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=IMAC and MOAC: Application to Phosphoproteomics=

Immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) are chromatography methods used to enhance proteins that have undergone phosphorylation for detection and characterization. Phosphorylation is one post translational modification (PTM) that is particularly difficult to detect and characterize in peptide fragments. Difficulties arise because the ratio of phosphorylated to nonphosphorylated peptides is very small. Because of this, the signal obtained from mass spectrometry analysis is especially weak. Various types of chromatography can be used to enrich samples to make the detection by mass spectrometry clearer.

With regards to the enrichment of proteins that have undergone phosphorylation, multiple methods of chromatography have been developed and optimized, most notably immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC). These processes take advantage of the affinity of phosphorylated species to metal ions (such as iron) or metal oxides (such as titanium dioxide, aluminum hydroxide, or zirconium dioxide), immobilized in a column, to separate the desired phosphopeptides from nonphosphorylated peptides. The phosphopeptides are washed from the column and transferred to a mass spectrometer for analysis.

Immobilized Metal Affinity Chromatography (IMAC):
Immobilized metal affinity chromatography is the most widely used method for enrichment of phosphoproteins. It is based on the affinity of the metal ion for the phosphate group, so once the sample is added to the column, the phosphopeptides stick to immobilized iron, and any nonphosphopeptides are washed out with a gradient solution of acetic acid and water. The phosphopeptides are then released from the IMAC beads with ammonium hydroxide (NH4OH) solution, and collected for separation and analysis. Studies have shown that IMAC has greatly improved the clarity in collected data, compared to results collected from experiments that did not use the enrichment step for phosphopeptides. However, this method presents disadvantages, the first one being the loss of sample in the IMAC column. The second drawback includes false positive results due to acidic amino acid residues (such as aspartic acid and glutamic acid) sticking to the column, along with true phosphopeptides. Also, there are multiple regeneration steps of stationary phase after each analysis. Problems with data accuracy arise from the differing affinities for the metal ions depending on the degree of phosphorylation of the peptide. The problem of false positive results due to acidic residue affinity for the metal ions has been addressed in recent scientific literature.

Synthetic chemistry methods have been proposed to try to solve the problem of unwanted acidic residues sticking to the IMAC beads in the columns. One way to approach this problem is to neutralize the acidic groups to reduce their attraction to the metal ions. This has been accomplished in two ways: methyl esterification and beta elimination of the phosphate group, plus an affinity tag to increase detection levels. These methods, though are sound in theory, do not work in practical application. They can cause many unfavorable side reactions, which make the sample more complex than the initial.

Another route to take when dealing with false positives due to highly acidic peptides is to treat a sample of the phosphorylated protein with alkaline phosphatase, a common dephosphorylating agent, and test it in parallel to the original sample with IMAC. If any peaks remain in the mass spectrum readout from the sample treated with alkaline phosphatase, it can be inferred that there are unwanted acidic residues present, and they have false positive effects on the original sample in IMAC. Although this solution greatly increases success in identifying false positives, the treatment with alkaline phosphatase is only a temporary solution because it includes extra sample preparation and procedures that are ultimately inefficient.

Metal Oxide Affinity Chromatography (MOAC):
The upgrade of the IMAC method, metal oxide affinity chromatography (MOAC), uses similar technique to IMAC, incorporating metal oxides, such as titanium dioxide TiO2, zirconium dioxide ZrO2, or aluminum hydroxide Al(OH)3, in place of metal ions. Metal oxides tend to have higher selectivity for phosphopeptides, making it easier to trap them in the column.

Metal Oxide Affinity Chromatography (MOAC):
Zhou et al. performed a study to examine the effects of ZrO2 nanoparticle beads for selectivity and efficiency for the enrichment of phosphoproteins. They chose ZrO2 for its high stability and amphoteric properties as a Lewis acid or base. Samples of α-casein (a model protein used in phosphoproteomics) were digested with trypsin, followed by either direct analysis with matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), or a ZrO2 enrichment step plus the analysis with MALDI-MS. Figure 1a and 1b show the improvement of detection when the ZrO2 enrichment step is employed prior to MS analysis. Figure 1(a) shows the 7 phosphopeptides detected strictly by MALDI-MS, while Figure 1(b) spectrum from the ZrO2 enriched digests reveals the same 7 found by direct MS analysis, plus an additional 10 phosphopeptides. Also noted is that the intensity of the phosphopeptides increases in 1(b) due to the absence of nonphosphorylated species.



Figure 1: MALDI-MS spectrum for purified phosphopeptides from tryptic digests of α-casein in (a) direct MS analysis and (b) analysis following enrichment with ZrO2 MOAC (Source: Zhou, Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis, 2007).

Comparison of IMAC and MOAC:
Zhou et al (2007) proposed another experiment to compare the efficacy of these two enrichment processes. The quality of ZrO2 beads used in MOAC was compared with that of traditional IMAC beads in two identical trials. Tryptic digests of β-casein and bovine serum albumin (BSA) were enriched with both ZrO2 and IMAC beads, and analyzed with MALDI-MS. Results are shown in Figure 2 (a-d). Judging from the drastic change in mass spectra in Figure 2(b-d), it is indicated that the enrichment with ZrO2 MOAC is more selective and specific than with IMAC methods. It is suggested that strong and specific bidentate-bridging coordination of phosphate groups to ZrO2 particles, and surface area and nanoscale effect may improve enrichment specificity and selectivity.



Figure 2: MALDI-MS spectrum for purified phosphopeptides from tryptic digests of BSA and β-casein in (a) direct MS analysis, (b, c) trapped peptides from ZrO2 nanoparticle enrichment in digest mixtures with molar ratios of 1:10 and 1:100, and (d) trapped peptides from IMAC enrichment (Source: Zhou, Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis, 2007).

Conclusions:
Based on experiments highlighted in recent experiments and literature, it can be concluded that MOAC is more efficient, cost-effective, and overall, more successful in enriching phosphopeptides for detection and analysis, than is IMAC. There are no extra steps to be taken when using MOAC, as there are for IMAC when problems of false positives arise. However, IMAC is still the most widely used method for enrichment, largely because MOAC technology is new enough that it has not yet been widely adopted.