Table 1.

Ubiquitin quantification by the LC-SRM analysis

Pepetide Names	Peptide Sequences	Synthetic peptides		SILAC peptides		SILAM peptides		Precursor Ions (m/z)					Product Ion (m/z)					RT (min)
Pepetide Names	Peptide Sequences	Labeled AA	Mass Shift (Da)	Labeled AA	Mass Shift (Da)	Labeled AA	Mass Shift (Da)	z	Native	Synthetic	SILAC	SILAM	Ion Name	Native	Synthetic	SILAC	SILAM	RT (min)
Ub (unmodified, TLS)	TLSDYNIQK	L2	7.0606	K9	8.0142	K9	6.0201	2	541.2798	544.8101	545.2869	544.2899	b1-2	215.1390	222.1996	215.1390	215.1390	13.1
Ub (unmodified, TLS)	TLSDYNIQK	L2	7.0606	K9	8.0142	K9	6.0201	2	541.2798	544.8101	545.2869	544.2899	y1-7	867.4207	867.4207	875.4349	873.4408	13.1
Ub (linear GG tag)	(GG)MQIFVK	I3	7.0606	K6	8.0142	K6	6.0201	2	440.2415	443.7718	444.2486	443.2515	b1-7	733.3702	740.4308	733.3702	733.3702	19.7
Ub (linear, oxidative)	(GG)M*QIFVK	I3	7.0606	K6	8.0142	K6	6.0201	2	448.2389	451.7692	452.2460	451.2490	b1-7	749.3651	756.4257	749.3651	749.3651	14.4
Ub (K6)	MQIFVK(GG)TLTGK	L8	7.0606	K6, K11	16.0284	K6, K11	12.0403	2	690.3894	693.9197	698.4036	696.4095	y1-8	1007.5884	1014.6490	1023.6168	1019.6287	21.7
Ub (K6 oxidative)	M*QIFVK(GG)TLTGK	L8	7.0606	K6, K11	16.0284	K6, K11	12.0403	2	698.3869	701.9172	706.4011	704.4070	y1-8	1007.5884	1014.6490	1023.6168	1019.6287	19.4
Ub (myc tagged K6)	LISEEDLGMQIFVK(GG)TLTGK	L16	7.0606	K14, K19	16.0284	K14, K19	12.0403	3	746.0680	748.4215	751.4108	750.0814	y2-17	1005.5142	1009.0446	1013.5284	1011.5344	28.9
Ub (myc-K6 oxidative)	LISEEDLGM*QIFVK(GG)TLTGK	L16	7.0606	K14, K19	16.0284	K14, K19	12.0403	3	751.4011	753.7546	756.7439	755.4145	y2-17	1013.5117	1017.0420	1021.5259	1019.5318	27.3
Ub (K11, yeast)	TLTGK(GG)TITLEVESSDTIDNVK	V20	6.0520	K5, K21	16.0284	K5, K21	12.0403	3	793.4148	795.4321	798.7576	797.4282	b2-20	1116.5657	1119.5917	1120.5728	1119.5758	22.1
Ub (K11, human)	TLTGK(GG)TITLEVEPSDTIENVK	V20	6.0520	K5, K21	16.0284	K5, K21	12.0403	3	801.4269	803.4442	806.7697	805.4403	y1-9	1002.5102	1008.5622	1010.5244	1008.5303	21.7
Ub (K27, yeast)	TITLEVESSDTIDNVK(GG)SK	V15	6.0520	K16, K18	16.0284	K16, K18	12.0403	3	698.3585	700.3758	703.7013	702.3719	y2-16	939.9682	942.9942	947.9824	945.9883	20.1
Ub (K27, human)	TITLEVEPSDTIENVK(GG)AK	V15	6.0520	K16, K18	16.0284	K16, K18	12.0403	3	701.0390	703.0563	706.3818	705.0524	y2-16	943.9889	947.0149	952.0031	950.0090	20.5
Ub (K29, yeast)	SK(GG)IQDK	GG	6.0520	K2, K6	16.0284	K2, K6	12.0403	2	416.7298	419.7558	424.7440	422.7499	y1-4	503.2824	503.2824	511.2966	509.3025	7.4
Ub (K29, human)	AK(GG)IQDK	GG	6.0520	K2, K6	16.0284	K2, K6	12.0403	2	408.7323	411.7583	416.7465	414.7525	y1-4	503.2824	503.2824	511.2966	509.3025	7.2
Ub (K33)	IQDK(GG)EGIPPDQQR	P9	6.0520	K4, R13	18.0225	K4	6.0201	3	546.6129	548.6302	552.6204	548.6196	y2-6	370.6879	373.7139	375.6921	370.6879	11.3
Ub (K48)	LIFAGK(GG)QLEDGR	L8	7.0606	K6, R12	18.0225	K6	6.0201	2	730.8964	734.4267	739.9077	733.9065	y2-10	617.8124	621.3427	626.8236	620.8224	19.5
Ub (K63)	TLSDYNIQK(GG)ESTLHLVLR	L17	7.0606	K9, R18	18.0225	K9	6.0201	3	748.7376	751.0911	754.7451	750.7443	y2-16	1015.5369	1019.0672	1024.5481	1018.5470	22.7

Labeled AA: the selected residue for stable isotopic labeling (e.g. L8, the eighth leucine residue); mass shift: the mass change due to isotopic labeling; synthetic peptides: chemically labeled during chemical reaction; SILAC peptides: metabolically labeled in SILAC experiments; SILAM peptides: metabolically labeled in SILAC mice; z: charge state; native: endogenous peptide ion without labeling; precursor ions: monoisotopic m/z values of native and labeled peptides; product ions, monoisotopic m/z values of monitored product ions in SRM; Ion name: the monitored ions in SRM (e.g. b1-2 is single charged b₂ ion); RT: experimental retention time in one setting of reverse liquid LC-SRM (the retention time may change in another experimental setting and needs to be defined when changing the LC column); The total Ub level was calculated by adding the amount of TLS peptide to that of the GG-linked K63 peptide.