TABLE 1.

Comparison of current AMPylation screening methods.

No	1	2	3	4	5
Platform	AP-MS	AP-MS	MS	AP-MS	NAPPA
Method	Click chemistry+MS	Fluorescent ATP+MS	Isotope labeling+MS	Anti-AMPylation antibody+MS	Click chemistry+NAPPA
Type of ATP	N6pATP	Fl-ATP	Isotope-labeled ATP	ATP	N6pATP
Source of substrate proteins	Cell lysate	Cell lysate	Cell lysate	Cell lysate	cell-free produced recombinant human proteins
Isolation of AMPylated proteins	Pull-down	Pull-down	No	Pull-down	No
Identification	MS	MS	MS	MS	Fluorescence
Potential target identified	1 known substrate for VopS (Cdc42)	6 for VopS, including 2 known substrates (Rac1 and Cdc42)	1 for Bep2	None reported	27 for VopS and 29 for IbpA, including 3 known substrates for each AMPylator (RhoA, Rac1 and Cdc42)
Advantages	Natural proteins, with PTMs	Natural proteins, with PTMs	Natural proteins, with PTMs	Natural proteins, with PTMs	No protein abundance issue
Possible reason of failed target identification	Low abundance in cell lysate; quality of capture molecules; loss of proteins during pull-down and sample preparation	Low abundance in cell lysate; quality of capture molecules; loss of proteins during pull-down and sample preparation	Isotopic toxicity; Low abundance in cell lysate; loss of proteins during pull-down and sample preparation	Low abundance in cell lysate; quality of anti-AMPylation antibodies; loss of proteins during pull-down and sample preparation	Protein may not be well-folded or with PTMs
Reference	Grammel M, et al.19	Lewallen DM, et al.16	Pieles K, et al.14	Hao Y, et al.17	Yu X, et al.10

^*AP-MS, affinity-purification mass spectrometry; NAPPA, Nucleic Acid Programmable Protein Arrays.