Strategies for the enrichment and identification of basic proteins in proteome projects

Abstract

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI . 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) -tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI . 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.

Recent developments in the determination of urinary cancer biomarkers by capillary electrophoresis

“…A number of CE modes have been developed for the separation and analysis of proteins: isotachophoresis (ITP) [112], isoelectric focusing (IEF) [103,104,106,113], electrokinetic chromatography (EKC) [114][115][116][117][118], affinity electrophoresis [119,120], and immunosubstraction CE (IS-CE) [121]. CE has been used for the diagnosis of such disorders as adenylosuccinase deficiency, 5-oxoprolinuria, Bence-Jones proteinuria, and nephrotic syndrome, and has been gaining importance for use in routine clinical analysis [122][123][124].…”

Prefractionation techniques in proteome analysis: The mining tools of the third millennium

“…Current approaches are qualified as incapable of having a whole vision of the proteome, even limited on structural aspects. For instance, strongly alkaline proteins are poorly represented using classical 2-DE, as underlined by Bae et al [14], and highly hydrophobic proteins cannot be properly solubilized and consequently not analyzed and/ or identified. Electrophoresis-based methods taken alone (still the most commonly used to date) are neither appropriate for polypeptides of masses lower than 5000 Da, nor effective for very alkaline proteins.…”

Proteomics for Protein Expression Profiling in Neuroscience

“…MS technology is not as limited in this regard. Two-dimensional electrophoresis is also limited in its ability to detect very basic proteins (pI > 10) (102). The assumptions have been challenged by a recent study (63).…”