\chapter{Supplementary Figures}
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\begin{figure} %Initial rates with different sugars
	\centerline{\includegraphics[width=0.8\columnwidth]{appendix0/NT-sug-dependence.eps}}
    \caption[Role of the nucleotide sugar in E10-32 cleavage]{Role of the nucleotide sugar in E10-32 cleavage. A series of time courses for E10-32 cleavage by wild-type OGT in the presence of different UDP-sugar donors. The N-terminal product (``N-term'', \textit{m/z} 569.2626; 2+) was quantified by integration of the EIC, and normalized by determining the ratio relative to the internal standard, N\textsuperscript{*}. Peptide sequences are provided in Table~\ref{tab:listofpeptides}. These experiments were conducted with 12 $\mu$M E10-32 and 18 $\mu$M wild-type OGT.}
    \label{fig:app-NT-sug-dependence}
\end{figure}
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\begin{figure} %Rate comparison for C9X
	\centerline{\includegraphics[width=0.9\columnwidth]{appendix0/C9X-relative-rates.eps}}
    \caption[Relative rates of cleavage for C9X peptides]{Relative rates of cleavage for C9X peptides. A series of time courses for X9E10-32 peptide cleavage by OGT (D554N). As all peptides produce the same C-terminal product (``C-term''), relative reaction rates were determined by monitoring the amount of C-term produced over time. EIC traces for ``C-term'' (\textit{m/z} 747.6698; 3+) were integrated and normalized by determining the ratio relative to the internal standard, N\textsuperscript{*}. Peptide sequences are provided in Table~\ref{tab:listofpeptides}. These experiments were conducted with 12 $\mu$M E10-32 and 20 $\mu$M OGT (D554N).}
    \label{fig:app-C9X-relative-rates}
\end{figure}
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\begin{figure} %Alignment of human HCF repeats
	\centerline{\includegraphics[width=0.9\columnwidth]{appendix0/Human-hcf-repeats.eps}}
    \caption[Primary sequence of HCF proteolytic repeats in \textit{Homo sapiens}]{Primary sequence of HCF proteolytic repeats in \textit{Homo sapiens}. The canonical 26 amino acid sequences for the six repeats are shown~\cite{Capotosti:2011kd}.}
    \label{fig:app-HCF-human-repeats}
\end{figure}
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\begin{figure}[b] %This is the WT time course
    \caption[Time course for E10-32 cleavage by wild-type OGT]{\textit{(On the following page)} Time course for E10-32 cleavage by wild-type OGT. Shown are: A (\textit{m/z} 845.1319; 4+), B (\textit{m/z} 747.6698; 3+), and C (\textit{m/z} 569.2626; 2+) (labeled as shown in Figure~\ref{fig:mechanism-hcf-peptide-1}), as well as two glycopeptide species, labeled BP (byproduct) and Int-1 (intermediate 1) (\textit{m/z} 895.9017; 4+), and an anhydropeptide [M-H\textsubscript{2}O], labeled Int-2 (intermediate 2) (\textit{m/z} 840.8784; 4+). There are two peaks for Int-2, indicated by red arrows. These peaks both have the same exact mass and the same fragmentation pattern (see Figure~\ref{fig:int-2-peaks-same} for MS/MS spectra). Plots represent a sum of EIC traces for all species. These experiments were conducted with 12 $\mu$M E10-32 and 12 $\mu$M wild-type OGT. Times indicated are the times between OGT addition and quenching. The y-axes are scaled such that the largest peak in each chromatogram is assigned as 100\% abundance. The use of ``Relative Abundance (\%)'' as a vertical axis is not without limitations; each species will not have an identical response signal, so peak heights are not directly comparable. However, this issue also makes a fixed vertical axis undesirable. Graphical representations must be considered with plotted data presented following displayed EICs. Plots of integrated EICs for each species are presented in Figure~\ref{fig:cleavage-reaction-plots}.}
    \label{fig:app-TC1-WT-E10}
\end{figure}
\begin{figure}[p]
    \centerline{\includegraphics[width=0.8\columnwidth]{appendix0/SI-Fig1.eps}}
\end{figure}
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\begin{figure}[b] %This is the 100 min D554N time course
    \caption[Time course for E10-32 cleavage by OGT (D554N), 100 minutes]{\textit{(On the following page)} Time course for E10-32 cleavage by OGT (D554N), 100 minutes. Shown are: A (\textit{m/z} 845.1319; 4+), B (\textit{m/z} 747.6698; 3+), and C (\textit{m/z} 569.2626; 2+) (labeled as shown in Figure ~\ref{fig:mechanism-hcf-peptide-1}), as well as a glycopeptide species, Int-1 (intermediate 1) (\textit{m/z} 895.9017; 4+), and an anhydropeptide [M-H\textsubscript{2}O], labeled Int-2 (intermediate 2) (\textit{m/z} 840.8784; 4+). There are two peaks for Int-2, indicated by red arrows. These peaks both have the same exact mass and the same fragmentation pattern (see Figure~\ref{fig:int-2-peaks-same} for MS/MS spectra). Plots represent a sum of EIC traces for all species. These experiments were conducted with 12 $\mu$M E10-32 and 12 $\mu$M OGT (D554N). Times indicated are the times between OGT addition and quenching. The y-axes are scaled such that the largest peak in each chromatogram is assigned as 100\% abundance. The use of ``Relative Abundance (\%)'' as a vertical axis is not without limitations; each species will not have an identical response signal, so peak heights are not directly comparable. However, this issue also makes a fixed vertical axis undesirable. Graphical representations must be considered with plotted data presented following displayed EICs. Plots of integrated EICs for each species are presented in Figure~\ref{fig:app-TC1-D554N-E10-graphs}.}
    \label{fig:app-TC1-D554N-E10}
\end{figure}
\begin{figure}[p]
    \centerline{\includegraphics[width=0.95\columnwidth]{appendix0/SI-Fig5.eps}}
\end{figure}
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\begin{figure} %These are the 100 min D554N time course graphs.
	\centerline{\includegraphics[width=0.9\columnwidth]{appendix0/SI-Fig6.eps}}
    \caption[Graphical representations of 100 minute time course for cleavage of E10-32 with stoichiometric OGT (D554N)]{Graphical representations of the 100 minute time course for cleavage of E10-32 with stoichiometric OGT (D554N). Integration of individual EICs (as shown in Figure~\ref{fig:app-TC1-D554N-E10}) provided quantitation of response units for each species at each time point. Response units are shown for species A (\textit{m/z} 845.1319; 4+), Intermediate 1 (Int-1, \textit{m/z} 895.9017; 4+), Intermediate 2 (Int-2, \textit{m/z} 840.8784; 4+) and products, B (\textit{m/z} 747.6698; 3+) and C (\textit{m/z} 569.2626; 2+). The 19 minute time point was excluded from all plots due to lower abundance of each species; the 45 minute time point was excluded in the product plot due to abnormally high signal. All values used are corrected based on the signal of an EIC from a blank run. Integration for Int-2 included both peaks in the EIC (see caption of Figure~\ref{fig:app-TC1-D554N-E10} for details). No decay fit for A is shown here due to sparse data at early time points.}
    \label{fig:app-TC1-D554N-E10-graphs}
\end{figure}
%\begin{figure}[p]
%\textit{(On the following page)}     
%\end{figure}
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\begin{figure}[b] %This is the 3 min D554N time course
    \caption[Time course for E10-32 cleavage by OGT (D554N), 3 minutes]{\textit{(On the following page)} Time course for E10-32 cleavage by OGT (D554N), 3 minutes. Shown are: A (\textit{m/z} 845.1319; 4+), B (\textit{m/z} 747.6698; 3+), and C (\textit{m/z} 569.2626; 2+), Int-1 (intermediate 1) (\textit{m/z} 895.9017; 4+), and Int-2 (intermediate 2) (\textit{m/z} 840.8784; 4+). There is little or no signal for B and C in this time frame. There are two peaks for Int-2, indicated by red arrows. These peaks both have the same exact mass and the same fragmentation pattern (see Figure~\ref{fig:int-2-peaks-same} for MS/MS spectra). Plots represent a sum of EIC traces for all species. These experiments were conducted with 3 $\mu$M E10-32 and 27 $\mu$M OGT (D554N). Times indicated are the times between OGT addition and quenching. The y-axes are scaled such that the largest peak in each chromatogram is assigned as 100\% abundance. The use of ``Relative Abundance (\%)'' as a vertical axis is not without limitations; each species will not have an identical response signal, so peak heights are not directly comparable. However, this issue also makes a fixed vertical axis undesirable. Graphical representations must be considered with plotted data presented following displayed EICs. Plots of integrated EICs for each species are presented in Figure~\ref{fig:app-TC2-D554N-E10-graphs}.}
    \label{fig:app-TC2-D554N-E10}
\end{figure}
\begin{figure}[p]
    \centerline{\includegraphics[width=0.8\columnwidth]{appendix0/SI-Fig7.eps}}
\end{figure}
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\begin{figure}[b] %These are the 3 min D554N time course graphs.
    \caption[Graphical representations of 3 minute time course for cleavage of E10-32 with excess OGT (D554N)]{\textit{(On the following page)} Graphical representations of the 3 minute time course for cleavage of E10-32 with excess OGT (D554N). Integration of individual EICs (as shown in Figure~\ref{fig:app-TC2-D554N-E10}) provided quantitation of response units for each species at each time point. For species A (\textit{m/z} 845.1319; 4+), a first-order exponential decay (of the form shown in Equation~\ref{first-order-decay}) was used to fit the data. The obtained rate constant is shown. Intermediate 1 (Int-1, \textit{m/z} 895.9017; 4+) forms rapidly, and towards the end begins to saturate before declining (as seen in longer time courses). Intermediate 2 (Int-2, \textit{m/z} 840.8784; 4+) forms at the expense of Intermediate 1, and appears to persist at the final time point. The values quantified include both peaks for Int-2, as they both decompose. The products, B (\textit{m/z} 747.6698; 3+) and C (\textit{m/z} 569.2626; 2+) form over time; however, in this short time course, the lag-phase between reaction initiation and initial product formation is evident. It appears that over the first minute only Intermediate 1 and 2 have been produced, with little decomposition of Intermediate 2. All values used are corrected based on the signal of an EIC from a blank run.}
    \label{fig:app-TC2-D554N-E10-graphs}
\end{figure}
\begin{figure}[p]
    \centerline{\includegraphics[width=1\columnwidth]{appendix0/SI-Fig8.eps}}
\end{figure}
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\begin{figure}[b] %This is the 30 min D554N time course
    \caption[Time course for E10-32 cleavage by OGT (D554N), 30 minutes]{\textit{(On the following page)} Time course for E10-32 cleavage by OGT (D554N), 30 minutes. LC traces showing EICs for each of: A (\textit{m/z} 845.1319; 4+), B (\textit{m/z} 747.6698; 3+), and C (\textit{m/z} 569.2626; 2+), Int-1 (intermediate 1) (\textit{m/z} 895.9017; 4+), and Int-2 (intermediate 2) (\textit{m/z} 840.8784; 4+). There are two peaks for Int-2, indicated by red arrows. These peaks both have the same exact mass and the same fragmentation pattern (see Figure~\ref{fig:int-2-peaks-same} for MS/MS spectra). Plots represent a sum of EIC traces for all species. These experiments were conducted with 3 $\mu$M E10-32 and 27 $\mu$M OGT (D554N). Times indicated are the times between OGT addition and quenching. The y-axes are scaled such that the largest peak in each chromatogram is assigned as 100\% abundance. The use of ``Relative Abundance (\%)'' as a vertical axis is not without limitations; each species will not have an identical response signal, so peak heights are not directly comparable. However, this issue also makes a fixed vertical axis undesirable. Graphical representations must be considered with plotted data presented following displayed EICs. Plots of integrated EICs for each species are presented in Figure~\ref{fig:app-TC3-D554N-E10-graphs}.}
    \label{fig:app-TC3-D554N-E10}
\end{figure}
\begin{figure}[p]
    \centerline{\includegraphics[width=1\columnwidth]{appendix0/SI-Fig9.eps}}
\end{figure}
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\begin{figure} %These are the 30 min D554N time course graphs.
	\centerline{\includegraphics[width=1\columnwidth]{appendix0/SI-Fig10.eps}}
    \caption[Graphical representations of 30 minute time course for cleavage of E10-32 with excess OGT (D554N)]{Graphical representations of the 30 minute time course for cleavage of E10-32 with excess OGT (D554N). Integration of individual EICs (as shown in Figure~\ref{fig:app-TC3-D554N-E10}) provided quantitation of response units for each species at each time point. Time points are widely separated so the changes in response units over time are more informative for some species than others (e.g., the accumulation of products), although the temporal information is clear and consistent with data previously presented. The masses of species are the same as in previous captions. Note the lag between reaction initiation and initial product formation. The values quantified include both peaks for Int-2, as they both decompose. All values used are corrected based on the signal of an EIC for a blank run.}
    \label{fig:app-TC3-D554N-E10-graphs}
\end{figure}
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%\textit{(On the following page)}  
%\end{figure}
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\begin{figure} %These are the 14 min D554N time course graphs.
	\centerline{\includegraphics[width=1\columnwidth]{appendix0/SI-Fig11.eps}}
    \caption[Graphical representations of 14 minute time course for cleavage of E10-32 with excess OGT (D554N)]{Graphical representations of the 14 minute time course for cleavage of E10-32 with excess OGT (D554N). Data is based on an intermediate length time course analogous to those shown in Figures~\ref{fig:app-TC2-D554N-E10} and~\ref{fig:app-TC3-D554N-E10}. Integration of individual EICs provided quantitation of response units for each species at each time. The masses of species are the same as in previous captions. Noteworthy is the decomposition of Int-1, which appears to occur from about 2 minutes to about 7 minutes. Int-2 forms steadily, but appears to reach a maximum between 10 and 15 minutes, consistent with data shown in Figure~\ref{fig:app-TC3-D554N-E10-graphs}. The integrated values include both peaks for Int-2. All values used are corrected based on the signal of an EIC for a blank run.}
    \label{fig:app-TC4-D554N-E10-graphs}
\end{figure}
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%   \textit{(On the following page)}  
%\end{figure}
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\begin{figure}[b] %This is MS/MS showing Int-2 vs A
    \caption[Intermediate 2 is a dehydrated form of E10-32 (A)]{\textit{(On the following page)} Intermediate 2 is a dehydrated form of E10-32 (A). (a) A comparison of the fragmentation pattern (HCD) for A (top) and Int-2 (bottom) shows many of the same fragment ions, however, the \textit{m/z} range from 600-950 shows key differences. A complete list of fragment ions observed by MS/MS for Int-2 and A are presented in Tables~\ref{tab:y-ion-table-HCD-Int-2}, \ref{tab:b-ion-table1-HCD-int-2}, \ref{tab:b-ion-table2-HCD-int-2}, \ref{tab:y-ion-table-CID-Int-2}, \ref{tab:b-ion-table-CID-int-2}, \ref{tab:b-ion-table-CID-int-2-1}, and Tables \ref{tab:y-ion-table-HCD-A}, \ref{tab:b-ion-table1-HCD-A}, \ref{tab:b-ion-table2-HCD-A}, respectively (with \textit{m/z} errors for fragment ions presented in Figure~\ref{fig:MSMS-error-plot}). A more completely annotated MS/MS spectrum for Int-2 is presented in Figure~\ref{fig:int-2-HCD-spectrum}. (b) In order to filter out dehydration artifacts resulting from numerous Ser/Thr residues in the C-terminal region of the HCF-short peptide, a side-by-side comparison between A and Int-2 was performed. A zoom of the 600-950 \textit{m/z} range reveals \textit{b} ions are dehydrated in Int-2, but not in A. Based on this comparison, we can localize the dehydration in Int-2 to the C\textsuperscript{9}E\textsuperscript{10} region of the peptide (HCF repeat numbering).}
    \label{fig:dehydration-not-cterm}
\end{figure}
\begin{figure}[p]
    \centerline{\includegraphics[width=1\columnwidth]{appendix0/SI-Fig12ab.eps}}
\end{figure}
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\begin{figure}[b] %retention times are the same for WT and mutant
    \caption[Different HCF peptides produce the same intermediates with both wild-type OGT and OGT (D554N)]{\textit{(On the following page)} Different HCF peptides produce the same intermediates with both wild-type OGT and OGT (D554N). LC traces showing retention times for glycopeptide and anhydropeptide intermediates formed in reactions of three different HCF peptides using either wild-type OGT or OGT (D554N). Traces were selected from time courses such that both anhydropeptides were of similar intensity. For E10-32 and E10V11-32 intermediates the vertical axis is normalized to a fixed intensity for each species; for R9E10-32 intermediates the vertical axis is normalized such that the tallest peak is 100\%, this is due to the low relative abundance of wild-type-produced intermediates, making them poorly visualized when at the same scale. The LC trace for the E10V11-32 glycopeptide is not shown because the abundance of glycopeptide intermediate was extremely low and a large quantity of glycopeptide byproduct was observed, making the trace uninformative. (To be more specific: due to these problems, the glycopeptide intermediate for this peptide sequence could only be assigned after it was identified in reactions with the mutant enzyme, whereas all other intermediates identified in the reactions with wild-type OGT were identified without reliance on results for the mutant enzyme.)}
    \label{fig:common-retention-times}
\end{figure}
\begin{figure}[p]
	\centerline{\includegraphics[width=1\columnwidth]{appendix0/SI-Fig15.eps}}
\end{figure}
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\begin{figure}[b] %ECD spectrum of Intermediate 1 (1/2)
    \caption[ECD MS/MS spectrum of Intermediate 1, with zooms]{\textit{(On the following page)} ECD MS/MS spectrum of Intermediate 1, with zooms. (a) Full ECD spectrum of Intermediate 1 \textit{m/z} 895.9017 (4+). The inset sequence shows the observed \textit{c} and \textit{z} ions graphically. Fragmentation N-terminal to proline is not observed by ECD (as would be the case for the \textit{c}\textsubscript{7} and \textit{c}\textsubscript{8} fragments). Further, due to the low efficiency of ECD fragmentation, most \textit{c} and \textit{z} ions are present at abundances too low to be meaningfully annotated on this spectrum. (b) Zoomed views of \textit{c} ions near the glycosylated glutamate residue. (c) Zoomed views of \textit{z} ions near the glycosylated glutamate residue. A full list of observed theoretical ions for Int-1 is supplied in Tables~\ref{tab:c-ion-table1-ECD-Int1}, \ref{tab:c-ion-table2-ECD-Int1}, \ref{tab:z-ion-table1-ECD-Int1}, \ref{tab:z-ion-table2-ECD-Int1}. Figures \ref{fig:ECD-spectrum-int1-site} and \ref{fig:glycopeptide-int-ECD} show additional zooms of ions in the region surrounding the GlcNAc-modified residue.}
    \label{fig:ECD-spectrum-int1-zooms}
\end{figure}
\begin{figure}[p]
	\centerline{\includegraphics[width=1\columnwidth]{appendix0/SI-Fig17.eps}}
\end{figure}
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\begin{figure} %ECD spectrum of Intermediate 1 (2/2)
	\centerline{\includegraphics[width=1\columnwidth]{appendix0/SI-Fig18.eps}}
    \caption[The GlcNAc moiety in Intermediate 1 is located at E11, not T12]{The GlcNAc moiety in Intermediate 1 is located at E11, not T12. Zoom of \textit{z} ions 21 and 22 from the ECD spectrum of Intermediate 1. Fragments corresponding to the \textit{z}\textsubscript{22}\textsuperscript{2+} and (\textit{z}\textsubscript{22}+1)\textsuperscript{2+} ions for a peptide in which a GlcNAc is modeled at residue E11 are observed; the analogous ions are not observed for a peptide in which the GlcNAc is modeled at T11.}
    \label{fig:ECD-spectrum-int1-site}
\end{figure}
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\begin{figure} %Error plots for MS/MS
	\centerline{\includegraphics[width=1\columnwidth]{appendix0/SI-Fig21.eps}}
    \caption[Errors in MS/MS fragment ions for A, Intermediate 1 and Intermediate 2]{Errors in MS/MS fragment ions for A, Intermediate 1 and Intermediate 2. A graphical representation of the mass errors (in ppm) between expected and observed fragment ions for each species evaluated by MS/MS in this study. Tabulated data for of all ions observed, for each species, are presented in Tables \ref{tab:y-ion-table-HCD-Int-2}, \ref{tab:b-ion-table1-HCD-int-2}, \ref{tab:b-ion-table2-HCD-int-2}, \ref{tab:y-ion-table-CID-Int-2}, \ref{tab:b-ion-table-CID-int-2}, \ref{tab:b-ion-table-CID-int-2-1}, \ref{tab:y-ion-table-HCD-A}, \ref{tab:b-ion-table1-HCD-A}, \ref{tab:b-ion-table2-HCD-A}, \ref{tab:c-ion-table1-ECD-Int1}, \ref{tab:c-ion-table2-ECD-Int1}, \ref{tab:z-ion-table1-ECD-Int1}, and \ref{tab:z-ion-table2-ECD-Int1}. The rms error value inset in each graph is the mean for all observed theoretical ions for that species. Rms error was calculated as: $x_{rms} = \sqrt{\frac{1}{n}(x_1^2+x_2^2+\ldots+x_n^2)}$. The maximum error tolerated for any ion reported was 10 ppm, however, the vast majority of ions were within 5 ppm of the theoretical \textit{m/z}.}
    \label{fig:MSMS-error-plot}
\end{figure}
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\begin{figure} %Calibration curve for E10 formation
	\centerline{\includegraphics[width=0.9\columnwidth]{appendix0/S-Figure-S14-pt2.eps}}
    \caption[Calibration curve for SID quantitation of N-term]{Calibration curve for SID quantitation of N-term. N-term and N\textsuperscript{*} were combined at discrete proportions and diluted into E10-32 reaction mixtures (lacking OGT) to account for matrix effects during measurement. Each concentration was measured in duplicate.}
    \label{fig:app-calibration-curve-SID}
\end{figure}
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