40 A Practical Guide to Cancer Systems Biology
On account of the Lewis acid–base character of TiO 2 , the phosphate groups
of peptides can efficiently bind to TiO 2 under acidic condition but desorbed
under alkaline condition. However, the acidic peptides rich in glutamic and
aspartic acids are often absorbed to the TiO 2 , leading to the non-specific
binding during phosphopeptide enrichment.^5 In 2008, hydroxyl acid-modified
metal oxide chromatography (HAMMOC) was developed as a highly efficient
method to enrich phosphopeptides.^6 An aliphatic hydroxy acid, such as
lactic acid, is used to increase the specificity of phosphopeptide enrichment
by competing with acidic non-phosphopeptides. Therefore, only phospho-
peptides can successfully bind to the TiO 2 due to its high affinity.6,7 In
comparison with other strategies of phosphopeptide enrichment, HAMMOC
is easy to perform, and the coverage on phosphopeptide identification can
be improved.^7
During the past few years, the detection of protein phosphorylation was
extraordinarily improved due to the rapid development in mass spectrometry
and proteomic technologies.1,2Based on the MS-based phosphoproteomics,
complex protein samples are enzymatically digested into peptide mixtures,
where the phosphopeptides are specifically enriched by affinity- or antibody-
based methods. The peptide samples can be separated by nano-scale liquid
chromatography and analyzed by mass spectrometry (nanoLC-MS/MS).^8
In this chapter, we describe a strategy to prepare samples for quantitative
phosphoproteomic study by combining many previously published methods
including phase transfer surfactant-aided trypsin digestion,^9 dimethyl label-
ing,^10 and HAMMOC.^7 Many processes can be performed with StageTip.^11
This strategy provides a simple and efficient way for sample preparation
before LC-MS/MS analysis.
- Protein extraction by phase-transfer surfactant (PTS)
buffer^9
Materials:
- Sodium deoxycholate (SDC) (CAS NO. 302-95-4)
- Sodium N-lauroylsarcosinate (SLS) (CAS NO. 137-16-6)
- Protease inhibitor cocktails (100X)
- Ser/Thr phosphatase inhibitor cocktails (100X)
- Tyr phosphatase inhibitor cocktails (100X)