The isolation and separation of biomolecules, such as proteins and peptides, has become of an increased interest during the past years. Some biomolecules need to be isolated as a last step of a biotechnological method for the production thereof, for example in the preparation of protein or peptide-based pharmaceutical compounds. Similarly there is also a need to separate biomolecules for analytical purposes in order to be able to quantify and identify the proteins and/or peptides present in a sample. A wide variety of methods are used for the detection and quantification of the separated proteins and/or peptides. For identification and characterization of separated proteins, mass spectrometry (MS) methods are commonly used as these methods are fast and require very small amounts of proteins and/or peptides.
In order to increase sensitivity in mass spectrometry methods, it is often necessary to reduce the complexity of the sample. The reason for this is that prominent proteins in a sample give rise to numerous identical peptide fragments which will be detected over and over again in the analyses, obscuring the identification of proteins/peptides in low copy numbers. There are a number of ways to achieve this; on a tissue, cell, or sub-cellular level.
A certain tissue is often isolated by excision or biopsy whereas cultured cells may be sorted into different populations by flow cytometry, e.g. through fluorescence-activated cell sorting (FACS). Cells from heterogenous tissue may also selectively be released by enzyme treatment. An example is the isolation of mesenchymal stem cells from the umbilical cord (Lechner et al., Volume 36, 2007, pp 405-406). Cells can further be fractionated into organelle fractions using density gradient ultra-centrifugation.
When proteins are extracted as a cell homogenate, its complexity can be reduced by different methods. Depletion of abundant proteins by antibodies is a common way, e.g. to remove albumin from plasma samples.
A number of chromatography methods are commonly applied to separate complex protein samples. Examples include ion-exchange chromatography where ions and polar molecules can be separated according to their affinity to the ion exchange material, separation based on hydrophobicity, affinity to metals, capture of expressed tags, e.g. his-tagged proteins, or separation by size.
Protein electrophoresis is a very common and versatile separation technology where proteins, or peptides, are separated according to their size (e.g. SDS-PAGE), intrinsic charge (e.g. isoelectric focusing, IEF), or a combination thereof (e.g. 2D-DIGE).
In SDS-PAGE, proteins are given a uniform negative charge by mixing with the anionic detergent sodium dodecyl sulfate (SDS), where the detergent will bind to proteins approximately in proportion to the protein mass. When these proteins are electrophoretically separated through a polyacrylamide gel, the separation will be based on their size only.
In isoelectric focusing (IEF), proteins are separated based on their overall intrinsic charge only, usually in an immobilized pH-gradient polyacrylamide gel. When a voltage is applied the proteins will migrate in the gradient pH-field until they reach a pH where their net charge is zero, i.e. their isoelectric point, pI.
2-dimensional electrophoresis uses IEF as the first separation method, directly followed by SDS-PAGE. This technique renders a protein map with very high resolution where also protein modifications such as phosphorylation or glycosylation can be seen. Proteins of interest are picked as small gel plugs and prepared for analyses, e.g. by MS.
Free flow electrophoresis is a similar technique to IEF, but liquid-based and continuous. A protein sample is continuously fed into a laminar buffer flow and a high voltage applied perpendicular to the flow. Separation of the sample occur based on charge density and pI, and the separated sample can be collected into different fractions. The resolution is dependent on flow speed, voltage and length of the sample cell. Arguably, the resolution is usually less good compared to IEF, where the latter can be performed in very narrow immobilized pH-ranges.
In several of the separation techniques mentioned above, apart from reducing the complexity, they may also serve as a tool to enrich certain protein populations. Partitioning of the separated sample into discrete fractions based on differences in size, hydrophobicity, pI, or buoyancy in a density gradient greatly increases the number of molecules with a specified quality. However, often molecules expressed in low copy numbers are still a problem to detect.
Selective capturing of molecules offers an advantage and has long been used in an industrial context or in research. Antibodies bound to a solid matrix, beads and/or magnetic substrates selectively capture their target antigens. Often these proteins are propagated in cells of different organisms using an expression system which give the proteins a tag that facilitates capturing. Alternatively, systems which are based on other strong protein-protein interactions are commonly used; e.g. biotin-streptavidin.
Posttranslational modifications (PTM) of proteins are very common regulators of their activity, and selectively sorting-out modified proteins from a non-modified protein population may offer means to a deeper understanding of their roles, or may be used diagnostically. There are a large number of PTMs; e.g. phosphorylation, glycosylation, alkylation, methylation, prenylation, or ubiquitination. Phosphorylation is the most studied modification and there are estimations that one third of the mammalian proteins may be phosphorylated some time in their life cycle. Capturing of phosphorylated proteins can be achieved using phospho-specific antibodies, or, more commonly, using immobilized metal affinity chromatography with chelated titanium ions (IMAC-Ti4+).
In isoelectric focusing (IEF), the separation takes place in a pH gradient that occupies the whole separation distance and is arranged so that the pH in the gradient increases from anode towards the cathode. While other alternatives also exist, the pH gradients required in isoelectric focusing are in practice generated in two different ways: with the aid of a solution of carrier ampholytes or with an immobilized pH gradient. IEF with carrier ampholytes usually gives less good resolution as there is a pH-drift in the generated pH field, in particular when the electrophoresis proceed over long time.
In the case of an immobilized pH gradient (IPG) the charged or chargeable groups generating the pH gradient are bound either to the wall of a capillary system or to a matrix, often a polyacrylamide gel, to become convection stabilization. The immobilized charged or chargeable groups used are normally a limited number of carboxylic groups or amino groups with different pK-values distributed within or close to the pH gradient, which is to be generated. The concentration of the charged or chargeable groups is varied along the separation distance in a manner causing the pH at which the wall or the gel matrix has a zero net charge to increase from the anode to the cathode. A commercially available example of a system for generation of immobilised pH gradients is the Immobiline II System™ (Amersham Biosciences, Uppsala, Sweden), wherein a pH gradient covalently attached to a polyacrylamide gel is formed. Immobilized pH gradients are truly stationary and today they are normally used together with carrier ampholytes. In this combination the immobilized gradient determines the resulting pH gradient, while the carrier ampholytes contribute with conductivity. A common problem with isoelectric focusing of proteins and/or peptides is that the focused proteins/peptides are unevenly distributed in the gel with poor resolution.
To solve this problem for proteins, a non-linear pH-gradient has been immobilized in IEF gels, for example Immobiline DryStrips pH 3-11 NL™ (Amersham Biosciences, Uppsala, Sweden). The pH gradient is flattened in the neutral pH area where the majority of proteins have their pI. This non-linear gradient works less well for focusing of peptides. The reason for this is the uneven distribution of tryptic peptides in a broad pH field. When subjecting the whole human proteome to theoretical trypsination and using pI prediction programs optimized for peptides in urea, which is the most common solute in IEF, the peptides collect principally in three pH ranges; pH 3.4-5.0, 5.2-6.8, and pH 7.8-10 (Bjellqvist et al. Electrophoresis, 1994, 15 (3-4), 529-539). To better resolve peptides in IEF gels, a stepwise, non-linear pH gradient has been described in WO2007/073293.
IEF on Immobiline DryStrips with a linear pH gradient of pH 3.7-4.9 followed by MS has been used to reveal an unparalleled number of unique peptide identifications from human cellular samples (Branca R. M., et al. HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics. Nature Methods, 2014 January; 11(1):59-62. Epub 2013 Nov. 17. PMID: 24240322).
In spite of the above there is still a need for improved methods and gels or strips for isoelectric focusing to enable reproducible isolation and enrichment of PTM peptides.