu-Leu.
Highlights
The amino acid sequence of the thioredoxin isolated observation that thioredoxin catalyzes the refolding of disulfrom rabbit bone marrow was determined by fide-containing proteins [12] is consistent with the high dehigh performance tandem mass spectrometry and fast gree of homology between the active sites of protein-disulfide atom bombardment mass spectrometry combined with isomerase and thioredoxin [13]. manual Edmandegradation
Tandem Mass Spectrometry-Tandem mass spectrometry was carried out by using all four sectors of the JEOL HXllO/HX110, an dithiol active center which function in anumber of oxidation- instrument of ElBlE2B2configuration
FHALSEKF C-terminal peptide been isolated from C. nephridii, and the latterw, hich contains 3 cysteine residues [16], is the only prokaryotic thioredoxin known to have more than two thiols
Summary
Phe-Asn-Asn-Val-Val-Phe-Ile-Glu-Val-Asp-Val-Asp-Fast Atom Bombardment Mass Spectrometry-FABMS2 of the Asp-Cys-Lys-Asp-Ile-Ala-Ala-Glu-Cys-Glu-Val-Lyspr-oteolytic peptides was carried out on the first (MS-1) of two mass C y s - M e t - P r o - T h r - P h e - G l n - P h e - P h e - L y s - L y s -spectrometers of a tandemhigh resolution mass spectrometer (JEOL Gly-Gln-Lys-Val-Gly-Glu-Phe-Ser-Gly-Ala-Asn-LysH-XllO/HX110) at an accelerating voltage of 10 kV and a resolution. The abbreviations used are: FABMS, fast atom bombardment mass spectrometry; CID, collision-induced dissociation; HPLC, high performance liquid chromatography; Xle, leucine or isoleucine; TPCK, L-l-tosylamido-2-phenylethyclhloromethyl ketone. Additional sequence information was obtained by subjecting HPLC fractions of proteolytic peptides to manual Edman degradations, followed byFABMS to determine the masses of the truncated peptides. The difference in molecular weight of each peptide in the mixture before and after each step of Edman degradation indicates the successive N-terminal amino acids in each peptide. The molecular indicate the fractions collected for analysis by FAB and CID mass weights of the peptides present in each fraction arethen spectrometry. VGEFSGANK quently, the molecular weights of these peptides alone permit 6 the unique alignment of the tryptic peptides
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