Absence of sugars in electrophoretically purified cytochrome b5 demonstrated by combined gas chromatography-mass spectrometry.

Abstract

The problem of determining small but significant amounts of carbohydrates, in purified proteins, has been studied using the membrane protein, cytochrome b5 . A newly developed method that involves direct gas chromatography-mass spectrometry of sugars obtained by hydrolysis of proteins purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) allows the identification and determination of small amounts of carbohydrates (e .g ., 20 I-Lg of glycoprotein containing a minimum of 0.1% monosaccharid e), even in the presence of relatively high amounts of impurities . Application of this method to cytochrome b5 fragments obtained by tryptic digestion from rat liver microsomes and purified by combined gel filtration and ion exchange chromatography, followed by SDS PAGE, has consistently yielded values below 0.07 mol of the individual sugars and aminosugars per mole cytochrome b 5 . It is concluded that cytochrome b 5 , at least its trypsin-released major aminoterminal fragment, is not constitutively glycosylated .In studies of the synthesis and modification of proteins, especially membrane proteins, the question of the glycosylation of specific proteins is of fundamental importance . Although efficient methods exist for the determination of sugar components present in proteins relatively rich in carbohydrates, the detection of carbohydrates in proteins that are low in relative sugar content and can be obtained in purified form only in limited amounts is still a methodological problem. To clarify the possible existence of sugars in certain proteins, a method has been developed that allows the unequivocal identification and determination of very small amounts of sugars in polypeptides separated and purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) . The value of this method is demonstrated using the example of the widespread membrane protein, cytochrome bs, which has been repeatedly reported to be glycosylated. Cytochrome b5 is a relatively wellcharacterized membrane constituent of endoplasmic reticulum (ER, e.g ., references 8 and 38) . Relatively smaller concentrations of this protein have also been found in isolated nuclear envelope (10, 21), outer mitochondrial membrane (9, 12), Golgi apparatus (18, 19; see, however, reference 33), and in several plasma membrane fractions (3, 4, 19; see, however, reference 34) . Preparation and purification (29), intramembrane orienta-THE JOURNAL OF CELL BIOLOGY " VOLUME 89 JUNE 1981 615-620 ©The Rockefeller University Press " 0021-9525/81/06/0615/06$1 .00 tion (31, 38), re-integration into membranes in vitro (7), immunological localization (9), and at least some functions (17) of this protein have been described. The complete amino acid sequence of the major tryptic fragment (31) as well as of whole cytochrome b5 (8) has been established .In spite of the numerous and intensive studies, it is still not clear whether cytochrome b5 is a glycoprotein. For human apocytochrome b5 prepared by the detergent method, Ozols (30) has...

Amino acid sequence of the N-terminal aggregation and cross-linking region (7S domain) of the alpha1 (IV) chain of human basement membrane collagen

“…The derivatives were separated by gas chromatography and identified by mass spectrometry [22] using xylose as internal standard. A Carlo Erba Fractovap 2101 for split and splitless injections with a quart capillary OV 1701 (30 in long, 0.35 mm wide), interfaced with a Varian CH 7 A mass spectrometer (datasystem SS 200 MS) was used.…”