Human 14 Multiple Affinity Removal System Spin Cartridge From Agilent

One of the barriers to an informative proteomic analysis of serum is the presence of a relatively small group of very abundant proteins. These proteins, which include albumin, immunoglobulins, complement proteins, and others, make up over 90% of the total protein content of serum. Since the concentration of the majority of these proteins doesn’t change appreciably or consistently with most disease states, they are essentially uninformative with regard to the identification for disease-relevant changes in the serum proteome. Hence, one of the most effective ways of dealing with these proteins is simply to remove them prior to serum analysis by means of affinity chromatography.

Agilent’s foray into the field of abundant serum protein depletion includes the Human 14 Multiple Affinity Removal System (MARS 14) spin cartridge. As indicated by the name, the spin cartridge is designed to remove 14 of the most abundant proteins from up to 10 ul of serum per run. These proteins are albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, and transthyretin. The cartridge is small, containing only 0.45 ml affinity matrix, and can nest inside a 1.5 ml centrifuge tube for use in standard laboratory microfuges. Although the cartridge, plus accessories and buffers, is a bit pricey at close to $2000, the ability to reuse it up to 200 times without loss of activity brings the cost of each depletion down to the very affordable neighborhood of $10 each.

The depletion protocol is very simple as it involves only two buffers: Buffer A (for serum dilution, cartridge equilibration, and wash) and Buffer B (for elution of bound abundant proteins). Serum, diluted 1:20 in Buffer A, is spin filtered through a 0.22 um device and applied to the cartridge. The cartridge is then centrifuged at 100 x g for 1 min. The cartridge is allowed to sit at room temperature for 5 min and then 2 x 400 ul washes with Buffer A are spun through at 100 x g for 2.5 min each. The three flow through fractions contain the serum proteins, now largely free of the 14 abundant proteins mentioned above, and can be pooled. The cartridge is regenerated by using a syringe to slowly push through 2.5 ml of Buffer B followed by 5 ml of Buffer A.

The effectiveness of the depletion is demonstrated in the figure to the left. The gel is a 4-12% NuPAGE bis-tris gel, run in MES buffer, and stained with SimplyBlue SafeStain (all from Invitrogen). Lane M contains MW markers; lane S contains 5 ug serum proteins; lanes D1 and D2 contain 5 ug each of depleted serum proteins from two successive runs of the column. It is evident that depletion has increased the relative abundance of many proteins as compared to an equivalent amount of undepleted serum. About half of the total protein content of serum is made up of albumin, which is the heavy band in lane S. Loading 5 ug serum only shows albumin, IgG, and a few other abundant proteins. After depletion, many of the lower abundant proteins are evident. I have used the MARS 14 cartridge to deplete over 20 serum samples and the reproducibility is remarkable.