HPLC for Fractionation
Agilent Technologies (www.agilent.com) is using multidimensional HPLC (MDLC) for fractionation and analysis of intact proteins, according to Alex Apffel, Ph.D., senior research scientist, molecular technology laboratories. This method extends Agilent's work in off-line multidimensional coupling strong anion exchange and reversed phase HPLC by directly splitting the flow from the second-dimension reversed phase separation, so it simultaneously collects fractions and ESI-TOF MS data on intact proteins.
Adding the prefractionation techniques increases dynamic-range capabilities and reduces sample complexity to create an integrated workflow to understand protein-expression patterns, Dr. Apffel explains. And, he continues, using orthogonal information, based on intact protein characteristics constrains the search for such attributes as hydrophobicity. Separations are performed in liquid phase, Dr. Apffel explains, for improved recovery, resolution, and easy automation.
"Compared to conventional 2-D gel electrophoresis, this method separates a broader range of proteins, and is fast, robust, automatable, reproducible, and easy to use."
In terms of applications, "the MS data can be used for protein characterization," Dr. Apffel adds and to compare and differentiate samples by deconvoluting partially separated peaks in the 2-D separation space. "Informatics and data visualization play a key role," he says, "in identifying key features for subsequent identification and analysis at the peptide level. Statistical comparison of multiple samples allows identification of significantly differentially expressed features.
"The challenge in fractionating and analyzing intact proteins is like finding a pico-needle in a stack of needles," because the dynamic range of protein expression can be very broad (1012).
"Proteomics extracts such vast quantities of data that sometimes we don't remember the question. Therefore, we need to ask specific questions," Dr. Apffel adds, to return to hypothesis-driven science. "For example, if identifying key proteins in a process is useful, that may be more important than identifying 100 proteins in a sample."
Novel ultrahigh-pressure split-less nano HPLC systems are resulting in dramatic increases in resolution and sensitivity for complex proteomic analysis, according to Frank Yang, Ph.D., president, Micro-Tech Scientific (www.microlc.com).
The challenge, Dr. Yang says, is to move beyond atta-mole detection and increase complex biological sample resolution using small diameter nano-HPLC columns with small particles. To that end, Micro-Tech has developed an ultrahigh-pressure splitless nano-HPLC system that operates at pressures of up to 15,000 psi (1,000 bars) with split-less gradient flow rates as low as 100 microliters per minute for columns with inner diameters of 25 to 150 microns.
"There are two ways to further improve resolution in any binary gradient system of a multidimensional processincrease column lengths or reduce particle size," he adds. In lab tests, he found that increasing column length from 15 cm to 100 cm and using 3-micron particles (rather than 5-micron particles) achieved a greater than two-fold increase in the number of peptides and proteins identified by increasing the operating pressure under the same gradient conditions.
For example, running a 15-cm column at 1,000 psi with a flow rate of 0.4 microliters/minute and a 120-minute analysis time identified 865 peptides and 191 proteins. Increasing the column length to 100 cm and the pressure to 8,000 psi increased the number of peptides identified to 1,217 and the number of proteins to 276. Then, increasing the analysis time to 356 minutes and reducing the acetonitrile gradient percent change from 60 to 35% resulted in the identification of 2,351 peptides and 465 proteins.
"The increased resolution of complex biological samples (mouse brain homogenate, P2 fraction) was demonstrated by using a 100-cm x 75-µm ID capillary column packing with 3-µm particles operated under 8,000-psi ultrahigh pressure," Dr. Yang elaborates.