A microbore high-performance liquid chromatography strategy for the purification of polypeptides for gas-phase sequence analysis. Structural studies on the murine transferrin receptor

Abstract

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 pl). 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)l reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.The receptor for the iron-binding protein transferrin is an integral membrane glycoprotein located on the surface of all proliferating mammalian cells [l]. Recent evidence indicates that the transferrin receptor is a dimer composed of two similar or identical polypeptide chains ( M , 95 000) joined by disulfide bonds [2 -41. The recent purification to homogeneity [5] of small quantities (200 pmol) of the murine transferrin receptor from NS-1 myeloma cells has led us to undertake a partial amino acid sequence analysis of this protein. The partial sequence information obtained in this manner has been used to construct the appropriate oligonucleotide probes, thereby permitting the primary structure determination of the murine transferrin receptor using recombinant DNA procedures [6].Direct amino acid sequence analysis of high-M, proteins often yields little meaningful sequence information. As this was the case with the murine transferrin receptor, we decided to fragment the parent molecule into smaller peptides (e. g. tryptic peptides), which are more suitable for sequence analysis. Crucial to the success of this strategy is the ability to purify to homogeneity submicrogram amounts of some of the resultant peptides, and to recover them in volumes suitable for gas-phase sequence analysis. To this end we demonstrate

Molecular cloning of a gene encoding an arabinogalactan-protein from pear (Pyrus communis) cell suspension culture.

“…The protein backbones were digested with thermolysin, and the products were separated on a C18 microbore HPLC column (Ultrasphere ODS, 2.1 x 250 mm) and eluted with a gradient of acetonitrile in 0.1% aqueous TFA. Individual peaks were repurified and sequenced (19). Amino acid analyses were performed as described by Simpson et al (20).…”

Complete amino acid sequence of plastocyanin from a green alga,Enteromorpha prolifera

“…Peptide mixtures resulting from enzymatic cleavage were fractionated by reversed-phase high-performance liquid chromatography (HPLC) on a Hewlett-Packard liquid chromatograph model 1090, fitted with a model 1040 diode array detector as described elsewhere [17].…”

Purification of proteins and peptides for sequence analysis using microcolumn liquid chromatography

“…Previously, we described a "multidimensional" RPLC strategy for peptide purification which was based on the sequential use of alternative stationary phase supports and mobile phase conditions such as pH and ionic buffer [1,9]. Central to this strategy is the ability to recover samples in high yield.…”

“…The ability to purify submicrogram amounts of protein and peptides to homogeneity is crucial to the success of any microsequencing strategy employing Edman degradation chemistry. Previously, we described a "multidimensional" RPLC strategy for peptide purification which was based on the sequential use of alternative stationary phase supports and mobile phase conditions such as pH and ionic buffer [1,9]. Central to this strategy is the ability to recover samples in high yield.…”