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Researchers Devise Array-Based 2D LC System for Plasma Depletion

NEW YORK (GenomeWeb) – Researchers at China's Fudan University have devised an array-based two-dimensional liquid chromatography system for depletion of high-abundance proteins in plasma samples prior to mass spec analysis.

Detailed in a paper published this week in Analytical Chemistry, the system could offer improvements to what is an essential step in many mass spec workflows.

A major challenge of mass spec analysis of plasma is the fact that high-abundance proteins tend to dominate the signal, suppressing that of the lower-abundance proteins that are often of interest in, for instance, disease biomarker research. Given this issue, mass spec workflows typically call for plasma samples to be processed to remove the highest abundance proteins prior to analysis.

A common approach for depleting plasma samples is use of immune-affinity columns, which use antibodies to a selection of high- abundance proteins to pull them out of samples. As the Analytical Chemistry authors noted, though, low-abundance proteins of interest are often attached to higher-abundance proteins, and so can be lost during this depletion step. Additionally, such columns deplete only the small selection of proteins targeted by the antibodies they contain.

Initially seeking to tackle these issues, the Fudan researchers developed an offline anion exchange chromatography/reversed phase LC system. Using this system for a study of the human liver proteome, they depleted dozens of high-abundance proteins from their samples.

The system's throughput was too low for routine use in discovery proteomic workflows, though, and so, aiming to improve on this first attempt, the researchers constructed a two-dimensional AEC/RPLC system featuring eight reverse-phase LC columns in its second dimension, allowing for parallel separations. Additionally, the columns used were conventional-sized columns, which allowed for a significant increase in the amount of sample that could be injected compared to the original system, which used capillary columns.

These changes brought the required depletion time down to four hours, which, they noted, was comparable to the time required for immune-depletion-based methods.

They used the system for analysis of human plasma, linking it to a nano Acquity UPLC system and a Thermo Fisher Scientific LTQ Orbitrap XL mass spec. In this analysis they identified a total of 1,332 proteins, including several proteins at very low abundances, such as C-X-C motif chemokine 9, androgen receptor, and inosine-5'-monophosphate dehydrogenase 2, all of which were present at levels of .01 ng per mL. Overall, they detected proteins across nine orders of dynamic range, with the depletion step removing 84 high-abundance proteins.