Phosphoprotein/phosphopeptide enrichment

1. Immunoprecipitation

最常用的是tyrosine的抗体，anti-phosphoserine and anti-phosphothreonine antibodies are not yet routinely employed in phosphoproteomics due to their low specificity

2. Metal oxide affinity chromatography（MOAC）金属氧化物亲和色谱，最常用的是TiOx2。

Titanium dioxide (TiO2) chromatography的原理是TiO2与溶液中的磷酸盐离子有亲和性。

Pinkse et al. [20] presented an on-line 2-D/LC strategy for phosphopeptide analysis using spherical TiO2 particles球形二氧化钛粒子 (Titansphere) in the first dimension and reversed-phase (RP) material in the second dimension.

Under this configuration phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions酸性环境 on a TiO2 column. Trifluoroacetic acid (TFA) in loading buffer protonates acidic residues使酸性残基质子化 and prevents adsorption吸附 of nonphosphorylated peptides to TiO2.

After washing step, nonphosphorylated peptides flow through.

Subsequently, phosphopeptides are desorbed (eluted洗脱) from the TiO2 column under alkaline conditions碱性环境, separated on the RP column, and analyzed by nano-flow LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometry). NH4OH eluent at pH 10.5 affords a high recovery of phosphopeptides. The pKa and pKb values for Titania are 4.4 and 7.7, respectively [21], so at high pH, Titania should become negatively charged and allow phosphopeptide elution洗脱.

3. Immobilized metal Ion chromatography (IMAC)固定金属离子色谱法

4. On-plate enrichment for MALDI-Ms

MS analysis of phosphopeptides

1. 离子源

ESI或MALDI

2. 碎裂模式

常用的是CID

Quantitative phosphoproteomics

1. stable-isotope labelling by amino acids in cell culture (SILAC)

2. isotope tagging reagents for relative and absolute quantification (iTRAQ)

3. tandem mass tags (TMT

4.absolute quantification (AQUA)-selected reaction monitoring analysis (SRM)

5. Label free

Bioinformatics

1. Ascore [96]

presented by Gygi's lab , which measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra . It re-analyzes phosphopeptide search engine results to assign a confidence value to each phosphorylated site.配合SEQUEST使用

2. PhosphoScore [97]

From an open-source assignment program that is compatible with phosphopeptide data from multiple MS levels. is an algorithm that takes into account both the match quality and normalized intensity of observed spectral peaks compared to a theoretical spectrum.

3. Mascot Delta Score (MD-score)

was validated and achieved similar sensitivity and specificity for phosphosite
localization as the Ascore

4. databases gathering information about phosphorylation sites

PHOSIDA (http://www.phosida.com)
Phospho.ELM [99]