Keli Ou

Improved high-performance liquid chromatography of amino acids derivatised with 9-fluorenylmethyl chloroformate

Abstract An improved high-performance liquid chromatography method for the separation of amino acids derivatised with 9-fluorenylmethyl chloroformate (Fmoc) is described. This method, in conjunction with a multi-tasking program, not only allows high automatic througput but also offers baseline resolution of the common Fmoc-amino acids from a linear acetonitrile gradient, greater chromatographic reproducibility over the life of the column (800 runs) and easy column regeneration. The methods described are shown to be suitable for analysis of hydrolysates of proteins from two-dimensional gels for protein identification purposes, and will also be useful for routine quality control and screening of biological samples.

Large-scale amino-acid analysis for proteome studies

Amino-acid analysis is a relatively new method for identification of proteins separated by two-dimensional gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes. This article describes modified amino-acid analysis methods for this purpose. Streamlined sample handling is a key feature of the process. To minimise sample manipulation, a single vial is used for hydrolysis and the protein hydrolysate on PVDF membrane is extracted by a one-step procedure. The hydrolysate should not be stored for long periods before analysis. Applications of the technique are presented to demonstrate the identification procedure. This approach is the most cost-effective and time-effective first step in mass protein screening for a large-scale proteome project.

High sample throughput phosphoamino acid analysis of proteins separated by one- and two-dimensional gel electrophoresis

Abstract Studies on protein phosphorylation usually involve radiolabelling techniques and visualisation on gels; this limits studies to those on tissues which can be 32 P labelled. With the advent of reproducible micropreparative two dimensional gel electrophoresis, the proteome (protein complement) of a subcellular fraction, cell or tissue can be displayed. We have investigated the sensitivity of limited protein hydrolysis to detect in vivo phosphorylation in proteins blotted from one or two-dimensional polyacrylamide gels onto polyvinylidene difluoride (PVDF) membranes. The method uses 9-fluorenylmethyl chloroformate (FMOC) derivatisation chemistry and a modified HPLC AMINOMATE system. Conditions were established for hydrolysis of the PVDF-blotted protein (5.7 M HCl at 110°C for 4 h) which resulted in the recovery of phosphoserine (Ser(P)), phosphothreonine (Thr(P)) and phosphotyrosine (Tyr(P)). The chromatography was carried out on the same system routinely used for amino acid compositional analysis using a gradient elution modified from that used for separation of 16 amino acids. A chromatographic window was designed where all 3 phosphoamino acids are separated with baseline resolution in the order of Ser(P), Thr(P) and Tyr(P), and eluted before the normal protein amino acids. The total separation time is 13 min and includes the elution of excess FMOC, its derivatives and incompletely hydrolysed peptides. The FMOC fluorescence of these three standard phosphoamino acids is linear in the range 10–100 pmol. The technique is shown to be successful in finding phosphoproteins separated by two-dimensional gel electrophoresis. The method is sensitive (30 pmol of a single site phosphorylated protein, 3.8 pmol of Ser(P) detected) and allows automated sample throughput. This presents an option for rapid screening of protein phosphorylation of large numbers of proteins separated by two-dimensional polyacrylamide gel electrophoresis.

Immunodetection and photostability of NADPH-protochlorophyllide oxidoreductase in Pinus pinea L.

Antibody against the light-dependent NADPH-protochlorophyllide oxidoreductase of oat was used to detect a protein of the same molecular weight in cotyledons of 40-day-old dark-grown seedlings of Pinus pinea L. Exposure of the seedlings to light resulted in a rapid decrease in protochlorophyllide content without the concomitant decrease in 38 kDa protein which is observed on transfer of dark-grown angiosperm seedlings to light. The stability of the light-dependent NADPH-protochlorophyllide oxidoreductase in pine in the absence of accumulated substrate is consistent with either (1) a different mechanism of regulation of chlorophyll synthesis in gymnosperms or (2) a higher proportion of stable extra-plastidic protein reacting with the antibody to the light-dependent NADPH-protochlorophyllide oxidoreductase than is the case in angiosperms.

Evidence for a Light-Independent Chlorophyll Biosynthetic Pathway in Angiosperm Seeds Germinated in Darkness

It is not generally recognised that during the course of evolution of seed plants, many angiosperms and gymnosperms have retained the capacity for light-independent (dark) chlorophyll (Chl)* synthesis exhibited by cyanobacteria, algae, bryophytes and pteridophytes (1,2,3). As a consequence the view that when greening or mature angiosperm leaves are transferred from light to darkness, Chl(ide) formation ceases immediately because of the cessation of Pch(lide) photoreduction, is still widely held (4). It is reinforced by the visual contrast between etiolated dark grown angiosperm seedlings and the bright green cotyledons of dark grown gymnosperms. This important phenotypic difference in the two groups of seed bearing plants is usually interpreted in terms of an absolute reqirement of angiosperms for light for Pch(lide) reduction, whereas in gymnosperms Chi is formed in light as well as darkness (5). In this context it is not surprising that early reports of trace amounts of Chi in dark grown angiosperm seedlings (6,7) and the accumulation of Chl by wheat embryos grafted onto pine megagametophytes (8) attracted little attention.

Susceptibility of Dark Chlorophyll Synthesis in Barley and Pine Seedlings to Inhibition by Gabaculine

Dark chlorophyll accumulation in pine (Pinus pinea and Pinus nigra) and barley (Hordeum vulgare) seedlings was inhibited by gabaculine (3-amino 2,3-dihydrobenzoic acid) (GAB). The inhibitory effect of GAB was overcome by the addition of 5-aminolevulinic acid (ALA). Since GAB specifically interferes with ALA synthesis via the C5 (but not Shemin) pathway we conclude that ALA for light-independent chlorophyll synthesis in angiosperms and gymnosperms is formed via this route.