Phosphorylated proteins and peptides are studied intensively in the field of proteomics, with good reason. All sorts of biological processes occurring in cells involve phosphorylation, including signalling, metabolism, programmed cell death (apoptosis) and cell differentiation. So, the isolation, measurement and identification of phosphorylated compounds is one of the top priorities for scientists in this area.

The classical method in large-scale studies involves enzymatic digestion of proteins, either in complex mixtures or following some degree of separation. The resulting peptide mixtures contain both phosphorylated (PPs) and non-phosphorylated peptides (NPs) and the two classes are generally separated to that the NPs do not interfere in the ensuing mass spectrometric analysis. This procedure is governed to some extent by the other compounds present in the mixture, such as enzymes, buffers and detergents, as well as the compounds likely to be present after purification, which might affect the subsequent analysis.

Some of the most popular PP/NP separations involve the use of chromatographic resins with a particular affinity for the phosphate group. Immobilised metal affinity chromatography (IMAC) and titanium dioxide and zirconium dioxide chromatography fall into this category. IMAC is the most popular but it suffers from interference from low-molecular-mass contaminants such as EDTA, alkaline metal salts and some detergents, so a pre-purification reversed-phase chromatographic step is employed. However, this extra step leads to sample losses and the risk of phosphopeptide loss.

In an effort to optimise phosphopeptide enrichment, two scientists from the University of Southern Denmark have compared iron(II)-based IMAC to TiO₂ chromatography for PP/NP separation. Martin Larsen and Soren Jensen chose TiO₂ because previous studies had shown it to be highly specific towards PPs, eliminating the need for a pre-purification step. In this new research, they tested the effects of additives on both techniques, using a peptide mixture prepared by tryptic digestion of a mixture of 12 standard proteins. The purified phosphopeptides were analysed by MALDI mass spectrometry.

In the first instance, the researchers confirmed that strong acid such as trifluoroacetic acid (TFA) gave reasonably efficient PP enrichment but the addition of phthalic acid to TFA prevented virtually all of the remaining NPs from binding to TiO₂. However, for more complex mixtures such as cell lysates, as opposed to standard proteins, extra NP excluders are required.

Several acids such as oxalic, gallic and phthalic acid were tested as NP excluders. Phthalic acid contaminates the HPLC column and the mass spectrometer, while other acids interacted with TiO₂, caused non-specific binding, or excluded some PPs. The most effective acid was glycolic acid, which trapped the majority of PPs.

Next, the effects of a range of detergents and surfactants on PP enrichment by IMAC and TiO₂ were studied. Inclusion of SDS, ASB14, Triton X-100 or RapiGest, which interfere with PP purification by IMAC, had no negative effects in TiO₂ chromatography. The intensities of the peptide mass spectrometric signals of all the additives were comparable to those obtained with TFA, confirming that the procedure is robust and reproducible. Indeed, the presence of SDS and RapiGest increased signal intensities, probably due to reduced binding of PPs to the plastic surfaces of the pipette tips or Eppendorf tubes.

The same reagents interfered with binding in IMAC, in which multiply phosphorylated peptides were bound more strongly than singly phosphorylated peptides, resulting in a skewed PP representation.

Phosphate-buffered saline had no effect on TiO₂ chromatography but led to very low PP recovery by IMAC. EDTA is often added to biological mixtures to trap magnesium ions, so inactivating kinase enzymes. However, it cannot be added prior to IMAC because it strips the resin of Fe(II) ions. With TiO₂, EDTA gave no such problems.

Further comparisons between TiO₂ and ZrO₂ chromatography, a lesser PP purification technique, showed that the former is more efficient. However, oxides sourced from different suppliers had different surface properties, affecting the PP binding affinities.

Overall, it is clear that TiO₂ chromatography is a better alternative to IMAC. It is far more tolerant of the normal chemicals encountered in protein isolation and proteomics analysis and eliminates the need for a RP-HPLC pre-purification step.

Related links:

• Department of Biochemistry and Molecular Biology, University of Southern Denmark
• Rapid Communications in Mass Spectrometry, 2007, 21, 3635-3645: "Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

Larsen - enriching phosphopeptides