Table 2.

Comparison of CuAAC reaction yields on/in live OVCAR5 cells between HPG–proteins and the biotin-azide 9 in the presence of the ligand 1 vs 3.

Pre-treatment	Protein	Intracellular GSH (µM)[f]	CuAAC with 1		CuAAC with 3
			Amount of 10 (pg)/µg proteins	Yield (%)[h]	Amount of 10 (pg)/µg proteins	Yield (%)[h]
Control 1[a]	Whole-cell		< 0.10	<0.2
Control 2[b]	Whole-cell		49.18 ± 0.95	100
(−)	Membrane		3.03 ± 0.25	6.2	8.83 ± 0.82	18.0
(−)	Cytosolic	1585 ± 29	0.34 ± 0.02	0.5	0.48± 0.05	0.8
Scraping[c]	Cytosolic	427 ± 17	1.09 ± 0.05	2.0	4.28 ± 0.28	8.5
NEM[d]	Cytosolic	N.D.[g]	2.08 ± 0.29	4.0	7.02 ± 0.42	14.1
BSO[e]	Cytosolic	4.9 ± 0.4	0.49± 0.05	0.8	1.04± 0.06	1.9

^[a]Control 1 (negative control), the CuAAC reaction was performed with the adhered cells without HPG incorporation into cellular proteins.

^[b]Control 2 (positive control), the CuAAC reaction was performed with the total extracted HPG-containing proteins from cell lysate under the optimal condition (see text).

^[c]Detached the cells with a cell scraper in cold PBS after HPG incorporation.

^[d]Adherent OVCAR5 cells were incubated in 100 µM NEM in PBS for 10 minutes after HPG incorporation.

^[e]Adherent OVCAR5 cells were incubated in RPMI-1640 medium containing 20 µM BSO for 24 hours before HPG incorporation.

^[f]Determined by LC-ESI-MS/MS upon alkylation of the cellular GSH with NEM (ESI†).

^[g]Not measurable with this method.

^[h]Yield (%) was determined by the amount of CuAAC reaction product 10 (pg) per proteins (µg) divided by the result obtained in control 2 (ESI†). All measured yields of cytosolic proteins were deducted by 0.2% due to the 2% contamination of cytosolic proteins by the membrane proteins that contain 18% of the product 10.