A microbore high-performance liquid chromatography strategy for the purification of polypeptides for gas-phase sequence analysis. Structural studies on the murine transferrin receptor.
We describe herein the use of reversed-phase high-performance liquid chromatography coupled with the novel application of short (10 cm or less) microbore columns (2 mm internal diameter) to fractionate and purify a number of tryptic peptides generated from approximately 200 pmol purified murine transferrin receptor. The use of reversed-phase microbore columns permits the recovery of submicrogram amounts of purified polypeptides in high yield (greater than 90%) in small eluent volumes (20-60 microliter). In this manner, purified polypeptides can be loaded directly onto the gas-phase sequencer without further manipulation. This procedure avoids sample loss, which frequently occurs with other forms of concentration (e.g. lyophilization, evaporation). The application of second-order-derivative ultraviolet spectroscopy, using a diode array detector, for the analysis of aromatic aminoacid-containing peptides in complex tryptic digests is described. N-terminal amino acid sequence analyses were performed on six tryptic peptides, yielding 105 unique assignments; this corresponds to approximately 14% of the molecule. A comparison of this amino acid sequence information with the primary structure of human transferrin receptor deduced from the mRNA sequence [Nature (Lond.) 311, 675-678 (1984); Cell 39, 267-274 (1984)] reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.
Grego
B
B
Van Driel
I R
IR
Stearne
P A
PA
Goding
J W
JW
Nice
E C
EC
Simpson
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D008565:Membrane Proteins / Q000302:isolation & purification; D051379:Mice; D010455:Peptides / Q000302:isolation & purification*; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014357:Trypsin
D008565:Membrane Proteins; D010455:Peptides; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D014357:Trypsin