Role of Intestinal Microbiota in Ulcerative Colitis

Intensity 50
0
C1
175.05
J. Holck et al. / Process Biochemistry 46 (2011) 1039–1049 1045
Z1
218.09
190.09
291.06
C2
351.07
Z2
454.13
C3
527.08
Z3
630.16
C4 806.23 C5
703.15
879.21
982.29
1098.3
1157.9
200 1200
m/z
Fig. 4. MS/MS high energy CID spectrum of the MS peak at 1157.9, H8 (Fig. 2). The mass is found to be 103 higher than DP6 (or 73 less than DP7). The spectrum is annotated
assuming that the extra 103 Da is located in the reducing end. In case it is located in the non-reduced end, the C and Z ions should be reversed.
Table 4
Identified compounds, peak identity and yields from HG permeate. Yield in mg/g
initial pectin. For peak identities, refer to (Fig. 2). Yields are given as means and
coefficient of variance were in the range of 1.3–3.1%.
Peak ID Structure Yield mg/g pectin
H1 galA2 5.8
H2 galA2 15.2
H5 galA3 24.0
H7 galA4 46.2
H9 galA5 37.1
H10 galA6 32.6
H11 galA7 25.7
H12 galA8 23.3
Total 201.1
than H12 will contain homogalacturonides with a DP above 8, and
thereby could add approximately 50 mg/g pectin. However, since
no verification by MS/MS was performed, these putative homogalacturonides
could not be taken into account in the total recovery.
Likewise it can be assumed, based on MS/MS data, that H3-4, 6, 8
(Fig. 2) roughly could add additional 50 mg/g pectin to the overall
yield, since these peaks also contained some galacturonic acid.
3.4. Purification of rhamnogalacturonan I oligosaccharides
Analogously to the procedure used for the HG permeate, the
RGI permeate was de-esterified by alkaline treatment and purified
by anion exchange chromatography (Fig. 5). Fractions were
collected, lyophilized and analyzed by MALDI-TOF MS and MS/MS
for structural determination. The major compound of peak R1 was
found to be galA 2rha 2gal 2 and the MS/MS data proved that the two
galactose molecules were evenly distributed, one on each rhamnose
(Fig. 6). The MS/MS spectrum illustrated a characteristic loss
involving an A type cross-ring cleavage of the rhamnose as discussed
in [43] (Fig. 7). No further studies were done to determine
which of the bonds were cleaved as this was not relevant for the
study. The residual ion, a cross ring fragment, is denoted CRFx, x
denoting the number of the cleaved monomer. These characteristic
CRF ions were seen in all the examined MS/MS spectra of the
RGI like compounds. Fraction R1 also contained trace amounts of
galA 2rha 2gal 3. Contrary to the major compound, which was found
mAU
80
60
40
20
0
Z4
Z5
Conductivity UV_235
R1
R2
R3
R4
R5
150 200 250 300 350 400 ml
mS/cm
Fig. 5. Elution profile of deesterified RGI permeate during ionic exchange chromatography
on a Source 15Q packed HR16/10 column using a linear gradient of
ammonium formate and UV detection at 235 nm. For peak codes, refer to Table 5.
to have one galactose on each rhamnose, the MS/MS spectrum
showed all 3 galactose units to be localized on a single rhamnose,
either the one located in the reducing end or the one next
to the non-reduced unsaturated galacturonic acid (the spectrum
revealed a mixture of both) (data not shown). R2 contained a
galA 2rha 2gal molecule. MS/MS data showed that this peak comprised
of a mixture of two kinds of structures with the galactose
on either the reducing-end rhamnose or on the internal rhamnose.
R3 was pure galA 2rha 2 with no traces of other compounds. R4 contained
mainly galA 3rha 3gal 3 with one galactose on each rhamnose.
Also trace amounts of putative galA 2rha 2gal 2 were observed, but
this structure was not confirmed by MS/MS analysis. R5 contained
galA 3rha 3gal 2 in a mixture of compounds with the unsubstituted
rhamnose in either the reducing end, the middle position, or next
to the non-reducing unsaturated galacturonic acid. The MS spectrum
of peak R6 revealed two different components. One of them
being galA 3rha 3, the other one being galA 3rha 2gal 2. MS/MS anal-
R6
25.0
20.0
15.0
10.0
5.0
0.0