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 f (μM)	CuAAC with 1		CuAAC with 3
			Amount of 10 (pg) per μg proteins	Yield h (%)	Amount of 10 (pg) per 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

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

^bControl 2 (positive control), the CuAAC reaction was performed with the total extracted HPG-containing proteins from cell lysate under the optimal conditions (see text).

^cDetached the cells with a cell scraper in cold PBS after HPG incorporation.

^dAdherent OVCAR5 cells were incubated in 100 μM NEM in PBS for 10 minutes after HPG incorporation.

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

^fDetermined by LC-ESI-MS/MS upon alkylation of the cellular GSH with NEM (ESI).

^gNot measurable with this method.

^hYield (%) was determined by the amount of CuAAC reaction product 10 (pg) per protein (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 the cytosolic proteins by the membrane proteins that contain 18% of the product 10.