Article Figures & Data
Figures
- FIG. 1.
SDS-PAGE analysis of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a (8% gel). Lane 1, molecular mass standard (AllBlue Precision Plus Protein standard; Bio-Rad); lanes 2 and 4, S-layer glycoprotein derived from continuous culture; lanes 3 and 5, S-layer glycoprotein derived from batch culture; lanes 2 and 3, Coomassie blue staining; lanes 4 and 5, periodic acid-Schiff staining. The amounts of total protein loaded per gel lane were 10 μg in lanes 2 and 3 and 25 μg in lane 4 and 5. The arrow indicates the nonglycosylated S-layer protein protomer.
- FIG. 2.
(+)IR-MALDI-orthogonal TOF mass spectrum of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a. The spectrum shows the average mass of the nonglycosylated species of SgsE at 94.46 kDa and those of the three inherently heterogeneic glycoprotein species at 101.66 kDa, 108.68 kDa, and 115.73 kDa.
- FIG. 3.
(A) (+)Nano-ESI-QTOF MS/MS spectrum of the doubly charged ions at m/z = 1,024.68, representing glycopeptide TFDEEVTTGSNITVVQ with one galactose and one rhamnose attached to the peptide backbone. (B) (+)Nano-ESI-QTOF MS/MS spectrum of the doubly charged ions at m/z = 872.53 originating from glycopeptide TFDEEVTTGSNITVVQ after β-elimination. The glycosylation site is displayed by the y9 ion and the b8 ion and a loss of water from the threonine-590 in the sequence of SgsE. #, sodiated ions.
- FIG. 4.
(+)Nano-ESI-QTOF mass spectrum of the released glycan chains of the S-layer glycoprotein from G. stearothermophilus NRS 2004/3a. The main peaks correspond to [M + 2H+2K]4+. Chain lengths vary between of 12 and 17 repeating units (RU) for both the glycan structure with two core rhamnoses (first series; RU = X) and the one with three core rhamnoses (second series; RU = X + Rha).
- FIG. 5.
(+)Nano-ESI-QTOF mass spectrum of glycopeptides belonging to the peptide ATLTSADVIRVD derived from the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a. The most abundant peaks derive from overlapping peaks of the two molecular ion series [M + 3H+K]4+ and [M + 2H+2Na]4+; all glycopeptides carry a single glycan chain attached to serine-794. The number of repeating units (RU) varies between 13 and 17.
Tables
- TABLE 1.
Nano-ESI-QTOF MS analysis of oligosaccharides released from the S-layer glycoprotein of G. stearothermophilus NRS 2004/3aa
RUb m/z [M + 2H+Na+K]4+ [M + 2H+2K]4+ [M + H+Na+2K]4+ Theorc Exptl Theorc Exptl Theorc Exptl 12 1,453.43 NDd 1,457.46 1,457.38 1,462.96 1,462.77 12 + Rha 1,489.97 1,489.94 1,494.00 1,493.94 1,499.49 1,499.36 13 1,563.04 1,563.07 1,567.07 1,566.93 1,572.56 1,571.78 13 + Rha 1,599.58 1,599.70 1,603.60 1,603.51 1,609.10 1,609.13 14 1,672.65 1,672.65 1,676.68 1,676.55 1,682.17 1,681.90 14 + Rha 1,709.18 1,709.13 1,713.21 1,712.97 1,718.71 1,718.48 15 1,782.26 1,782.22 1,786.28 1,786.14 1,791.78 1,791.82 15 + Rha 1,818.79 1,818.96 1,822.82 1,822.78 1,828.31 1,827.90 16 1,891.86 1,891.47 1,895.89 1,895.87 1,901.39 1,901.15 16 + Rha 1,928.40 1,928.13 1,932.43 1,932.52 1,937.92 1,937.80 17 2,001.47 ND 2,005.50 2,005.34 2,010.99 ND 17 + Rha 2,038.01 ND 2,042.03 2,042.39 2,047.53 ND ↵ a Data show the theoretical (Theor) and experimentally determined (Exptl) average m/z values of quadruply charged ions containing different numbers of Na+ and K+ ions.
↵ b RU designates the number of trisaccharide repeating units, with the terminal rhamnose of the repeating unit at the nonreducing end being O-methylated. The core structure consists of two rhamnoses and one galactose. An additional rhamnose in the core is indicated by “+ Rha.”
↵ c Theoretical molecular masses were calculated using average masses.
↵ d ND, not determined.
- TABLE 2.
NanoESI-QTOF MS analysis of the S-layer glycopeptide ATLTSADVIRVD of G. stearothermophilus NRS 2004/3aa
RUb Mass M theorc M exptl 13 7,428.44 NDd 13 + Rha 7,574.59 ND 14 7,866.87 7,866.25 14 + Rha 8,013.01 8,013.41 15 8,305.30 8,305.05 15 + Rha 8,451.44 8,450.85 16 8,743.73 8,743.57 16 + Rha 8,889.87 8,890.21 17 9,182.16 9,181.53 17 + Rha 9,328.30 ND ↵ a Data show the theoretical (Theor) and experimentally determined (Exptl) average molecular masses.
↵ b RU designates the number of trisaccharide repeating units, with the terminal rhamnose of the repeating unit at the nonreducing end being O-methylated. The core structure consists of two rhamnoses and one galactose. An additional rhamnose in the core is indicated by “+ Rha.”
↵ c Theoretical molecular masses were calculated using average masses.
↵ d ND, not determined.
