Table 2.

Ubiquitin-AQUA/PRM analysis of ubiquitin fragments with GG modifications in lysine residues

Amounts are shown in fmol, and the percentage for each fragment is shown in parentheses.

Ubiquitin	Detected fragmenta
	TITLEb	Totalc	Lys6(GG)	Lys11(GG)	Lys27(GG)	Lys29(GG)	Lys33(GG)	Lys48(GG)	Lys63(GG)	M1(GG)	Unmodifiedd
	fmol
WT	234.4	236.1	0.04	1.65	NDe	0.0003	0.07	115.2	0.08	ND	25.3
		(100)	(<0.1)	(0.7)	(<0.1)	(<0.1)	(<0.1)	(49.1)	(<0.1)	(<0.1)	(50.0)
K48R	62.4	65.5	0.15	3.05	ND	0.002	0.01	NAf	0.09	ND	37.6
		(100)	(0.2)	(4.7)	(<0.1)	(<0.1)	(<0.1)		(0.1)	(<0.1)	(95.0)

^a Three independent reaction products were analyzed as described previously (33), and the averages are shown.

^b The amounts of the TITLE (TITLEVESSDTIDNVLK) fragment were determined as internal controls.

^c Total ubiquitin levels were assessed as the sum of TITLE, Lys¹¹(GG), and Lys²⁷(GG) fragments. Because the TITLE fragment is between Lys¹¹ and Lys²⁷, generation of these fragments is mutually exclusive.

^d The amounts of unmodified ubiquitin were calculated by subtraction of the amount of total GG-modified fragments from that of total ubiquitin.

^e ND, not detected.

^f NA, not applicable because of the mutation.