Table 1.
Advantages and disadvantages of S-palmitoylation detection and identification methods.
| METHOD | ADVANTAGES | DISADVANTAGES | EXAMPLES OF IDENTIFIED PROTEINS | REFERENCE |
|---|---|---|---|---|
| METABOLIC LABELING | ||||
| Metabolic radioactive labeling | • Sensitive and effective • Allows monitoring S-PALM turnover |
• Can be utilized only for a single protein • The S-PALM/unpalmitoylated protein ratio cannot be determined • Does not provide information on the exact site of S-PALM •Radioactive • Hazardous and time-consuming |
Ankyrin, Gαi, α2A-adrenergic receptor, H-Ras, N-Ras, p21N-ras BACE1, HTT, GluK2, 5-HT1A, D1R, 5-HT1B, 5-HT3A, 5-HT4, and 5-HT7 | Schmidt et al., 1979; Magee et al., 1987; Staufenbiel, 1987; Degtyarev et al., 1994; Kennedy and Limbird, 1994; Bizzozero, 1995; Magee et al., 1995; Jin et al., 1999; Baker et al., 2003; Papoucheva et al., 2004; Kvachnina et al., 2005, 2009; Ponimaskin et al., 2005; Renner et al., 2007; Veit et al., 2008; Copits and Swanson, 2013 |
| Metabolic non-radioactive labeling | • Allows robust in-gel visualization • Allows global profiling of S-PALM proteins • Allows monitoring S-PALM turnover |
• Highlights endogenously biotinylated proteins • Cannot be used to study palmitoylation in higher organisms •Only in vitro studies • Non-specific (other fatty-acylated proteins are labeled) • Does not provide information on the exact site of S-palmitoylation |
APP, SNAP23, RAP2B, calnexin, D2R GNAQ, and flotillin 1 | Drisdel and Green, 2004; Roth et al., 2006; Charron et al., 2009a,b; Hannoush and Arenas-Ramirez, 2009; Martin and Cravatt, 2009; Fukata and Fukata, 2010; Yount et al., 2011; Ebersole et al., 2015 |
| Click chemistry-proximity ligation assay | • Allows visualization of single S-PALM protein • Provides information on S-PALM protein subcellular localization • Can be used to investigate colocalization of S-PALM protein with other proteins |
•Can only be used on fixed samples • Cannot be applied to cells that are transfected with plasmids that encode tagging proteins (e.g., GFP) • Can generate non-specific signals from endogenous biotin • Does not provide information on the exact site of S-PALM |
Wnt, Sonic Hedgehog, H-Ras, | Gao and Hannoush, 2014 |
| BIOCHEMICAL TOOLS | ||||
| Acyl-biotin exchange assay | • Provides large-scale profiling of protein S-PALM • Allows quantitative analysis of S-PALM proteins • Both individual proteins and total protein extracts can be analyzed |
• Does not provide information on the exact site of S-PALM •Requires multiple reactions and purification of samples, which can cause substantial sample loss •Incomplete blockade of free Cys residues can result in false positives •Indirect method • Insufficient hydrolysis of thioester by hydroxylamine or inefficient biotin labeling may generate false negatives |
LIM kinase, AMPA, NMDA NR2A and NR2B receptor subunits, 5HT3A, SNAP-25, PSD-95, APP, VGLUT1, small GTPase Ras, MBP, D3R, D4R | Wan et al., 2007; Kang et al., 2008; Sato et al., 2009; Emmer et al., 2011; Ho et al., 2011; Hemsley et al., 2013; George et al., 2015; Zhang and Kim, 2016; Zhang et al., 2016 |
| Palmitoyl Protein Identification and Site Characterization | • Provides large-scale profiling of protein S-PALM • Allows quantitative analysis of modified proteins • Both single proteins and total protein extracts can be analyzed • Allows identification and S-PALM site characterization |
•Requires multiple reactions and purification of samples, which can cause substantial sample loss •Incomplete blockade of free Cys residues can result in false positives •Indirect method • Insufficient thioester hydrolysis by hydroxylamine or inefficient biotin labeling may generate false negatives |
PSD-95, SNAP 25, NOS, Fasn, PLP, NMDA, glutamate receptor, AMPA, Ddah1, Gja1, AP2A2, ADAM10, AP2, or Ahna | Yang et al., 2010; Collins et al., 2017 |
| Resin-assisted capture of S-acylated proteins | • Provides large-scale profiling of protein S-PALM • Allows quantitative analysis of modified proteins •Allows for S-PALM site identification |
•Requires multiple reactions and purification of samples, which can cause substantial sample loss •Incomplete blockade of free Cys residues can result in false positives •Indirect method |
Gαs, Gα11, H-ras, Uba1, SNAP23, Sec61b | Forrester et al., 2010, 2011 |
| Acyl-PEG exchange | • Allows visualization of single S-PALM protein • Allows visualization of a number of S-PALM sites |
•Requires multiple reactions and purification of samples, which can cause substantial sample loss •Incomplete blockade of free Cys residues can result in false positives •Indirect method |
IFITM3, | Percher et al., 2016, 2017 |