Enrichment and quantification strategies used to study cAMP-dependent phosphoproteins using phosphoproteomics

Phosphopeptide enrichment
32P-labeling and 2D gel electrophoresisCells 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 spectrometryChu et al., 2004
 HAMMOC resinPhosphoproteins are enriched using resins with hydroxy acid–modified metal oxidesImamura et al., 2017
 MOAC matrixPhosphoproteins 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 resinPhosphoproteins 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 phosphoproteinsde Graaf et al., 2014; Golkowski et al., 2016; Williams et al., 2016; Beltejar et al., 2017; Isobe et al., 2017
Protein quantification
 Label-free quantificationAll 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 proteinLundby et al., 2013; de Graaf et al., 2014; Beltejar et al., 2017
 SILAC labelingCells 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 massesGolkowski et al., 2016; Williams et al., 2016; Imamura et al., 2017
 TMT labelingProteins 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 samplesImamura 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.