On first glance, a post translational modification is just another amino acid. However, for several modifications, there are complications.

• acid labile modifications do not survive chromatography conditions.
• glycosylation is unstable-the carbohydrate chain also fragments giving complex data; there are laboratories which specialize in this work.
• phosphorylation is a modification which has its own fragmentation in addition to peptide fragmentation.
• the modified protein may be only a small fraction of the total amount of protein-this is a routine problem with phosphorylation.
• the peptide with the modified amino acid may not show in the analysis.

Phosphorylation site analysis by mass spectrometry

History

Mass spectrometry has looked for phosphorylation sites ever since it has been used to sequence proteins. The success rate has been increasing which along with increased cost of using radio-isotopes has caused a reduction in the use of P-32 to look for phosphorylation sites.

Why it is difficult to identify phosphorylated peptides by mass spectrometry

1. stoichiometry of phosphorylation is often low e.g 0.1% so the peptide of interest is 1/1000 that of other peptides, and hence easily overlooked in a mixture
2. phosphopeptides lose phosphoric acid in the mass spectrometer and become peptides which do not fragment well.

A solution to problem 1 is to enrich phosphopeptides. Over the years numerous methods have been published to isolate phosphopeptides. Many methods showed isolation of phosphopeptides from casein, which is not representative of interesting phosphoproteins. Only a few methods have seen much use.

IMAC
Immobilized Metal Affinity Chromatography (IMAC) has been the most widely used method. Commercially available pipet tips with metal chelating resin were one of the methods which was not successful for typical samples. A number of laboratories have been successful making their own IMAC columns. A major problem for IMAC is that acidic peptides are often purified with phosphopeptides; esterification of glutamate and aspartate residues reduced the problem but caused its own complications. The biggest problem is that the technique is successful for some people but not others, even in the same lab.

Titanium dioxide
In 2005 (1) titanium dioxide was promoted as a chromatography resin to isolate phosphopeptides. Although some studies suggest IMAC in the hands of some can find more phosphopeptides, titanium dioxide chromatography seems to work adequately for most people and thus is more productive. Also some people report that titanium dioxide is superior. It is the method used locally to identify phosphorylation sites for the Cell Migration Consortium. Like IMAC, it binds non-phosphorylated peptides. TFA, dihydroxybenzoic acid (DHB) and lactic acid (2) are used by investigators to improve the specificity of titanium dioxide chromatography.

Ion exchange
Strong cation exchange chromatography has been used by some, by itself and in combination with titanium dioxide chromatography, to isolate phosphopeptides.

1.   M.R. Larsen, T.E. Thingholm, O.N. Jensen, P. Roepstorff, T.J.D. Jørgensen (2005) Molec. Cellular Proteomics 4 873-886

2. N. Sugiyama, T. Masuda, K. Shinoda, A. Nakamura, M. Tomita, and Y. Ishihama Molec. Cellular Proteomics 6 1103-1109, 2007