TABLE 3.

Loss of Arg PTM in vivo due to site-directed mutagenesis of Mmp10^a

Mutationb	Peak area (AU) for peptide 1c		Loss of PTM for peptide 1 (%)	Peak area (AU) for peptide 2c		Loss of PTM for peptide 2d (%)	Mean loss of PTM (%)	Relative protein expressione
	r275	R275		r275	R275
None	37	0	0	ND	ND	NA	0	(1)
G140,144,146 → A	220	37	14.0	5.8	1.2	17.5	15.8	32
N212D213 → AA	96	52	35.0	ND	ND	NA	35.0	0.4
H375 → G	340	3.2	0.9	10	0.062	0.6	0.8	28
H375 → F	310	3.3	1.1	7.6	ND	ND	0.5	ND
H375 → F, ΔN371	160	100	38.0	3.8	3.4	47	42.5	9

^aND, not detected; NA, not applicable.

^bThe mutated mmpX genes were expressed in a plasmid with the PhmvA promoter in the ΔmmpX background. The wild-type gene expressed in the same plasmid served as a positive control. All mutations were verified by Sanger sequencing of the plasmids. All the variants also had a His tag attached to the N terminus to allow confirmation of expression using MALDI.

^cPeptide 1 is MGNALPGRR²⁷⁵ARGPNEPGGIRF; peptide 2 is PGRR²⁷⁵ARGPNEPGGIRF. “R” indicates the presence of unmethylated arginine. “r” indicates the presence of 5-C-(S)-methylarginine. The peak area is multiplied by 10⁷ to obtain the reported values, which are presented in arbitrary units (AU).

^dLoss of PTM = R²⁷⁵/(R²⁷⁵ + r²⁷⁵). The mean is calculated by averaging results from both peptides 1 and 2 if available. If a peptide was not detected, the value was taken as zero.

^eProtein expression was quantified by Western blotting (Fig. S12). The band intensity for the recombinant native Mmp10 was set at 1. Relative protein expression = band intensity of mutated Mmp10/band intensity of native Mmp10.