Two-dimensional electrophoresis as a complementary method of isolating peptide fragments of cleaved proteins for internal sequencing.
To determine the primary structure of proteins, usually proteolytic enzyme digests are separated by reversed-phase high-performance liquid chromatography (HPLC) and each fraction is collected and sequenced. The results obtained by different cleavages are combined to reveal the entire sequence. However, there are many N-terminal-blocked proteins and/or intact proteins or their particular fragments that are not eluted from HPLC columns. Internal fragments of such proteins were successfully isolated by the use of two-dimensional electrophoresis, after digestion. Electroblotted spots were easily sequenced to identify those difficult fragments which could not be obtained using HPLC.
Nokihara
K
K
Tokyo University of Agriculture and Technology, Japan.
Kuriki
T
T
Morita
N
N
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D008071:Lipoprotein Lipase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D010669:Phenylthiohydantoin; D011506:Proteins / Q000737:chemistry*; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011506:Proteins; D012709:Serum Albumin; D010669:Phenylthiohydantoin; D008071:Lipoprotein Lipase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase