Protein identification and protein characterization by high-performance time-of-flight mass spectrometry.
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Amino Acid Sequence↗
Biomedical subjects
W M Hines
Publications and source records attributed to W M Hines.
Accurate mass measurements using MALDI-TOF with delayed extraction.
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is now an essential tool in biopolymer analysis. Sensitivity and mass range are unsurpassed, but mass measurement accuracy and resolution have been limited. With delayed extraction and a reflecting analyzer, mass measurements using MALDI-TOF can be made with an accuracy of a few parts per million (ppm). It is possible to distinguish Lys from Gln in peptides, and to determine the elemental composition of smaller molecules (mass 100-500). In database searching strategies, a smaller mass window, resulting from an increase in mass accuracy, greatly decreases the number of possible candidates. Mass measurement accuracy with errors less than 5 ppm is demonstrated on a mixture of 12 peptides ranging in mass from ca. 900 to 3700 Da. Mass measurements on 13 peaks in an unseparated tryptic digest of myoglobin gave results with an overall average error less than 3.5 ppm, with a maximum error of 7 ppm.
Adrenocorticotropic Hormone↗
Primary structures for a mammalian cellular and serum copper amine oxidase.
The 6-hydroxydopa quinone-containing active site peptide from bovine serum amine oxidase has been found to be highly homologous to a segment of a cloned human kidney amiloride-binding protein (Barbry, P., Champe, M., Chassande, O., Munemitsu, S., Champigny, G., Lingueglia, E., Maes, P., Frelin, C., Tartar, A., Ullrich, A., and Lazdunski, M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7347-7351). Additionally, a second 38-residue tryptic peptide (peptide XI) isolated from bovine serum amine oxidase shows 82% identity with a portion near the carboxyl terminus of the human kidney amiloride-binding protein. When an extended active site peptide was isolated from porcine kidney diamine oxidase (Janes, S. M., Palcic, M. M., Scaman, C. H., Smith, A. J., Brown, D. E., Dooley, D. M., Mure, M., and Klinman, J. P. (1992) Biochemistry 31, 12147-12154), it was found to be fully contained in the human kidney amiloride-binding protein. Examination of amiloride binding to bovine serum amine oxidase and porcine kidney diamine oxidase reveals dissociation constants of 196 and 9.1 microM, respectively. Taken together, these findings indicate that the cDNA isolated for human kidney amiloride-binding protein encodes a human kidney diamine oxidase. Two oligonucleotides, based on the tryptic peptide XI and active-site peptide of bovine serum amine oxidase, were used to amplify a portion of cDNA from a commercial bovine liver cDNA library through the use of the polymerase chain reaction. A full-length clone (2.7 kilobase pairs) for bovine serum amine oxidase was subsequently obtained through screening of the same cDNA library with the amplified 0.7-kilobase pair cDNA. These studies provide the first primary sequences for a mammalian cellular and serum copper amine oxidase. Computer alignment of amine oxidase cDNA-derived protein sequences reveals three conserved histidine residues, which are likely to be ligands to copper.
Amine Oxidase (Copper-Containing)↗
The sequence and reactive site of ecotin. A general inhibitor of pancreatic serine proteases from Escherichia coli.
Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unusual in its ability to inhibit chymotrypsin, trypsin, and elastase. To address the structural basis of its broad specificity, the gene for ecotin has been cloned and its sequence determined. A promoter of the 17-base pair spacing class was identified, and the probable transcriptional start site lies 18 base pairs upstream from a ribosome binding locus. The gene is followed by a series of conserved repetitive extragenic palindromic sequences. Ecotin has a signal peptide of 20 amino acids which confirms its periplasmic localization. Sequence analyses by Edman degradation and mass spectrometry confirmed 71% of the deduced protein sequence of calculated monomeric molecular mass 16,096 Da. Comparisons of the primary structure for the 142-amino acid protein with the major classes of serine protease inhibitors suggest that ecotin is a novel inhibitor. The reactive site of ecotin was determined to be Met84 for its complexes with chymotrypsin, trypsin, and elastase. The scissile Met84-Met85 bond lies within a disulfide-bonded protein segment similar to other classes of inhibitors.
Amino Acid Sequence↗
Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping.
Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.
Acrylic Resins↗