TABLE 2.

Relative quantification of hydroxyproline residues in the hinge-region tryptic peptide (HYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR – 4136.8899 Da) of plant produced IgA1 co-infiltrated with Nb-P4H overexpression and silencing constructs.

A		Hyp residues							av. #Hyp	% change
		0	1	2	3	4	5	6
pEAQ-IgA1	Nb-IgA1	26.3	27.9	23.9	16.0	4.8	1.1	0.1	1.49	100
	+Nb-P4H1 overexpression	9.6	12.5	17.2	25.4	25.5	8.0	1.7	2.75	195
	+Nb-P4H4 overexpression	32.0	28.4	19.9	13.7	4.8	1.2	0.1	1.35	90
	+Nb-P4H9 overexpression	20.7	23.5	21.8	20.9	9.7	2.9	0.3	1.85	127
	+Nb-P4H10 overexpression	18.0	23.1	27.4	19.6	8.3	3.1	0.5	1.88	130
B		Hyp residues							av. #Hyp	% change
		0	1	2	3	4	5	6
pEAQ-IgA1	Nb-IgA1	26.3	27.9	23.9	16.0	4.8	1.1	0.1	1.49	100
	+Nb-P4H10-RNAi	35.2	31.0	19.7	6.6	4.0	1.9	1.7	1.26	83
	+Nb-P4H1 and P4H10-RNAi	31.5	31.0	20.5	10.8	4.2	1.4	0.7	1.32	87
pPT2M-IgA1	Nb-IgA1	32.2	30.7	18.8	11.4	5.2	1.5	0.3	1.33	100
	+Nb-P4H1-RNAi	23.1	30.5	23.6	14.4	6.1	1.9	0.4	1.57	119
	+Nb-P4H10-RNAi	36.9	31.5	17.1	8.9	4.1	1.3	0.2	1.17	88
	+Nb-P4H1 and P4H10-RNAi	50.5	27.0	13.7	6.0	2.5	0.4	0.0	0.84	64

Panel (A) includes the results showing the effect of overexpression of all 4 Nb-P4H proteins (fused to RFP or GFP) on hydroxyproline formation. Panel (B) shows relative quantification results using silencing constructs for Nb-P4H1 and Nb-P4H10. The two different expression vectors (pPT2M and pEAQ, which display maximal expression at different time points) for recombinant production of IgA1 are evaluated separately. Values are presented in percent (%) using the summed peak areas of all observed charge states. The first four isotopic peaks were considered for each of the charge states; in case of ammonium adduct formation only the monoisotopic peak was considered. The average number of Hyp residues per peptide is listed below (av. #Hyp) together with percent increase or decrease (%change) compared to the wild IgA1 construct (Nb-IgA1). The corresponding mass spectra can be found in Supplementary Figure 6 and in Figure 5.