In-Depth Analysis of Protein Phosphorylation by Multidimensional Ion Exchange Chromatography and Mass Spectrometry - LC-MS/MS in Proteomics: Methods and Applications - page 114

time-of-flight (Q-TOF) mass spectrometer (Waters/Micromass
UK Ltd, Manchester, UK). In our example, we used a BEH
C 18 100-

μ ×

100-mm column (Waters/Micromass UK Ltd,
Manchester, UK).
1. LC-MS/MS mobile phases: solution A (0.1% FA in
LC-MS grade water) and solution B (0.1% FA in LC-MS
grade ACN).
2. Sample resuspension solution: 0.1% TFA.

m

3. Methods

Protein phosphorylation is a reversible and dynamic process that
regulates essential biological functions in health and disease. The
identification of phosphorylation sites can provide mechanistic
information at the molecular level and serve as read-outs of
pathway activities. In this sense, mass spectrometry combined
with phosphopeptide enrichment methodologies has emerged as
a powerful method for phosphoproteomic studies ( 9 , 10 ) .
Among the numerous phosphopeptide enrichment strate-
gies available, IMAC and TiO 2 have become the most popular
techniques due to their simplicity and great selectivity to iso-
late phosphopeptides. IMAC is based on electrostatic interactions
between the positive charge of the immobilised metal ions (such
as Fe(III) or Ga(III)) and the negative charge of phosphopep-
tides ( 11 ) . Although non-specific recovery of peptides containing
acidic amino acids represents a problem, pH/acid-controlled con-
ditions raise the specificity of IMAC ( 12 ) . TiO 2 instead has shown
higher selectivity for phosphopeptides than IMAC, with lower
unspecific binding, and a higher recovery of less acidic (mainly
monophosphorylated) peptides ( 13 , 14 ) . These findings suggest
that the complementarity of the two approaches may have poten-
tial for comprehensive phosphoproteomic profiling. This concept
was applied by Thingholm and colleagues to develop a combined
approach named 'sequential elution from IMAC' (SIMAC) ( 8 ) ,
which is based on differential elution of phosphopeptides accord-
ing to their acidity from the IMAC material using acidic and basic
elution conditions. The less acidic fractions eluted from IMAC
(containing a larger proportion of monophosphopeptides) are
then further enriched using TiO 2 chromatography.
The SIMAC method produces three fractions per sample.
This is often insufficient fractionation when the aim is to per-
form an in-depth analysis of the phosphoproteome. We propose
therefore a combination of SCX sample fractionation followed by
phosphopeptide enrichment by SIMAC in order to more com-