PubMed Health⌕ Search

PubMed · 10900454

Proteome profiling-pitfalls and progress.

Abstract

In this review we examine the current state of analytical methods in proteomics. The conventional methodology using two-dimensional electrophoresis gels and mass spectrometry is discussed, with particular reference to the advantages and shortcomings thereof. Two recently published methods which offer an alternative approach are presented and discussed, with emphasis on how they can provide information not available via two-dimensional gel electrophoresis. These two methods are the isotope-coded affinity tags approach of Gygi et al. and the two-dimensional liquid chromatography-tandem mass spectrometry approach as presented by Link et al. We conclude that both of these new techniques represent significant advances in analytical methodology for proteome analysis. Furthermore, we believe that in the future biological research will continue to be enhanced by the continuation of such developments in proteomic analytical technology.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

P A Haynes, J R Yates. 2000-06-30. Proteome profiling-pitfalls and progress.. https://doi.org/10.1002/1097-0061(20000630)17%3A2%3C81%3A%3Aaid-yea22%3E3.0.co%3B2-z

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

Intracellular metabolism and bioactivity of quercetin and its in vivo metabolites.

Understanding the cellular effects of flavonoid metabolites is important for predicting which dietary flavonoids might be most beneficial in vivo. Here we investigate the bioactivity in dermal fibroblasts of the major reported in vivo metabolites of quercetin, i.e. 3'-O-methyl quercetin, 4'-O-methyl quercetin and quercetin 7-O-beta-D-glucuronide, relative to that of quercetin, in terms of their further metabolism and their resulting cytotoxic and/or cytoprotective effects in the absence and presence of oxidative stress. Uptake experiments indicate that exposure to quercetin led to the generation of two novel cellular metabolites, one characterized as a 2'-glutathionyl quercetin conjugate and another product with similar spectral characteristics but 1 mass unit lower, putatively a quinone/quinone methide. A similar product was identified in cells exposed to 3'-O-methyl quercetin, but not in the lysates of those exposed to its 4'-O-methyl counterpart, suggesting that its formation is related to oxidative metabolism. There was no uptake or metabolism of quercetin 7-O-beta-D-glucuronide by fibroblasts. Formation of oxidative metabolites may explain the observed concentration-dependent toxicity of quercetin and 3'-O-methyl quercetin, whereas the formation of a 2'-glutathionyl quercetin conjugate is interpreted as a detoxification step. Both O -methylated metabolites conferred less protection than quercetin against peroxide-induced damage, and quercetin glucuronide was ineffective. The ability to modulate cellular toxicity paralleled the ability of the compounds to decrease the level of peroxide-induced caspase-3 activation. Our data suggest that the actions of quercetin and its metabolites in vivo are mediated by intracellular metabolites.

Chromatography, Liquid↗

Modeling the performance of pilot-scale countercurrent chromatography: scale-up predictions and experimental verification of erythromycin separation.

Biosynthesis of polyketide antibiotics, such as erythromycin A (EA), can result in the formation of analogues of the main compound that are chemically and structurally extremely similar. The large-scale purification of these antibiotics by conventional high-performance liquid chromatography (HPLC) can be prohibitively expensive due to the large volume of both solvent and adsorbent required. This study examines the feasibility of using a novel pilot-scale countercurrent chromatography (CCC) machine as an alternative to HPLC. CCC is a low-pressure (typically <4000 kN m(-2)) liquid-liquid chromatographic technique that allows the separation of solutes on the basis of their partitioning between two immiscible liquid phases. The effects of mobile phase flow rate, column rotational speed, and sample injection volume on the attainable yield and purity of EA were investigated. Our results show that, at a mobile phase flow rate of 40 mL min(-1), a rotational speed of 1200 rpm, and an injection volume of 100 mL (10 g total erythromycin), EA could be satisfactorily fractionated with a purity of approximately 92% (w/w) and a recovery yield of approximately 100% (w/w). The total solute throughput was estimated to be 0.41 kg day(-1). More importantly, we demonstrated simple and predictive linear scale-up of the CCC separation based on data obtained from a single laboratory-scale CCC chromatogram, and verified this experimentally. The retention time and peak width of the target compound at the pilot scale could be predicted to within 4% for operation at a range of mobile-phase flow rates and injection volumes. This predictable nature of CCC separations, unlike HPLC methods, can greatly reduce process development times and enable a complete process-scale operating scenario to be planned.

Chromatography, Liquid↗

Chromatographic performance of large-pore versus small-pore columns in micellar liquid chromatography.

Micellar liquid chromatography (MLC) is useful in bioanalysis because proteinaceous biofluids can be directly injected onto the column. The technique has been limited in part because of the apparently weak eluting power of micellar mobile phases. It has recently been shown [Anal. Chem. 72 (2000) 294] that this may be overcome by the use of large pore size stationary phases. In this work, large-pore (1000 A) C(18) stationary phases were evaluated relative to conventional small-pore (100 A) C(18) stationary phases for the direct sample injection of drugs in plasma. Furthermore, the difference between the large and small pore phases in gradient elution separations of mixtures of widely varying hydrophobicities was investigated. Large-pore stationary phases were found to be very effective for eluting moderately to highly hydrophobic compounds such as ibuprofen, crotamiton, propranolol, and dodecanophenone, which were highly retained on the small-pore stationary phases typically used in MLC. The advantages of direct introduction of biological samples (drugs in plasma) and rapid column re-equilibration after gradient elution in MLC were maintained with large-pore phases. Finally, recoveries, precision, linearity, and detection limits for the determination of quinidine and DPC 961 in spiked bovine plasma were somewhat better using MLC with wide pore phases.

Chromatography, Liquid↗