Advances in Microscale Protein Fractionation and Analysis

Fractionation is an essential step in the identification of proteins and their subsequent structural characterization. This information, in turn, is vital to understanding protein participation and function in normal and pathological cellular processes. The vast range in protein abundance (at least 6 orders of magnitude in dynamic range), differences in solubility, and relative amenability to separation by established methods (which are often time consuming and manually intensive) have created the need for new approaches to fractionation that can overcome these limitations. Specifically, problems that should be addressed include:

  • More efficient concentration, separation, and capture of low-abundance proteins, which can remain undetected in the presence of large-abundance proteins
  • Reduction in sample complexity by prefractionation to increase the potential for detecting a larger complement of proteins within a sample
  • Separation of proteins that tend to be insoluble in conventional gel separation buffers such as hydrophobic and membrane proteins
  • Concentration of fractions and minimization of losses due to manual handling or other loss-prone manipulations
  • Reduction in fractionation processing time, which greatly impacts R&D productivity.

One approach that has proven to be effective in meeting these challenges is liquid phase or free-flow isoelectric focusing (IEF). This technique establishes a pH gradient within a liquid buffer causing dissolved ampholytic molecules, such as proteins, to migrate until they reach a pH within the gradient corresponding to their isoelectric point (pI)—i.e., they have no net charge and therefore accumulate. Such an approach has proven fruitful in concentrating and/or purifying proteins with differing pIs. Accordingly, IEF provides a means for fractionating proteins that cannot be well separated by size or affinity. Because it is an orthogonal method, IEF can also be employed as an adjunct to enhance separation and reduce fraction complexity in conjunction with other protein fractionation techniques.

In performing protein fractionation, it is necessary to determine whether the sample has been properly conditioned (qualification) and is within the appropriate concentration range (quantification). Quantification is important in research planning, such as determining how much of each fractionated protein analyte is available for follow-up experiments. While satisfactory quantification can be achieved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), there are considerable limitations associated with this method, in particular, tedious and time-consuming manual sample processing. For these reasons, it is highly desirable to rapidly obtain relevant analytical information so as not to impede the pace of the work flow.

An elegant solution that meets these requirements is an analytical technique that utilizes electrophoretic-based separations within the confines of a microfluidic chip. The microscale dimensions of such chips enable the rapid and accurate analysis of very small sample aliquots of either protein or RNA. Recently, two instruments have been developed that employ the two different adaptations of electrophoresis indicated for the respective fractionation and analysis of microscale samples: the MicroRotofor system for protein fractionation and the Experion system for protein or RNA analysis (Bio-Rad Laboratories, Hercules, CA).

MicroRotofor protein separation system

Figure 1 - MicroRotofor system. The system is assembled according to the protocol outlined in the instruction manual and filled with 2.5 mL of a properly adjusted IEF ampholytic buffer (pH 3–10) containing 2.5 μg–2.5 mg of total protein to be fractionated. The apparatus is then placed in the chassis prior to separation. (Replacement of the focusing chamber is recommended after 4–5 runs with the same protein.) The system fractionates the sample into 10 discrete 200–250 μL fractions while applying continuous cooling in order to maintain protein integrity. Sample temperature can be held at ~20 °C or ~10 °C, corresponding to denaturing or nondenaturing conditions. After fractionation is complete, the focusing chamber is moved to an internal harvesting station, containing 10 harvesting needles aligned to penetrate the focusing chamber at each fraction location. Each fraction is drawn into its respective needle, and transferred by means of vacuum aspiration, into the appropriate lane in the harvesting tray located directly beneath the harvesting station cradle. The trays can be reused 5–10 times and then discarded.

Figure 2 - MicroRotofor free-flow IEF—enhancement of protein fractionation. A MicroRotofor separation is considered, in comparison with other protein fractionation techniques, to be an orthogonal method, i.e., it employs a different separation mechanism. More effective separations can generally be achieved with two orthogonal methods employed in sequence than with any single technique applied iteratively. The work flow depicted shows a variety of orthogonal methods that can be enhanced by an initial MicroRotofor fractionation. In effect, the 10-band MicroRotofor fractionation becomes the first dimension of a second-dimension or higher multidimensional separation scheme, with the downstream dimensions selected to suit the specific application.

Advances in current tools for proteomics research have enhanced the ability to work with smaller sample volumes and protein amounts. The MicroRotofor cell was designed to meet this requirement. Like the Rotofor® and MiniRotofor cells (Bio-Rad), the MicroRotofor cell utilizes liquid-phase IEF, but on microscale protein samples (~2.5 mL). In addition to reducing sample complexity for follow-up fractionations and/or experimentation, MicroRotofor IEF separations can be employed to enrich the low-abundance component of a protein sample, thereby increasing the effective sample load and/or dynamic range of detection. The system’s high-resolution capability facilitates the fractionation of proteins that are typically difficult to separate, such as closely related isoforms. Separations can be run under denaturing or nondenaturing conditions. Figure 1 illustrates the system components and outlines the IEF protocol. Figure 2 gives an overview of a number of separation techniques that can be enhanced by coupling with a MicroRotofor-based fractionation.

Applications

Figure 3- Enrichment of mouse brain proteins using the MicroRotofor cell. Comparison of 2-D gels of rat brain: a) unfractionated total protein (500 μg/gel), b) MicroRotofor fraction 5 (pH 7.5, 300 μg/gel), c) enlarged region (square) of unfractionated total protein gel in the same pH range as the MicroRotofor fraction shown, and d) enlarged region (square) of MicroRotofor fraction showing enrichment of many spots. Protein fractionation in the MicroRotofor cell improves the 2-D resolution of low-abundance proteins that are not clearly detectable in the unfractionated sample regardless of sample load. Increased sample loads of unfractionated sample simply lead to increased streaking, which reduces resolution and obscures low-abundance proteins.

The utility of a MicroRotofor fractionation in combination with other orthogonal separation mechanisms is demonstrated here.1 A proteomics application work flow was modified by first using the system on an E. coli lysate. Portions of the 10-band MicroRotofor fractionation were harvested and subjected to 1-D SDS-PAGE followed by in-gel tryptic digestion of selected bands and LC-MS-MS analysis of the digest peptides. The results indicated (data not shown) that each of the 10 fractions produced 10–50 protein bands in the follow-up SDS-PAGE separation, and that 2–5 proteins were identified from each band. It is estimated that 500 or more proteins could be identified by this approach, which is comparable to the multidimensional protein identification technology (MudPIT) approach but is less complicated. Comparison with a conventional 2-D PAGE separation confirms that the multiplexed MicroRotofor/SDS-PAGE approach gave equal or better results in the number of proteins detected. Moreover, the free-flow IEF fractionation minimized sample loss due to insolubility problems; eliminated the need to concentrate the crude protein extract prior to separation; and enabled the use of minigels for the SDS-PAGE step, simplifying and accelerating the work flow upstream of the LC-MS-MS analysis.

In a separate enrichment experiment, two samples of mouse brain protein were separated with 2-D gels. One sample was fractioned using the MicroRotofor. Comparison of the pH 7.5 Micro-Rotofor fraction with the same pH region of the unfractionated gel demonstrated enrichment of spots attributable to low-abundance proteins in the sample. This result was independent of sample load (Figure 3).

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