Table 3 Summary of site-specific modification analysis by mass spectrometry of non-cross-linked, hydroxylated and glycosylated residues in type I collagen from controls (MC and EV) and KO clones.

From: Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes

		Site occupancy (%)
		MC	EV	KO-1	KO-2	KO-3
α1(I) K87	Lys	1.9 ± 0.1	1.7 ± 0.1	5.6 ± 0.1***^,###	5.5 ± 0.2***^,###	3.8 ± 0.0***^,###
	Hyl	3.9 ± 0.2	3.2 ± 0.1	12.3 ± 1.2***^,###	11.9 ± 0.2***^,###	5.1 ± 0.1***^,###
	G-Hyl	7.7 ± 0.2	7.8 ± 0.1	10.5 ± 0.3***^,###	10.4 ± 0.3***^,###	7.3 ± 0.7
	GG-Hyl	86.4 ± 0.1	87.2 ± 0.1	71.6 ± 0.5***^,###	72.3 ± 0.6***^,###	83.8 ± 0.6***^,###
α1(I) K99	Lys	69.4 ± 0.4	70.5 ± 0.3	70.4 ± 0.1***^,###	70.9 ± 0.3***^,###	67.3 ± 0.1***^,###
	Hyl	20.0 ± 0.4	19.7 ± 0.3	22.1 ± 0.1***^,###	21.4 ± 0.4***^,###	25.0 ± 0.1***^,###
	G-Hyl	6.9 ± 0.1	6.4 ± 0.1	5.5 ± 0.1***^,###	5.6 ± 0.1***^,###	4.9 ± 0.1***^,###
	GG-Hyl	3.7 ± 0.1	3.4 ± 0.0	2.0 ± 0.0*	2.1 ± 0.0***	2.8 ± 0.0***^,###
α1(I) K174	Lys	53.3 ± 0.9	52.8 ± 0.3	64.3 ± 0.3***^,###	64.6 ± 0.3***^,###	57.7 ± 0.2***^,###
	Hyl	41.2 ± 0.4	42.3 ± 0.1	33.1 ± 0.2***^,###	32.6 ± 0.2***^,###	38.4 ± 0.2***^,###
	G-Hyl	3.1 ± 0.2	2.8 ± 0.2	1.9 ± 0.1***^,###	2.0 ± 0.0***^,##	2.5 ± 0.0*
	GG-Hyl	2.4 ± 0.3	2.1 ± 0.1	0.7 ± 0.1***^,###	0.8 ± 0.1***^,###	1.3 ± 0.0***^,##
α1(I) K219	Lys	85.4 ± 0.2	87.0 ± 0.2	83.6 ± 0.3***^,###	83.3 ± 0.3***^,###	81.3 ± 0.2***^,###
α1(I) K219	Hyl	14.6 ± 0.2	13.0 ± 0.2	16.4 ± 0.3***^,###	16.7 ± 0.3***^,###	18.7 ± 0.2***^,###
α1(I) K564	Lys	66.6 ± 1.2	67.2 ± 0.3	73.6 ± 0.5***^,###	72.5 ± 0.1***^,###	67.5 ± 0.1
	Hyl	24.1 ± 0.8	24.7 ± 0.2	22.3 ± 0.4^###	23.1 ± 0.0^##	27.3 ± 0.2***^,###
	G-Hyl	4.3 ± 0.3	4.0 ± 0.2	2.6 ± 0.1***^,###	2.8 ± 0.1***^,###	3.0 ± 0.1***^,##
	GG-Hyl	4.9 ± 0.2	4.2 ± 0.2	1.5 ± 0.0***^,###	1.6 ± 0.2***^,###	2.2 ± 0.0***^,###
α2(I) K87	Lys	8.4 ± 0.1	7.2 ± 0.2	7.2 ± 0.1***	7.2 ± 0.1***	4.1 ± 0.0***^,###
α2(I) K87	Hyl	91.6 ± 0.1	92.8 ± 0.2	92.8 ± 0.1***	92.8 ± 0.1***	95.9 ± 0.0***^,###
α2(I) K174	Lys	36.0 ± 1.4	35.6 ± 1.3	48.7 ± 0.3***^,###	49.4 ± 0.4***^,###	39.8 ± 0.6**^,##
	Hyl	6.2 ± 0.4	6.0 ± 0.4	8.0 ± 0.3***^,###	8.1 ± 0.4***^,###	4.6 ± 0.0*^,##
	G-Hyl	37.8 ± 0.3	38.4 ± 0.5	33.1 ± 0.7***^,###	31.9 ± 0.9***^,###	35.4 ± 0.1**^,##
	GG-Hyl	20.0 ± 1.3	20.2 ± 0.6	10.2 ± 0.1***^,###	10.6 ± 0.1***^,###	20.1 ± 0.5
α2(I) K219	Lys	34.1 ± 0.5	34.3 ± 0.4	54.7 ± 0.5***^,###	54.9 ± 0.2***^,###	46.7 ± 0.2***^,###
	Hyl	62.2 ± 0.7	62.0 ± 0.1	43.9 ± 0.5***^,###	43.7 ± 0.3***^,###	50.6 ± 0.3***^,###
	G-Hyl	0.6 ± 0.3	0.7 ± 0.1	0.4 ± 0.1	0.3 ± 0.1	0.5 ± 0.0
	GG-Hyl	3.2 ± 0.4	3.0 ± 0.4	1.1 ± 0.1***^,##	1.1 ± 0.1***^,###	2.3 ± 0.0*
α1(I) K918/930	Lys + Lys	2.1 ± 0.1	1.3 ± 0.0	3.4 ± 0.2***^,###	4.6 ± 0.2***^,###	5.2 ± 0.1***^,###
	Lys + Hyl	10.8 ± 0.1	9.5 ± 0.2	10.8 ± 0.1^###	11.6 ± 0.1***^,###	12.6 ± 0.0***^,###
	Hyl + Hyl	87.1 ± 0.0	89.2 ± 0.2	85.8 ± 0.3***^,###	83.8 ± 0.2***^,###	82.2 ± 0.1***^,###
α2(I) K933	Lys	0.3 ± 0.2	0.3 ± 0.2	1.3 ± 0.1***^,###	1.2 ± 0.1***^,###	0.7 ± 0.1*
α2(I) K933	Hyl	99.7 ± 0.2	99.7 ± 0.2	98.7 ± 0.1***^,###	98.8 ± 0.1***^,###	99.3 ± 0.1*
α1(I) K9^N	Lys	44.6 ± 0.5	48.0 ± 0.3	100 ± 0.0***^,###	100 ± 0.0***^,###	100 ± 0.0***^,###
α1(I) K9^N	Hyl	55.4 ± 0.5	52.0 ± 0.3	0.0 ± 0.0***^,###	0.0 ± 0.0***^,###	0.0 ± 0.0***^,###
α1(I) K16^C	Lys	51.9 ± 0.2	43.2 ± 0.3	100 ± 0.0***^,###	100 ± 0.0***^,###	100 ± 0.0***^,###
α1(I) K16^C	Hyl	48.1 ± 0.2	56.8 ± 0.3	0.0 ± 0.0***^,###	0.0 ± 0.0***^,###	0.0 ± 0.0***^,###
α2(I) K5^N	Lys	78.6 ± 0.8	77.3 ± 1.1	100 ± 0.0***^,###	100 ± 0.0***^,##	100 ± 0.0***^,###
α2(I) K5^N	Hyl	21.4 ± 0.8	22.7 ± 1.1	0.0 ± 0.0***^,###	0.0 ± 0.0***^,##	0.0 ± 0.0***^,###

Lys hydroxylation and its glycosylation (%) represents the relative levels of Lys, Hyl, G-Hyl, and GG-Hyl (Lys + Hyl + G-Hyl + GG-Hyl = 100%).
Values represent mean ± S.D. (n = 3) of triplicate analysis for each group. Statistical differences were determined by Kruskal–Wallis one-way analysis of variance and means comparison with the controls by Dunnett’s method.
Lys lysine, Hyl hydroxylysine, G- galactosyl-, GG- glucosylgalactosyl, MC MC3T3-E1, EV empty vector, KO knock-out.
*p < 0.05, **p < 0.01, and ***p < 0.001 between MC and KO; ^##p < 0.01 and ^###p < 0.001 between EV and KO, respectively.

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Table 3 Summary of site-specific modification analysis by mass spectrometry of non-cross-linked, hydroxylated and glycosylated residues in type I collagen from controls (MC and EV) and KO clones.

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