Advances in gas chromatographic mass spectrometric protein sequencing. 2—Application to membrane proteins

Abstract

The primary structure of the integral membrane protein bacteriorhodopsin was determined by an efficient combination of gas chromatographic mass spectrometric techniques with the Edman degradation. This combination of methodologies circumvented many of the experimental difficulties associated with the insolubility of bacteriorhodopsin and its primary degradation fragments in aqueous buffers. Specifically, in the gas chromatographic mass spectrometric analysis of the cyanogen bromide peptides derived from bacteriorhodopsin, it has been possible to identify homoserine-containing peptides which served as a starting point for the construction of C-terminal sequences. In most cases this C-terminal sequence constructed from the gas chromatographic mass spectrometric peptides overlapped the N-terminal sequence derived in an Edman degradation experiment, thereby completing the structure of the fragment. Furthermore, the specific identification of methionine-containing peptides required to establish the order of the cyanogen bromide fragments was accomplished by direct analysis of the complex mixtures generated by partial hydrolysis of segments of the protein. These data made it possible to determine the sequence of a large portion of bacteriorhodopsin solely from cyanogen bromide cleavage, one of the few specific reactions compatible with the solubility properties of this hydrophobic protein. Finally, the gas chromatographic mass spectrometric sequence data have been used to assign or confirm amino acids where the Edman data was ambiguous. These gas chromatographic mass spectrometric techniques resulted in an efficient and reliable determination of the complete sequence of this membrane protein which is 248 amino acids long.

Primary structure of monkey osteocalcin

“…The complete amino acid sequence of monkey osteocalcin (Figure 2) was determined by an efficient combination of GC-MS and Edman methods. We have previously demonstrated that the joint use of these entirely independent and complementary sequencing approaches is more efficient than either method used alone (Biemann, 1980;Herlihy et al, 1981). Automatic Edman degradation was carried out successfully for 46 steps before washout of the COOH-terminal tripeptide.…”

Structural characterization of recombinant hepatitis B surface antigen protein by mass spectrometry

“…The problems encountered during attempts to sequence the particle-associated S protein are common to figure 4: Hydropathic score (Kyte & Doolittle, 1982) of HBsAg determined by using a window of six in the prplot routine of the protein identification resource. structural studies of other integral membrane proteins (Asbeck et al, 1969;Gerber et al, 1979;Herlihy et al, 1981). The 5 protein (Figure 1) is exceedingly hydrophobic with an abundance of Pro, Trp, Phe, Leu, and lie contained in three hydrophobic domains (approximately residues 6-30, 78-113, and 168-226, Figure 4), two of which likely span the lipid bilayer of the particle.…”

Advances in gas chromatographic mass spectrometric protein sequencing. 3—Automated interpretation of the mass spectra of the polyamino alcohol derivatives

“…However, in order to prevent an abundant ion at high mass from carrying an inordinate amount of weight in the final score calulation, only ions whose original relative intensities are less than 40% are multiplied by the weighting factor, and any weighted intensity a The amino acid composition specified for this fragment was: Gly(l), Va1(2), Ser(1). Asp (2). Pro (2).…”