Enrichment and quantification strategies used to study cAMP-dependent phosphoproteins using phosphoproteomics
| Technique | Description | References |
|---|---|---|
| Phosphopeptide enrichment | ||
| 32P-labeling and 2D gel electrophoresis | Cells are incubated with radioactive phosphate prior to protein isolation. Cellular proteins are separated using 2D gel electrophoresis, and labeled bands corresponding to phosphoproteins are isolated for mass spectrometry | Chu et al., 2004 |
| HAMMOC resin | Phosphoproteins are enriched using resins with hydroxy acid–modified metal oxides | Imamura et al., 2017 |
| MOAC matrix | Phosphoproteins are enriched using a matrix of metal oxides or hydroxides (e.g., titanium dioxide) | Lundby et al., 2013; Williams et al., 2016; Isobe et al., 2017 |
| IMAC resin | Phosphoproteins are enriched using IDA or NTA resins with associated metal ions. Because the metal ions are positively charged, they interact with and retain negatively charged phosphate groups on phosphoproteins | de Graaf et al., 2014; Golkowski et al., 2016; Williams et al., 2016; Beltejar et al., 2017; Isobe et al., 2017 |
| Protein quantification | ||
| Label-free quantification | All samples are prepared for mass spectrometry and individually analyzed by LC-MS/MS. Quantification is based on the comparison of peak intensity of the same peptide or the spectral count of the same protein | Lundby et al., 2013; de Graaf et al., 2014; Beltejar et al., 2017 |
| SILAC labeling | Cells are incubated with isotopically labeled amino acids prior to protein isolation. Samples are combined for LC-MS/MS analysis, allowing for relative quantitation of protein content between up to three samples based on their differential masses | Golkowski et al., 2016; Williams et al., 2016; Imamura et al., 2017 |
| TMT labeling | Proteins from up to 16 samples are digested for mass spectrometry and then labeled with isobaric chemical tags allowing for relative quantitation of protein content between samples | Imamura et al., 2017 |
HAMMOC, hydroxy acid–modified metal oxide chromatography; IDA, iminodiacetic acid; IMAC, immobilized metal-ion affinity chromatography; MOAC, metal oxide affinity chromatography; LC-MS/MS, liquid chromatography tandem mass spectrometry; NTA, nitrilotriacetic acid; SILAC, stable isotope labeling with amino acids in cell culture; TMT, tandem mass tag.