Massspectrometric analyses of transmembrane proteins in human erythrocyte membrane.
It is difficult to understand the functional mechanisms of integral membrane proteins without having protein chemical information on these proteins. Although there have been many attempts to identify functionally important amino acids in membrane proteins, chemically and enzymatically cleaved peptides of integral membrane proteins have been difficult to handle because of their hydrophobic properties. In the present study, we have applied an analytical method to transmembrane proteins combining amino acid sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography with electrospray ionization (LC/ESI) mass spectrometry. We could analyze most (97%) of the tryptic fragments of the transmembrane domains of band 3 as well as other minor membrane proteins. The peptide mapping of the transmembrane domain of band 3 was completed and the peptide mapping information allowed us to identify the fragments containing lysine residues susceptible to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and to 2,4-dinitrofluorobenzene (DNFB). This method should be applicable to membrane proteins not only in erythrocyte membranes but also in other membranes.
Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Yoshito
Y
Abe
Tomohito
T
Chaen
Xiu Ri
XR
Jin
Tomohiro
T
Hamasaki
Naotaka
N
Hamasaki
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008565:Membrane Proteins; D010455:Peptides; D008239:Lysine