\chapter{OGT catalyzes aspartate-to-isoaspartate isomerization via side chain glycosylation} \label{ch:aspartate}
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\begin{quote}
``But that's just how it is. You don't see it until you do, and then you wonder why you didn't see it all along.''\newline\\
-- Roger Howe (Yale Mathematics)
\end{quote}
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\noindent Apart from minor modifications, a large portion of this chapter originally appeared as~\cite{Janetzko:2017ab}:\\%\newline\\
``Aspartate Glycosylation Triggers Isomerization to Isoaspartate''
John Janetzko and Suzanne Walker
\textit{Journal of the American Chemical Society}. Volume 139, Number 9: 3332--3335. 2017.\newline\\
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Contributions: I performed all experiments described in this chapter.
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\section*{Abstract}
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OGT is an essential human enzyme that glycosylates numerous nuclear and cytoplasmic proteins on serine and threonine. It also cleaves Host cell factor-1 (HCF-1) by a mechanism in which the first step involves glycosylation on glutamate. Replacing glutamate with aspartate in an HCF-1 proteolytic repeat was shown to prevent peptide backbone cleavage, but whether aspartate glycosylation occurred was not examined. In this chapter I will describe the finding that OGT glycosylates aspartate much faster than it glycosylates glutamate in an otherwise identical model peptide substrate; moreover, once formed, the glycosyl aspartate reacts further to form a succinimide intermediate that hydrolyzes to produce the corresponding isoaspartyl peptide. Aspartate-to-isoaspartate isomerization in proteins occurs in cells, but was previously thought to occur exclusively via non-enzymatic means. These findings suggest that there may also be an enzyme-catalyzed component. In addition to OGT, enzymes that may catalyze aspartate-to-isoaspartate isomerization would include PARPs, enzymes known to ribosylate aspartate residues in the process of poly(ADP-ribosyl)ation.
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\section{Introduction}
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As discussed in earlier chapters, OGT is an essential mammalian enzyme known to catalyze two distinct post-translational modifications (PTMs) using the same active site~\cite{Janetzko:2014fy,Levine:2016du}. One is the transfer of \textit{N}-acetylglucosamine to Ser/Thr residues of nuclear and cytoplasmic proteins~\cite{Bond:2015iy,Hardiville:2016jc}, and the other is cleavage of Host cell factor-1 (HCF-1)~\cite{Capotosti:2011kd}, a transcriptional co-regulator found in several chromatin-associated complexes~\cite{Parker:2014eo,Tyagi:2007cf}. Ser/Thr \textit{O}-GlcNAcylation affects localization, stability, and enzymatic activity of a wide variety of proteins and may serve to link cell state to glucose levels~\cite{Bond:2015iy,Hardiville:2016jc}; HCF-1 cleavage is required for cell cycle progression~\cite{Capotosti:2011kd,Julien:2003in,Mangone:2010cr}. HCF-1 cleavage occurs via glycosylation on glutamate followed by on-enzyme formation of an internal pyroglutamate that undergoes spontaneous hydrolysis \cite{Janetzko:2016er}. Glycosylation is a common feature of both OGT-catalyzed PTMs. Nevertheless, the discovery that OGT can glycosylate glutamate was surprising because there are major steric and electronic differences between Ser/Thr and Glu side chains. Given OGT’s capacity to glycosylate glutamate, we wondered whether it could also glycosylate aspartate.\par
%
%Here I present my findings that OGT indeed catalyzes aspartate glycosylation, and show that this modification leads to isomerization of the aspartyl residue to isoaspartate (isoAsp). Isoaspartate formation is a physiologically relevant phenomenon thought to occur spontaneously as proteins age~\cite{Qin:2015cs}; this is the first report of enzyme-catalyzed isoaspartate formation. These findings raise the possibility that some Asp to isoAsp isomerization events in cells my be the result of enzymatic modification of aspartate side chains.
%
\section{A model substrate for aspartate glycosylation}
%
Glycosylation on aspartate was assessed using a variant of the peptide previously employed to investigate the mechanism of HCF-1 cleavage by OGT~\cite{Janetzko:2016er}. The cleavage peptide, E10-32 (Discussed in Chapter \ref{ch:mechanism}; Figure \ref{fig:aspartate-peptide} and Table  \ref{tab:listofpeptides}), contains a threonine-rich region that binds to the tetratricopeptide repeat domain of OGT and a cleavage region consisting of ``CET'', with the site of cleavage being the amide bond between Cys9 and Glu10 (HCF-1 repeat numbering)~\cite{Lazarus:2013bq}. It was demonstrated previously that the residues adjacent to the glutamate can be varied without abolishing cleavage (see Chapters \ref{ch:hcfiscleaved} and \ref{ch:mechanism}); however, the glutamate itself is indispensable~\cite{Capotosti:2011kd,Janetzko:2016er,Kapuria:2016gy,Lazarus:2013bq}.\par
%
\begin{figure}
	\centerline{\includegraphics[width=0.75\columnwidth]{aspartate/peptide-scheme.eps}}
	\caption[Aspartate glycosylation, and related, substrates]{Aspartate glycosylation, and related, substrates. A complete list of peptides used in these studies is provided in Table \ref{tab:listofpeptides}.}
	\label{fig:aspartate-peptide}
\end{figure}
%
I replaced glutamate with aspartate (D10-32) and incubated this peptide with UDP-GlcNAc and wild-type OGT for varying amounts of time before analyzing the reaction mixture by high-resolution LC-MS (henceforth, unless otherwise specified, ``OGT'' implies the use of wild-type enzyme). A glycopeptide [M+GlcNAc, 892.3978 (z=4+)] formed within a minute of initiating the reaction and continued to accumulate for approximately fifteen minutes before decreasing. When aspartate was replaced with alanine (A10-32), this glycopeptide was not observed (Figure \ref{fig:aspartate-is-glyco}). We therefore concluded that glycosylation had occurred on the aspartate side chain.\par
%
\begin{figure}
	\centerline{\includegraphics[width=0.8\columnwidth]{aspartate/S-Figure-S1.eps}}
	\caption[A glycopeptide intermediate forms with D10-32, but not A10-32]{A glycopeptide intermediate forms with D10-32, but not A10-32. (a) EIC traces for the expected [M+GlcNAc] with D10-32 (left) and A10-32 (right); Vertical axes are normalized to 2.83$\times10^5$ counts. It is noteworthy that with all peptides used a gradual accumulation of several glycopeptides is observed, which we attribute to glycosylation within the T-rich region. (b) Graphical representation of short time courses performed (with representative EIC traces shown in part a) with A10-32 and D10-32. D10-32 shows rapid formation of a glycopeptide (GP) that appears to remain at a steady-state level over 15 minutes. Quantities are expressed as a ratio relative to an internal standard, N\textsuperscript{*}.}
	\label{fig:aspartate-is-glyco}
\end{figure}
%
It is worth noting, as mentioned briefly in the caption to Figure \ref{fig:aspartate-is-glyco}, that the signal observed in the [M+GlcNAc] EIC increases linearly over time for all peptides tested. Furthermore, the signal becomes diffuse after fifteen minutes due to numerous species that appear to be only partly resolved by LC, combined with the low absolute abundance of the glycopeptide species.\par
%
\section{Identifying and characterizing a succinimidyl peptide}
%
The decrease in glycopeptide abundance over time suggested that this species was being converted to other species. Further analysis of the reaction mixtures revealed the appearance of an anhydropeptide [M-H\textsubscript{2}O, \textit{m/z} 837.1253 (z=4+)] as well as a peptide with the same exact mass (isobaric) as the starting material but with a different retention time by LC (henceforth S.M. isomer)(Figure \ref{fig:aspartate-APandisomer}). The formation of both species was dependent on the presence of both OGT and UDP-GlcNAc (Figure \ref{fig:aspartate-OGT-UDPG-req}).\par
%
\begin{figure}
	\centerline{\includegraphics[width=1\columnwidth]{aspartate/S-Figure-S2-v1.eps}}
	\caption[Treatment of D10-32 with OGT leads to the formation of an anhydropeptide and a new species isobaric with starting material]{Treatment of D10-32 with OGT leads to the formation of an anhydropeptide and a new species isobaric with starting material. (left) EIC traces for D10-32 show consumption of starting material over time, and the formation of a new species isobaric with starting material, but having a different retention time (S.M. isomer). Vertical axes are normalized to 1.33$\times10^7$ counts. (right) EIC traces show the appearance of an anhydropeptide species. This species disappears over time. Vertical axes are normalized to 1.31$\times10^6$ counts.}
	\label{fig:aspartate-APandisomer}
\end{figure}
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\begin{figure}
	\centerline{\includegraphics[width=0.85\columnwidth]{aspartate/S-Figure-S3_v2.eps}}
	\caption[Reaction of D10-32 requires both UDP-GlcNAc and OGT]{Reaction of D10-32 requires both UDP-GlcNAc and OGT (a) EIC traces for D10-32 starting material (841.6279 \textit{m/z}). Vertical axes are scaled to 4$\times10^7$ counts. Reactions were conducted under standard isomerization conditions, except UDP-GlcNAc was omitted from the top two reactions. (b) Anhydropeptide formation (measured by integration of EIC for 837.1253 \textit{m/z}) is observed only for reactions where both UDP-GlcNAc and OGT are included.}
	\label{fig:aspartate-OGT-UDPG-req}
\end{figure}
%
A time course, conducted under substoichiometric conditions, showed that glycopeptide appeared before anhydropeptide, which in turn appeared before the starting material isomer (Figure \ref{fig:aspartate-order-species}). From these observations we concluded that the anhydropeptide had formed from the glycosyl aspartate, and we considered three pathways by which a glycosyl aspartate could progress to an anhydropeptide (Figure \ref{fig:aspartate-to-anhydro}).\par
%
\begin{figure}
	\centerline{\includegraphics[width=0.8\columnwidth]{aspartate/S-Figure-S4-v1.eps}}
	\caption[Temporal sequence for species produced during reaction of D10-32 with OGT]{Temporal sequence for species produced during reaction of D10-32 with OGT. Under standard reaction conditions with wild-type OGT, D10-32 consumption is concomitant with the formation of glycopeptide, anhydropeptide and an isobaric species (S.M. isomer), in that order. (a) A graphical representation of a ratio of integrated ion areas for D10-32/S.M. isomer (EIC 841.6279 \textit{m/z}) and anhydropeptide (EIC 837.1253 \textit{m/z}) relative to the internal standard N\textsuperscript{*} (EIC 574.2668 \textit{m/z}). (b) A zoom of data collected from zero to eight minutes of reaction of D10-32 with OGT, showing only integrated ion areas for glycopeptide (EIC 892.3978 \textit{m/z}) and anhydropeptide (EIC 837.1253 \textit{m/z}) relative to the internal standard N\textsuperscript{*} (EIC 574.2668 \textit{m/z}).}
	\label{fig:aspartate-order-species}
\end{figure}
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\begin{figure}
	\centerline{\includegraphics[width=0.65\columnwidth]{aspartate/glyco-asp-anhydro.eps}}
	\caption[Three possible mechanisms for anhydropeptide formation from a glycosyl aspartate]{Three possible mechanisms for anhydropeptide formation from a glycosyl aspartate. Pathway ``a'' involves trapping by the cysteine thiol, N-terminal to the glycosyl ester; pathway ``b'' involves attack of the C-terminal amide nitrogen, while pathway ``c'' involves attack of the N-terminal amide nitrogen. The result of pathway ``b'' is a succinimide, compared to pathway ``c'', which affords an \textit{N}-acyl-$\beta$-lactam. The product of pathway ``c'' is likely hydrolytically unstable~\cite{Vidya:2003ji}, with the potential to be attacked by water at either the endocyclic carbonyl to regenerate an aspartic acid residue, or at the exocyclic carbonyl to affect peptide backbone scission, the latter of which is not observed when D10-32 is treated with OGT/UDP-GlcNAc.}
	\label{fig:aspartate-to-anhydro}
\end{figure}
%
Pathway ``c'' seemed unlikely as it required the formation of a strained $\beta$-lactam ring (see Figure \ref{fig:aspartate-to-anhydro} caption). To test whether the anhydropeptide was a cyclic thioester (pathway ``a''), I replaced Cys9 with alanine (A9D10-32), serine (S9D10-32) and arginine (R9D10-32). These substitutions were previously shown to be compatible with glutamate glycosylation and cleavage~\cite{Janetzko:2016er,Lazarus:2013bq}. For each peptide we observed both an anhydropeptide and a peak isobaric with the corresponding starting material (Figure \ref{fig:succinimide}). Therefore, the anhydropeptide observed in reactions with D10-32 was not a cyclic thioester. The other pathway (pathway ``b'') leads to a succinimide. Succinimides have precedent in biological systems, where they are known to form spontaneously from certain aspartic acid (by dehydration) or asparagine residues (by deamidation) \cite{Athmer:2002dl,Geiger:1987ti,Grossenbacher:1993kc,Klaene:2014hx,Matthews:2017eh,McFadden:1987tz,Stephenson:q8KOOn2Y,Stevenson:1992tv}.\par
%
\begin{figure}
	\centerline{\includegraphics[width=0.75\columnwidth]{aspartate/S-Figure-S6.eps}}
	\caption[Cysteine 9 is not required for the formation of an anhydropeptide or an isobaric starting material species]{Cysteine 9 is not required for the formation of an anhydropeptide or an isobaric starting material species. EIC traces corresponding to [M-H\textsubscript{2}O] and [M] for C9X (X = A, S, R) variants are shown. Each peptide shows the formation of an anhydropeptide and a peak isobaric with the corresponding starting material. Vertical axes are normalized to the maximum number of counts observed for each given species. Horizontal axes are retention time in minutes. Reactions were conducted under standard isomerization conditions.}
	\label{fig:succinimide}
\end{figure}
%
Succinimides are known to be hydrolytically unstable under physiological conditions (t$_{\frac{1}{2}}$ = $\approx$ 138 min at pH 7.4 and 37 $^{\circ}$C)\cite{Geiger:1987ti,McFadden:1987tz}; hydrolysis can occur at either of the two endocyclic carbonyl groups to produce either an aspartic acid or isoaspartic acid species (Figure \ref{fig:asp-or-isoasp}), however the regiochemistry of this attack favors the formation of isoaspartate by between 3:1 to 5:1~\cite{Guttler:2013er,Konuklar:2003bu}.\par
%
\begin{figure}
	\centerline{\includegraphics[width=0.7\columnwidth]{aspartate/asp-isoasp-scheme.eps}}
	\caption[Succinimide hydrolysis can produce isoaspartate or aspartate]{Succinimide hydrolysis can produce isoaspartate or aspartate. Succinimide hydrolysis can occur at either C1 or C2 to produce isoaspartate and aspartate, respectively. The oxygen atom incorporated from bulk water is shown in red.}
	\label{fig:asp-or-isoasp}
\end{figure}
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\section{Characterizing an isoaspartyl peptide}
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\subsection{\textsuperscript{18}O incorporation supports succinimide and isoaspartate formation}
%
As isoaspartate and aspartate are isomeric, hydrolysis of a succinimide would explain the isobaric peaks observed in reactions of the D10-containing peptides with OGT. This would also be consistent with the fact that the isobaric species formed last in these reactions (Figure \ref{fig:aspartate-order-species}). Incorporation of \textsuperscript{18}O from bulk water is commonly employed to demonstrate the presence of a succinimide within a polypeptide \cite{Grossenbacher:1993kc,Liu:2012ks,Terashima:2007kt}.\par
%
Incorporation of \textsuperscript{18}O can be monitored through changes in the isotope distribution for a given charge state of the peptide \cite{Johnson:2004be}. For each \textsuperscript{18}O atom to replace an \textsuperscript{16}O atom there is a +$\frac{2}{z}$ shift in the isotope envelope, where \textit{z} is the charge state of the peptide, as shown in Figure~\ref{fig:D10-isotope-envelope}.
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%Figure below shows shift 100%
%
\begin{figure}
	\centerline{\includegraphics[width=0.8\columnwidth]{aspartate/isotope-envelope-shift-1.eps}}
	\caption[Calculated isotope envelope for D10-32 (z = 4+)]{Calculated isotope envelopes for D10-32. Theoretical model for sequential \textsuperscript{18}O incorporation into D10-32. Incorporation is modeled at 100 atom\% abundance, and shown for the quadruply charged ion (z=4+).}
	\label{fig:D10-isotope-envelope}
\end{figure}
%
% 18O incorporation model (@67%)
%
\begin{figure}
	\centerline{\includegraphics[width=0.95\columnwidth]{aspartate/S-Figure-S7.eps}}
	\caption[Theoretical model for \textsuperscript{18}O incorporation into D10-32]{Theoretical model for \textsuperscript{18}O incorporation into D10-32. A model for \textsuperscript{18}O incorporation (at 67 atom\% abundance, see section \ref{method:asp18O-model}) was constructed to obtain expected $\frac{A+n}{A+0}$ ratios.}
	\label{fig:18o-model}
\end{figure}
%
I reasoned that I could accurately quantify any changes in the isotope distribution by measuring the ratio of each isotopomer peak (A+n) relative to the monoisotopic peak (A+0) over time. Since the experimental resolution for the quadruply charged state of the D10-32 peptide (\textit{m/z} 841.62794, monoisotopic), was 38,506 (see section \ref{method:asp18O-model}), this meant that the maximum $\Delta$m resolvable under these conditions would be 0.02186 Da (Equation \ref{MS-resolving}, where R is the resolution (at FWHM), $\Delta$m is the difference (\textit{m/z}) between two peaks and M is the \textit{m/z} for the observed peak). Since this resolution in insufficient to resolve the mass difference between \textsuperscript{16}O+2$\times$\textsuperscript{13}C and \textsuperscript{18}O+2$\times$\textsuperscript{12}C ($\Delta$m = 0.002466 Da), we expect to observe accidental coincidence in the mass spectrum, rather than peak-splitting.
%
\begin{equation} \label{MS-resolving}
	R = \frac{M}{\Delta m}
\end{equation}
%
Under the experimental conditions employed, the maximum isotope enrichment of \textsuperscript{18}O would be 67 atom\% (see section \ref{method:asp18O}). As such, I constructed a model isotopic distribution where zero, one or two molar equivalents of \textsuperscript{18}O had been incorporated into the D10-32 peptide (at this abundance), and determined the expected $\frac{A+n}{A+0}$ ratios (Figure~\ref{fig:18o-model}).
%
I then conducted several time course experiments using \textsuperscript{18}O-water. With wild-type OGT, under single-turnover conditions, a clear shift in the isotope envelope was observed (Figure \ref{fig:18o-incorporation}). 
%
\begin{figure}
	\centerline{\includegraphics[width=0.45\columnwidth]{aspartate/18o-incorporation-D10.eps}}
	\caption[Observed \textsuperscript{18}O incorporation into D10-32]{Observed \textsuperscript{18}O incorporation into D10-32. A comparison of the isotope envelope for the quadruply charged state of D10-32 in \textsuperscript{18}O-water, without OGT (top), or after 45 minutes with wild-type OGT (bottom).}
	\label{fig:18o-incorporation}
\end{figure}
%
\textsuperscript{18}O incorporation is time-dependent, and required catalytically active OGT, as evidenced by the lack of incorporation when OGT (K842M) was employed (Figure \ref{fig:18o-incorporation-plots}). OGT (K842M) is compromised in it's ability to activate UDP-GlcNAc and does not allow for glycosyltransfer~\cite{Schimpl:2012ka}. Unsurprisingly, the use of wild-type OGT and \textsuperscript{16}O-water also showed no shift in the D10-32 isotope envelope. When E10-32 was reacted with wild-type OGT in the presence of \textsuperscript{18}O-water, no shift in the isotope envelope was observed (Figure \ref{fig:no-18o-E10}). This finding suggested that while both D10-32 and E10-32 form glycosyl ester intermediates, the glutamyl ester does not revert to starting material (via direct hydrolysis) to any appreciable extent. Presumably the same is true for the aspartyl GlcNAc ester, which lends further support to the notion that hydrolysis occurs from the succinimide species, rather than the glycosyl ester.
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\begin{figure}
	\centerline{\includegraphics[width=0.8\columnwidth]{aspartate/incorporation-plots-2.eps}}
	\caption[\textsuperscript{18}O incorporation requires active OGT]{\textsuperscript{18}O incorporation requires active OGT. Plots of $\frac{A+n}{A+0}$ ratios over time for D10-32 treated with wild-type OGT and \textsuperscript{16}O water (bottom left), with OGT (K842M) and \textsuperscript{18}O water (bottom right), and with wild-type OGT and \textsuperscript{18}O water (top left). Experiments were run in duplicate and all points are mean $\pm$ s.e.m.}
	\label{fig:18o-incorporation-plots}
\end{figure}
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\begin{figure}
	\centerline{\includegraphics[width=0.8\columnwidth]{aspartate/18O-incorporation-E10.eps}}
	\caption[\textsuperscript{18}O incorporation does not occur for E10-32]{\textsuperscript{18}O incorporation does not occur for E10-32. Reaction of E10-32 with wild-type OGT in either \textsuperscript{16}O or \textsuperscript{18}O water shows no \textsuperscript{18}O incorporation into the starting material peak. Reactions were conducted once, and run under single turnover conditions for 90 minutes. Theoretical incorporation ratios are for 67 atom\% \textsuperscript{18}O.}
	\label{fig:no-18o-E10}
\end{figure}
%
I compared the observed $\frac{A+n}{A+0}$ ratios to theoretical values for \textsuperscript{18}O incorporation at various levels of isotope enrichment. Under my experimental conditions, the abundance of \textsuperscript{18}O is 67\%, which means that for each molar equivalent of water to be incorporated/exchanged, 67\% would be \textsuperscript{18}O and 33\% would be \textsuperscript{16}O. Indeed, $\frac{A+n}{A+0}$ ratios for the incorporation of one \textsuperscript{18}O atom fit well to experimental data (Figure \ref{fig:fit-model}) suggesting that under these reaction conditions a single \textsuperscript{18}O was incorporated into the D10-32 peptide.\par
%
\begin{figure}
	\centerline{\includegraphics[width=0.95\columnwidth]{aspartate/model-fitting.eps}}
	\caption[Fitting experimental \textsuperscript{18}O incorporation to a theoretical model]{Fitting experimental \textsuperscript{18}O incorporation to a theoretical model. (a) Modeled isotope patterns are shown for D10-32 with 0\% \textsuperscript{18}O, 67\% \textsuperscript{18}O and 97\% \textsuperscript{18}O, along side the experimentally observed pattern for D10-32 after treatment with wild-type OGT and \textsuperscript{18}O-water for 90 minutes. The y-axis represents the fraction that a given isotopomer comprises relative to the sum of the first six isotopomers (A+0 through A+5). (b) Residuals, determined as the difference between the experimental isotopomer abundance at 90 minutes and the calculated abundance expected for 67 atom\% incorporation of a single \textsuperscript{18}O atom. Residuals are expressed as a \% error, which was computed by dividing the obtained residual by the experimental value and expressing it as a percentage.}
	\label{fig:fit-model}
\end{figure}
%
%MS/MS for D10
%
\begin{figure}[b]
    \caption[HCD MS/MS reveals position of \textsuperscript{18}O incorporation into D10-32 starting material isomer]{\textit{(On the following page)} HCD MS/MS reveals position of \textsuperscript{18}O incorporation into D10-32 starting material isomer. (a) 30 eV HCD spectrum of D10-32 S.M. isomer formed by treating D10-32 with wild-type OGT for 90 minutes in \textsuperscript{18}O water. Major \textit{b} and \textit{y} ions are labeled. The peptide sequence of D10-32 is shown and annotated to highlight the differences observed for \textit{b} and \textit{y} ions when this species is formed in \textsuperscript{16}O- vs. \textsuperscript{18}O-water. (b) Selected zooms of \textit{b} ions near the site of incorporation. A full list of ions observed by HCD is provided in Tables \ref{tab:y-ion-table-HCD-isoD10-32},~\ref{tab:b-ion-table1-HCD-isoD10-32},~\ref{tab:b-ion-table2-HCD-isoD10-32}. Vertical axes are not shown; each ion was plotted with the same vertical scaling for both \textsuperscript{16}O and \textsuperscript{18}O samples. Horizontal axes are \textit{m/z}.}
    \label{fig:MSMS-D10}
\end{figure}
\begin{figure}[p]
	\centerline{\includegraphics[width=0.95\columnwidth]{aspartate/S-Figure-S10-v2.eps}}
\end{figure}
%
%MS/MS (qTOF) for D10
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.95\columnwidth]{aspartate/CID-MSMS.eps}}
    \caption[CID MS/MS reveals position of \textsuperscript{18}O incorporation into D10-32 starting material isomer]{CID MS/MS reveals position of \textsuperscript{18}O incorporation into D10-32 starting material isomer. (a) CID spectra or D10-32 S.M. isomer at 2, 25 and 28 eV show that greater than 25 eV is required for fragmentation of the major 4+ ion (marked with a dark blue diamond). (b) A comparison of D10-32 S.M. isomer formed by treating D10-32 with wild-type OGT for 90 minutes in \textsuperscript{16}O-water (top) or \textsuperscript{18}O-water (bottom). Horizontal axes are \textit{m/z}, Vertical axes are normalized. A full list of ions observed by CID is provided in Tables~\ref{tab:y-ion-table-CID-isoD10-32},~\ref{tab:b-ion-table1-CID-isoD10-32}, and~\ref{tab:b-ion-table2-CID-isoD10-32}.}
    \label{fig:MSMS2-D10}
\end{figure}
%
I mapped the site of \textsuperscript{18}O incorporation using MS/MS (Figures \ref{fig:MSMS-D10} and \ref{fig:MSMS2-D10}, Tables \ref{tab:y-ion-table-HCD-isoD10-32}, \ref{tab:b-ion-table1-HCD-isoD10-32}, \ref{tab:b-ion-table2-HCD-isoD10-32}, \ref{tab:y-ion-table-CID-isoD10-32}, \ref{tab:b-ion-table1-CID-isoD10-32}, \ref{tab:b-ion-table2-CID-isoD10-32}). Compared to fragment ions for reactions carried out in \textsuperscript{16}O water, no shift was observed for \textit{b} ions up to \textit{b}\textsubscript{9} or \textit{y} ions up to \textit{y}\textsubscript{11}, which was as far as we could detect; however, a systematic shift of +2 Da was observed for each ion from \textit{b}\textsubscript{12} through \textit{b}\textsubscript{20}. These data showed that a single \textsuperscript{18}O atom had been incorporated within the tripeptide sequence spanning residues 9 to 11 (Figure \ref{fig:MSMS-D10}). Taken together with other data, we concluded that \textsuperscript{18}O had been incorporated into the side chain of Asp10 via hydrolysis of a succinimide produced from a glycosyl aspartate.\par
%
%
\begin{table} %Y ions HCD
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & C-term. & \textit{y}\textsuperscript{+} obs. (\textit{m/z}) & Error (ppm) \\
        \hline
        H(amidated) & 1 & 155.0926 & 0.64 \\ \hline
        Q & 2 & 283.1510 & 1.06 \\ \hline
        G & 3 & 340.1723 & 1.47 \\ \hline
        A & 4 & 411.2096 & 0.73 \\ \hline
        M & 5 & 542.2501 & 0.55 \\ \hline
        N & 6 & 656.2922 & 1.68 \\ \hline
        S & 7 & 743.3243 & 1.35 \\ \hline
		T & 8 & 844.3715 & 1.78 \\ \hline
		A & 9 & 915.4083 & 1.97 \\ \hline
		T & 10 & 1016.4527 & 5.02 \\ \hline
		T & 11 & 1117.5010 & 4.03 \\ \hline
        \end{tabular}
        \caption[List of \textit{y} ions for isoD10-32 by HCD]{List of \textit{y} ions for isoD10-32 by HCD. isoD10-32 (S.M. isomer formed by reaction of D10-32 with OGT/UDP-GlcNAc).}
        \label{tab:y-ion-table-HCD-isoD10-32}
    \end{center}
\end{table}
%
\begin{table} %B ions HCD (1/2)
    \begin{center}
        \begin{tabular}{|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{b}\textsuperscript{+} obs. (\textit{m/z}) & Error (ppm) \\
        \hline
        Y & 1 & -- & -- \\ \hline
        V & 2 & 263.1389 & 0.38 \\ \hline
        R & 3 & 419.2399 & 0.48 \\ \hline
        V & 4 & 518.3092 & 1.35 \\ \hline
        C & 5 & 621.3170 & 1.13 \\ \hline
        S & 6 & 708.3501 & 0.42 \\ \hline
        N & 7 & 822.3917 & 1.22 \\ \hline
		P & 8 & Ambiguous & -- \\ \hline
		P & 9 & Ambiguous & -- \\ \hline
		C & 10 & Interference with \textit{y}\textsubscript{10}+ & -- \\ \hline
		D & 11 & Ambiguous & -- \\ \hline
       	\end{tabular}
        \caption[List of singly-charged \textit{b} ions for isoD10-32 by HCD]{List of singly-charged \textit{b} ions for isoD10-32 by HCD. isoD10-32 (S.M. isomer formed by reaction of D10-32 with OGT/UDP-GlcNAc).}
        \label{tab:b-ion-table1-HCD-isoD10-32}
    \end{center}
\end{table}
%
\begin{table} %B ions HCD (2/2)
    \begin{center}
        \begin{tabular}{|c|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{b}\textsuperscript{2+} obs. (\textit{m/z}) & Error (ppm)\textsuperscript{$\dagger$} & Mass shift (Da)\textsuperscript{$\ddagger$}\\
        \hline
        N & 7  & 411.6997	& 0.73		& -- \\ \hline
        P & 8  & 460.2260	& 0.87		& -- \\ \hline
        P & 9  & 508.7533	& 1.18		& -- \\ \hline
        C & 10 & Ambiguous	& --		& -- \\ \hline
        D & 11 & Ambiguous	& --		& -- \\ \hline
        T & 12 & 669.2836	& 1479.89	& 1.98 \\ \hline
        H & 13 & 737.8244	& 1357.58	& 2.00 \\ \hline
		Q & 14 & 801.8534 	& 1248.67	& 2.00 \\ \hline
		T & 15 & 852.3778 	& 1175.27	& 2.00 \\ \hline
		G & 16 & 880.8879	& 1136.39	& 2.00 \\ \hline
		T & 17 & 931.4098 	& 1072.64	& 2.00 \\ \hline
		T & 18 & 981.9353 	& 1019.13	& 2.00 \\ \hline
		N & 19 & 1038.9545 	& 960.93	& 1.99 \\
		\hline \hline
		Residue & N-term. & \textit{b}\textsuperscript{3+} obs. (\textit{m/z}) & Error (ppm)\textsuperscript{$\dagger$} & Mass shift (Da)\textsuperscript{$\ddagger$}\\
		\hline
		T & 20 & 726.6568	& 918.88	& 2.00 	\\ \hline
       	\end{tabular}
        \caption[List of multiply-charged \textit{b} ions for isoD10-32 by HCD]{List of multiply-charged \textit{b} ions for isoD10-32 by HCD. isoD10-32 (S.M. isomer formed by reaction of D10-32 with OGT/UDP-GlcNAc).~\textsuperscript{$\dagger$}Error is measured as the difference in \textit{m/z} relative to predicted \textit{b} ions, expressed in ppm.~\textsuperscript{$\ddagger$}Mass shift measured as the difference in the monoisotopic peak within an isotope envelope between isoD10-32 peptide produced in \textsuperscript{16}O-water and \textsuperscript{18}O-water}
        \label{tab:b-ion-table2-HCD-isoD10-32}
    \end{center}
\end{table}
%
\begin{table} %Y ions QTOF
    \begin{center}
        \begin{tabular}{|c|c|c|c|c|c|}
        \hline
        Residue & C-term. & \textit{y}\textsuperscript{+} obs. (\textit{m/z}) & Error (ppm) & \textit{y}\textsuperscript{2+} obs. (\textit{m/z}) & Error (ppm) \\
        \hline
        H(amidated) & 1 	& --			& -- 	& -- 		& -- \\ \hline
        Q 			& 2 	& --			& -- 	& -- 		& -- \\ \hline
        G 			& 3 	& 340.1735		& 2.06 	& --		& -- \\ \hline
        A 			& 4 	& 411.2108		& 2.19 	& --		& -- \\ \hline
        M 			& 5 	& 542.2505		& 0.18 	& --		& -- \\ \hline
        N 			& 6 	& 656.2941		& 1.22 	& --		& -- \\ \hline
        S 			& 7 	& 743.3257		& 2.58 	& --		& -- \\ \hline
		T 			& 8 	& 844.3737		& 2.47 	& --		& -- \\ \hline
		A 			& 9 	& 915.4116		& 4.41 	& 458.2085 	& 0.44 \\ \hline
		T 			& 10 	& 1016.4561 	& 4.13 	& 508.7343 	& 3.54 \\ \hline
		T 			& 11 	& 1117.5062 	& 1.29 	& 559.2561 	& 0.54 \\ \hline
        \end{tabular}
        \caption[List of \textit{y} ions for isoD10-32 by CID]{List of \textit{y} ions for isoD10-32 by CID. isoD10-32 (S.M. isomer formed by reaction of D10-32 with OGT/UDP-GlcNAc).}
        \label{tab:y-ion-table-CID-isoD10-32}
    \end{center}
\end{table}
%
\begin{sidewaystable} %B ions QTOF
    \begin{center}
        \begin{tabular}{|c|c|c|c|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{b}\textsuperscript{2+} obs. (\textit{m/z}) 	& Error (ppm)\textsuperscript{$\dagger$} 	& Mass shift (Da)\textsuperscript{$\ddagger$} & \textit{b}\textsuperscript{3+} obs. (\textit{m/z}) 	& Error (ppm)\textsuperscript{$\dagger$} 	& Mass shift (Da)\textsuperscript{$\ddagger$}\\
        \hline
    %    T		& 18 	& --			& --		& -- 		& --			& --		& -- 			\\ \hline
        N		& 19 	& 1037.9571		& 0.00		& 2.00 		& --			& --		& --			\\ \hline
        T		& 20 	& 1088.4815		& 0.55		& 2.00 		& 725.9908		& 1.52		& 2.00			\\ \hline
        A		& 21 	& 1124.0000		& 0.44		& 2.00 		& 749.6701		& 1.73		& 2.00			\\ \hline
        T		& 22 	& 1174.5367		& 11.41		& 2.00 		& 783.3510		& 0.38		& 2.00			\\ \hline
        T		& 23 	& 1225.0385		& 7.10		& 2.00 		& Ambiguous		& --		& --			\\ \hline
        A		& 24 	& 1260.5637		& 1.59		& 2.00 		& Ambiguous		& --		& --			\\ \hline
        T		& 25 	& --			& --		& -- 		& 874.3946		& 1.03		& 2.00 			\\ \hline
        S		& 26 	& --			& --		& -- 		& Ambiguous		& --		& -- 			\\ \hline
        N		& 27 	& --			& --		& -- 		& 941.4193		& 1.27		& 2.00 			\\ \hline
        M		& 28 	& --			& --		& -- 		& 985.1014		& 0.81		& 2.00 			\\ \hline
        A		& 29 	& --			& --		& -- 		& 1008.7813		& 1.59		& 2.00 			\\ \hline
    %    G		& 30 	& --			& --		& -- 		& --			& --		& -- 			\\ \hline
        \end{tabular}
        \caption[List of \textit{b} ions for isoD10-32 by CID]{List of \textit{b} ions for isoD10-32 by CID. isoD10-32 (S.M. isomer formed by reaction of D10-32 with OGT/UDP-GlcNAc).~\textsuperscript{$\dagger$}Error is measured as the difference in \textit{m/z} relative to predicted \textit{b} ions, expressed in ppm.~\textsuperscript{$\ddagger$}Mass shift measured as the difference in the monoisotopic peak within an isotope envelope between isoD10-32 peptide produced in \textsuperscript{16}O-water and \textsuperscript{18}O-water.}
        \label{tab:b-ion-table1-CID-isoD10-32}
    \end{center}
\end{sidewaystable}
%
\begin{table} %B ions QTOF (2/2)
    \begin{center}
        \begin{tabular}{|c|c|c|c|c|}
        \hline
        Residue & N-term. & \textit{b}\textsuperscript{3+}--H\textsubscript{2}O obs. (\textit{m/z}) 	& Error (ppm)\textsuperscript{$\dagger$} 	& Mass shift (Da)\textsuperscript{$\ddagger$}\\
        \hline
%        T		& 18 	& --			& --		& -- 		\\ \hline
%        N		& 19 	& --			& --		& --		\\ \hline
        T		& 20 	& 719.9888		& 3.61		& 2.00		\\ \hline
        A		& 21 	& 743.6646		& 0.81		& 2.00		\\ \hline
        T		& 22 	& 777.3516		& 4.89		& 2.00		\\ \hline
        T		& 23 	& 811.0291		& 1.60		& 2.00		\\ \hline
        A		& 24 	& 834.7090		& 0.48		& 2.00		\\ \hline
        T		& 25 	& 868.3880		& 4.61		& 2.00		\\ \hline
        S		& 26 	& 897.4034		& 0.89		& 2.00		\\ \hline
        N		& 27 	& 935.4134		& 3.74		& 2.00		\\ \hline
%        M		& 28 	& --			& --		& --		\\ \hline
%        A		& 29 	& --			& --		& --		\\ \hline
%        G		& 30 	& --			& --		& -- 		\\ \hline
        \end{tabular}
        \caption[List of dehydrated \textit{b} ions for isoD10-32 by CID]{List of dehydrated \textit{b} ions for isoD10-32 by CID. isoD10-32 (S.M. isomer formed by reaction of D10-32 with OGT/UDP-GlcNAc).~\textsuperscript{$\dagger$}Error is measured as the difference in \textit{m/z} relative to predicted \textit{b} ions, expressed in ppm.~\textsuperscript{$\ddagger$}Mass shift measured as the difference in the monoisotopic peak within an isotope envelope between isoD10-32 peptide produced in \textsuperscript{16}O-water and \textsuperscript{18}O-water.}
        \label{tab:b-ion-table2-CID-isoD10-32}
    \end{center}
\end{table}
%
%
% KEEP GOING FROM HERE!
%								
%
Knowing that a single \textsuperscript{18}O atom was being incorporated from bulk solution, we reasoned that the first-order rate constant for succinimide hydrolysis could be obtained from the rate of \textsuperscript{18}O incorporation. I divided the observed $\frac{A+n}{A+0}$ ratio at each time point by the theoretical $\frac{A+n}{A+0}$ expected for the addition of one molar equivalent of \textsuperscript{18}O in order to obtain a fractional conversion. I then plotted conversions measured from $\frac{A+3}{A+0}$ and $\frac{A+4}{A+0}$ as a function of time, and fit the conversions to a linear regression. This data yielded an average first-order rate constant of 0.0039 min\textsuperscript{--1} (t$_{\frac{1}{2}}$ $\approx$ 177 min) (see Figure \ref{fig:18o-incorporation-linear-rate}). These values are comparable to rates previously measured for the spontaneous hydrolysis of succinimides in solution under similar conditions~\cite{Geiger:1987ti,McFadden:1987tz}. From these data it is unclear whether hydrolysis of the succinimide occurs while bound to OGT or not; however, the rate of hydrolysis is consistent with it being a non-enzymatic process.\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.80\columnwidth]{aspartate/S-Figure-S11-1.eps}}
	\caption[Rate of \textsuperscript{18}O incorporation into D10-32]{Rate of \textsuperscript{18}O incorporation into D10-32. The rate of succinimide hydrolysis was determined by the rate of \textsuperscript{18}O incorporation into the starting material and isomer peaks. The ratio of isotopomers at each time point relative to the expected ratio for one molar equivalent of \textsuperscript{18}O incorporation (at 67 atom\%) was used to obtain conversions. Time courses were conducted twice, on separate days, so data sets were plotted independently. The average slope between the four linear regressions affords a \textit{k\textsubscript{1}} of 0.0039 min\textsuperscript{--1}, corresponding to a t$_{\frac{1}{2}}$ $\approx$ 177 min.}
	\label{fig:18o-incorporation-linear-rate}
\end{figure}
%
\subsection{LC-MS/MS and PIMT provide further evidence}
%
To confirm that the isobaric peak observed in these reactions was an isoaspartyl peptide, as would be expected from hydrolysis of a succinimide intermediate, I obtained an authentic peptide standard, isoD10-32 (Table \ref{tab:listofpeptides}). This peptide had the same retention time as the isobaric peptide peak observed in the OGT reactions with D10-32 (Figure \ref{fig:isomer-LC-isoasp}).\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.75\columnwidth]{aspartate/S-Figure-S12.eps}}
	\caption[The S.M. isomer peak (D10-32) has the same LC retention time as an authentic isoD10-32 peptide]{The S.M. isomer peak (D10-32) has the same LC retention time as an authentic isoD10-32 peptide. EIC traces corresponding to the D10-32 starting material show that an authentic isoD10-32 peptide has a different retention time than starting material (S.M.), but the same retention time as the S.M. isomer peak that forms upon reaction with OGT. Vertical axes are normalized to 1.15$\times10^7$ counts.}
	\label{fig:isomer-LC-isoasp}
\end{figure}
%
Isoaspartate and aspartate cannot be readily distinguished in standard mass spectrometry experiments since they have the same \textit{m/z}~\cite{Eakin:2014eo}. Several other methods have had success distinguishing Asp and isoAsp~\cite{Carr:1991it}, including: Edman degradation~\cite{Weber1967vs}, site-specific \textsuperscript{3}H-incorporation~\cite{DiDonato:1986wu,Stevenson:1992tv}, carboxypeptidase Y cleavage~\cite{Johnson:1990we}, enzymatic methylation~\cite{Clarke:1985ja,Furuchi:2010do,Griffith:2001hy}, and nuclear magnetic resonance (NMR) spectroscopy~\cite{Chazin:1989uv}. In addition, several tandem mass spectrometry-based methods have shown the ability to distinguish isoaspartate from aspartate within a polypeptide~\cite{DeGraanWeber:2016iv,Lehmann:2000gz,Li:2012bh,OConnor:2006dg}. These methods employ ETD/ECD-based fragmentation to detect a unique ion pair, $c'$+57 and $z^\bullet$--57, which is produced when an isoaspartate is present. I obtained ETD spectra for both D10-32 and isoD10-32 and qualitatively evaluated these spectra for differences. Unfortunately there were no fragments unique to one species, and no notable differences in fragment ion intensity.\par
%
Seeking an alternative route to characterize the starting material isomer species, I turned to protein isoaspartyl methyl transferase (PIMT), an enzyme that repairs isoaspartate-containing proteins by selectively transferring a methyl group from SAM to the carboxylic acid of isoaspartate. Methylation promotes succinimide formation, and even though succinimide hydrolysis favors the re-formation of an isoaspartate, repeated cycles of methylation and succinimide hydrolysis eventually convert isoaspartate back to aspartate (Figure \ref{fig:PIMT-rxn})~\cite{Furuchi:2010do,Griffith:2001hy}. In the past, PIMT has been used to demonstrate the presence of an isoaspartate by quantifying the SAM-derived byproducts produced in the reaction, either methanol or \textit{S}-Adenosyl-\textsc{l}-homocysteine (AdoHcy; SAH)~\cite{Carlson:2000fg}. More recently, the use of PIMT in conjunction with \textsuperscript{18}O labeling has been used to identify isoaspartate residues within several polypeptides~\cite{Liu:2012ks}.
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.9\columnwidth]{aspartate/PIMT-rxn-scheme.eps}}
	\caption[PIMT catalyzes isoaspartate methylation, producing a succinimide that hydrolyzes to a mixture of aspartate and isoaspartate]{PIMT catalyzes isoaspartate methylation, producing a succinimide that hydrolyzes to a mixture of aspartate and isoaspartate. PIMT catalyzes SAM-dependent isoaspartate methylation, which leads to succinimide formation. Subsequent succinimide hydrolysis leads to a mixture of aspartate and isoaspartate. The example shown is for the dipeptide ``DG''.}
	\label{fig:PIMT-rxn}
\end{figure}
%
I treated D10-32 and isoD10-32 with PIMT and SAM, and only isoD10-32 reacted (Figure~\ref{fig:PIMT-is-selective}). Next, I treated a sample of D10-32, which had previously been incubated with wild-type OGT and UDP-GlcNAc to produce the putative isoaspartyl peptide, with PIMT/SAM; the peptide was consumed rapidly concomitant with a substantial increase in succinimide abundance (Figure~\ref{fig:isomer-is-isoasp}). Therefore, we concluded that OGT catalyzes isomerization of D10-32 to isoD10-32.\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.95\columnwidth]{aspartate/PIMT-specificity-1.eps}}
	\caption[PIMT methylates isoaspartate, but not aspartate]{PIMT methylates isoaspartate, but not aspartate. (a) D10-32 is not methylated by PIMT; there is no significant anhydropeptide observed for both --PIMT and +PIMT samples. (b) isoD10-32 is methylated by PIMT; the consumption of the isoaspartyl peptide (left) is concomitant with the production of anhydropeptide (right). Vertical axes are normalized to 1.03$\times10^7$ counts (isoD10-32 starting material) and 2.08$\times10^6$ counts (anhydropeptide).}
	\label{fig:PIMT-is-selective}
\end{figure}
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.8\columnwidth]{aspartate/PIMT-reacts-after-OGT.eps}}
	\caption[The starting material isomer is an isoaspartate-containing peptide]{The starting material isomer is an isoaspartate-containing peptide. (a) EIC traces for 841.6279 (4+), corresponding to D10-32 starting material and isomer. Traces are after treatment with wild-type OGT alone (top) or with wild-type OGT followed by PIMT (bottom). (b) Normalized succinimide abundance for OGT and PIMT treated D10-32 (left), pure isoD10-32 (middle) or pure D10-32 (right); abundances were measured by integrating the respective EIC traces for succinimide, 837.1253 (4+). The retention time of the anhydropeptide species formed by PIMT and OGT are identical.}
	\label{fig:isomer-is-isoasp}
\end{figure}
%
\subsection{Mechanistic considerations}
%
The fact that D10-32 was entirely consumed, rather than converted to an equilibrium mixture of aspartyl and isoaspartyl peptides, suggested that isoD10-32 was likely a poor substrate for OGT. In order to test whether it reacted, I conducted a time-course experiment with isoD10-32 and wild-type OGT and compared it to one of D10-32 under identical conditions. While D10-32 was consumed, isoD10-32 was modified by OGT with poor efficiency (Figure~\ref{fig:asp-vs-isoasp}). In addition, the amount of succinimide produced from isoD10-32 was at least an order of magnitude lower than that produced from D10-32 (Figure~\ref{fig:asp-vs-isoasp}).\par
%
\begin{figure}[ht]
	\centerline{\includegraphics[width=0.95\columnwidth]{aspartate/asp-vs-isoasp.eps}}
	\caption[isoD10-32 is a poor substrate for OGT]{isoD10-32 is a poor substrate for OGT. (a) Plot of D10-32 or isoD10-32 abundance, relative to N\textsuperscript{*}, determined from integrated EIC traces [841.6279 (4+)] and [574.2668 (2+)], respectively. (b) Normalized succinimide abundance, as measured by integration of EIC traces for succinimide, [837.1253 (4+)].}
	\label{fig:asp-vs-isoasp}
\end{figure}
%
Though these data show that OGT can glycosylate aspartate, in addition to serine/threonine and glutamate, whether OGT glycosylates any cellular proteins on aspartate is currently unknown. In order to assess the feasibility of aspartate glycosylation, I compared the rates of glycosylation on serine, glutamate and aspartate under identical conditions. Using three nearly identical peptide substrates with different glycosyl acceptor residues (S10-35, D10-32, and E10-32), I measured the rate of starting material disappearance as a proxy for the rate of glycosylation. The serine peptide reacted approximately 5-fold faster than the aspartyl peptide and at least 100-fold faster than the glutamyl peptide (Figure~\ref{fig:rate-comparison}). Based on structural data supported by measurements of reaction rates for an OGT mutant, OGT (D554N), we previously proposed that glycosylation on glutamate is disfavored by a repulsive interaction with aspartate 554 in OGT~\cite{Janetzko:2016er}. The nucleophile in D10-32 would not approach the D554 carboxylate as closely, which may explain its faster rate of glycosylation compared with E10. This notion is corroborated by previous findings that an E10D mutation enhanced the ability to co-immunoprecipitate an HCF substrate with OGT~\cite{Bhuiyan:2015fg}. Given that glycosylation on aspartate is relatively fast for a model peptide substrate, the possibility exists that OGT may glycosylate some proteins on aspartate.\par
%
\begin{figure}[b]
	\caption[Comparison of glycosyl acceptor abilities]{\textit{(On the following page)} Comparison of glycosyl acceptor abilities. (a) A plot of S10-35 starting material over time shows rapid consumption when serine is the acceptor. Data was fit with to a one-phase decay (see Equation~\ref{first-order-decay}). (b) A plot of D10-32 starting material over time shows consumption of an Asp-containing acceptor to be $\approx5\times$ slower than for a serine-containing acceptor. Reactant concentrations were inferred from fractional abundances relative to an untreated sample, and scaled according to the initial reactant concentration (12 $\mu$M). Data was fit with to a one-phase decay (Equation~\ref{first-order-decay}). (c) A plot of E10-32 over time reveals slow consumption of starting material; after more than 200 minutes, the amount of starting material consumed was on the order of $1-2\times$ [E]\textsubscript{T} (left). Starting material was measured by relative abundance to known amount used in the reaction. The N-terminal cleavage fragment (``N-term'') produced in the E10-32 cleavage reaction (right) was quantified by stable isotope dilution (SID)~\cite{Liebler:2013ka} using an isotopically labeled N-term internal standard (N\textsuperscript{*}, see Table~\ref{tab:listofpeptides}). A calibration curve was generated, and concentrations were determined by extrapolation (Figure~\ref{fig:app-calibration-curve-SID}). After 200 minutes, the concentration of N-term was measured to be $\approx$ 1 $\mu$M, consistent with the amount of starting material that had been consumed.}
	\label{fig:rate-comparison}
\end{figure}
\begin{figure}[p]
	\centerline{\includegraphics[width=0.95\columnwidth]{aspartate/S-Figure-S14-1.eps}}
\end{figure}
%
\section{Conclusions}
%
Until now isoaspartate formation was known only as a non-enzymatic process associated with aging proteins~\cite{Geiger:1987ti,Guttler:2013er,Qin:2015cs,Stephenson:q8KOOn2Y}. Our findings show that OGT can catalyze aspartate-to-isoaspartate isomerization in a model substrate, and one might expect that other enzymes that glycosylate aspartate might also promote this process as long as the local sequence permits succinimide formation~\cite{Clarke:1987bb}. While we do not yet know if OGT modifies aspartate in any cellular proteins, we note that PARPs naturally glycosylate aspartic acid residues~\cite{Daniels:2014kz,Hassa:2006iz,Martello:2016bq,Vyas:2014cq,Zhang:2013iu}; this activity may, therefore, lead to succinimide formation in some cases. If so, it is worth considering whether isoaspartate formation is always an unwanted side reaction or may serve other functions~\cite{Artigues:1990tg,Biterge:2014gi,Corti:2011ba,vanLoo:2008kw}.
%
\section*{Acknowledgments}
%
I am grateful for support from the National Science and Engineering Research Council (NSERC) of Canada as a PGS-M and PGS-D3 fellowship recipient. This work was funding by a National Institutes of Health grant (R01 GM094263) to Suzanne Walker. I thank David J. Vocadlo (Simon Fraser University, Vancouver, BC, Canada) for providing UDP-5\textit{S}GlcNAc. Sunia A. Trauger, Bogdan Budnik and John Neveu provided mass spectrometry assistance at various times during this work. Additional thanks to Michael B. Lazarus, Sara E. S. Martin and Zebulon G. Levine for their thoughtful discussions pertaining to this work and for critical reading of the \textit{J. Am. Chem. Soc.} manuscript.\par
%
\newpage
%
\section{Methods} \label{sec:aspartate-methods}
%
\subsection{Reagents and General Methods}
%
UDP-GlcNAc was obtained from Promega as a 100 mM solution in water. THP was obtained from Novagen as a 0.5 M solution in water. Calf alkaline phosphatase (CIP) was obtained from New England Biolabs. \textsuperscript{18}O-water (97 atom\%) was obtained from Cambridge isotope laboratories. PIMT and SAM were obtained from the Isoquant\textsuperscript{\textregistered} isoaspartate detection kit (Promega). Peptides were obtained from Biomatik (Cambridge, ON, Canada); their purity was evaluated by HPLC and mass spectrometry, and they were used as received. Lyophilized peptides were dissolved in water to $\approx5$ mM and added to reactions according to the final concentration desired. Peptide concentrations were determined by measuring A\textsubscript{274} ($\lambda$\textsubscript{max}, tyrosine) in water, assuming an extinction coefficient of 1400 M\textsuperscript{–-1}$\cdot$cm\textsuperscript{–-1}. A complete list of peptides used in this study is provided in Table \ref{tab:listofpeptides}. As the model peptide employed for these studies was derived from an HCF-1 cleavage substrate peptide, numbering begins from the first valine in the sequence (underlined in Table \ref{tab:listofpeptides}) in order to remain consistent with the numbering convention used in the HCF-1 literature.\par
%
\subsection{General LC-MS Methods}
%
General measurements of peptides were performed on a q-Exactive Plus FT-mass spectrometer (Thermo Scientific\textsuperscript{TM}) equipped with an Ultimate 3000 uHPLC. Reaction mixture components were separated using a 33 minute reverse phase HPLC method (Table \ref{tab:gradmethod1}); the last half of the LC method was empirically optimized to wash the column and re-equilibrate between runs to maintain column performance. with a Kinetex\textsuperscript{\textregistered} C18 (2.6 $\mu$m particle size, 100 \AA  ~pore size, 2.1 mm ID, 150 mm length) at a constant flow rate of 0.200 mL/min and column temperature of 35 $^{\circ}$C.  5 $\mu$L of sample was injected, unless otherwise specified. Detection on the q-Exactive Plus mass spectrometer was performed between 300–-2000 \textit{m/z}, using an acquisition target of 3$\times$10$^{6}$, maximum IT of 200 ms at a resolution of 70,000 for MS and 35,000 for MS/MS data. For MS/MS analysis, dynamic exclusion was turned off and a global inclusion list was used to maximize S/N for the targeted analysis. A collision energy of 30 eV and an isolation window of 6 \textit{m/z} was used for higher-energy collision-induced dissociation (HCD).\par
%
Additional measurements of peptides were collected on a Bruker Impact HD q-ToF mass spectrometer coupled to an Agilent 1290 uHPLC. Sample components were separated using a 45 minute reverse phase HPLC method (Table \ref{tab:gradmethod2}) with a Waters XBridge Peptide BEH C18 column (5 $\mu$m particle size, 300 \AA ~pore size, 1.0 mm ID, 100 mm length) at a constant flow rate of 0.100 mL/min with ambient column temperature. 10 $\mu$L of sample was injected, unless otherwise specified. A collision energy of 28 eV and an isolation window of 6 \textit{m/z} was used for collision-induced dissociation (CID). The source and ToF parameters were optimized for the transmission of higher molecular weight ions. A counter current drying gas flow rate of 12 L/min, and a nebulizing gas pressure of 45 bar were used. Funnel 1 was set to 150 V, and funnel 2 was set to 36 V, with a hexapole RF of 300 V\textsubscript{p--p}. A calibration segment was present at the beginning and end of the run for \textit{m/z} calibration with sodium formate cluster using the Bruker HPC algorithm for the curve fitting.\par
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Unless otherwise stated, extracted ion chromatograms displayed are for the indicated \textit{m/z} $\pm5$ ppm tolerance.\par
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\subsection{Purification of OGT}
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Wild-type OGT, OGT (K842M), OGT (D554N) and hOGT4.5 were expressed and purified as outlined in section~\ref{sec:ogt-purification-1}.
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\subsection{\textit{In vitro} peptide isomerization experiments} \label{ss:isomerization}
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Unless specified otherwise, reactions were conducted under the conditions outlined below, where the substrate:enzyme ratio is 10:1.\par

Isomerization reactions were conducted as follows: 20 $\mu$L reaction volume with components having the following final concentrations: 1 mM UDP-GlcNAc, 1 mM THP, 12 $\mu$M peptide substrate, 5 $\mu$M N* (internal standard; Table \ref{tab:listofpeptides}), 0.1 U CIP and 1.2 $\mu$M OGT; the reaction buffer contained 20 mM MgCl\textsubscript{2}, 20 mM Tris and 150 mM NaCl at pH 7.4. OGT was added last and reactions were mixed by pipetting then incubated at 37 $^{\circ}$C in a thermomixer to ensure uniform heating. After the desired reaction time, reactions were quenched by addition of four volumes (80 $\mu$L) of ice-cold methanol supplemented with 0.1\% formic acid. Reactions were then centrifuged at 16,100$\times$g for 10 minutes to remove any precipitated protein (4 $^{\circ}$C), and transferred to fresh tubes. Samples were concentrated to dryness using a speedvac (T = 30 $^{\circ}$C; $\approx$45--60 minutes). Samples were re-suspended in 100 $\mu$L 0.1\% formic acid, mixed by vortexing, and centrifuged for 10 minutes at 16,100$\times$g (4 $^{\circ}$C) to remove any insoluble material. Samples were then transferred to LC vials, and maintained at 4 $^{\circ}$C until they were analyzed.
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\subsection{\textit{In vitro} single turnover (STO) isomerization reactions} \label{ss:STO-isomerization}
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Isomerization reactions were conducted as follows: 20 $\mu$L reaction volume with components having the following final concentrations: 1 mM UDP-GlcNAc, 1 mM THP, 12 $\mu$M peptide substrate, 5 $\mu$M N* (internal standard; Table \ref{tab:listofpeptides}), 0.1 U CIP and 20 $\mu$M OGT; the reaction buffer contained 20 mM MgCl\textsubscript{2}, 20 mM Tris and 150 mM NaCl at pH 7.4. OGT was added last and reactions were mixed by pipetting then incubated at 37 $^{\circ}$C in a thermomixer to ensure uniform heating. After the desired reaction time, reactions were quenched by addition of four volumes (80 $\mu$L) of ice-cold methanol supplemented with 0.1\% formic acid. Reactions were then centrifuged at 16,100$\times$g for 10 minutes to remove any precipitated protein (4 $^{\circ}$C), and transferred to fresh tubes. Samples were concentrated to dryness using a speedvac (T = 30 $^{\circ}$C; $\approx$45--60 minutes). Samples were re-suspended in 100 $\mu$L 0.1\% formic acid, mixed by vortexing, and centrifuged for 10 minutes at 16,100$\times$g (4 $^{\circ}$C) to remove any insoluble material. Samples were then transferred to LC vials, and maintained at 4 $^{\circ}$C until they were analyzed.
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\subsection{\textsuperscript{18}O incorporation experiments} \label{method:asp18O}
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\textsuperscript{18}O experiments were conducted under single turnover conditions, as described in section~\ref{ss:STO-isomerization}. Each experimental condition (\textsuperscript{18}O-water \& wild-type OGT; \textsuperscript{16}O-water \& wild-type OGT; \textsuperscript{18}O-water \& OGT (K842M) were conducted twice. Stock reagents were diluted into \textsuperscript{16}O or \textsuperscript{18}O water, to give $\approx$ 100 atom\% and 67 atom\% abundance, respectively.
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\subsection{Isotope modeling} \label{method:asp18O-model}
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The experimental data was fit according to a least-squares minimization of residuals to determine the proportion of zero, one or two \textsuperscript{18}O incorporations that best represented the observed population. Analysis was performed using MatLab using a custom written script. The best fit corresponded to 0.64\% zero, 96.9\% one, and 2.46\% two incorporations.\par
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Peak area for individual isotopomers was obtained by EIC integration. Isotopomer abundances were then converted to ratios relative to the monoisotopic peak (A+0). Using Bruker Data Analysis software, the relative ratios of isotopomers expected for the incorporation of zero, one or two \textsuperscript{18}O atoms (determined at 67 atom\% abundance) were calculated. The Web-based server MS-Isotope (UCSF) can also be used to compute these ratios. A charge envelope corresponding to the quadruply charged species for the D10-32 peptide was modeled at the observed resolution: 38,506 (q-Exactive). \textsuperscript{18}O incorporation rates (reaction conversion) were measured indirectly as the change in ratio of $\frac{A+n}{A+0}$ over time relative to the ratio expected for the addition of one molar equivalent of \textsuperscript{18}O into the peptide. In order to ensure that the incorporation of a single molar equivalent of 67 atom\% abundance \textsuperscript{18}O was consistent with experimental data, the observed isotopomer ratios for experimental data (90 minute treatment with wild-type OGT and \textsuperscript{18}O water) were fit to the expected 67 atom\% incorporation model, and residuals were determined.
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\subsection{\textit{In vitro} cleavage/glycosylation experiments}
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Cleavage (E10-32) and glycosylation (S10-35) reactions were conducted under the same conditions outlined isomerization reactions (see section \ref{ss:isomerization}) wherein the substrate:enzyme ratio is 10:1.
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\subsection{\textit{In vitro} PIMT reactions}
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PIMT assays were conducted by adding 0.2 $\mu$L of SAM (1 mM stock) and 10 $\mu$L of PIMT (used as obtained from Promega) directly to isomerization reactions (conducted as described in section \ref{ss:isomerization}). Reactions were incubating for 30 minutes at 30 $^{\circ}$C with a thermomixer. As in the case of OGT reactions, reactions were quenched with the addition of four volumes (120 $\mu$L) ice-cold methanol supplemented with 0.1\% formic acid. Reactions were then centrifuged at 16,100$\times$g for 10 minutes to remove any precipitated protein (4 $^{\circ}$C), and transferred to fresh tubes. Samples were concentrated to dryness using a speedvac (T = 30 $^{\circ}$C; $\approx$45--60 minutes). Samples were re-suspended in 100 $\mu$L 0.1\% formic acid, mixed by vortexing, and centrifuged for 10 minutes at 16,100$\times$g (4 $^{\circ}$C) to remove any insoluble material. Samples were then transferred to LC vials, and maintained at 4 $^{\circ}$C until they were analyzed.
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\subsection{MS/MS of reaction-derived isoD10-32 for mapping the site of \textsuperscript{18}O incorporation}
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Samples for MS/MS were prepared as described in section \ref{method:asp18O}. MS\textsuperscript{2} spectra examined were for the peak corresponding to isoD10-32 (S.M. isomer) formed after a 90 minute reaction of D10-32 with wild-type OGT in the presence of either \textsuperscript{16}O or \textsuperscript{18}O water. Spectra were measured on both q-Exactive (HCD) and q-ToF (CID) mass spectrometers. With both instruments, we isolated the quadruply-charged species 841.6279 \textit{m/z}. With the q-ToF, ion isolation was verified using a 2 eV MS\textsuperscript{2} energy. Appreciable parent ion remained at 25 eV collision energy; however, the ion was almost entirely fragmented at 28 eV (See Figure~\ref{fig:MSMS2-D10}). There was no incorporation of \textsuperscript{18}O post-reaction, as determined by comparing samples resuspended in \textsuperscript{16}O vs. \textsuperscript{18}O water supplemented with 0.1\% formic acid prior to MS-analysis. Data were manually inspected for predicted fragment ions (as determined using the UCSF MS-Product server) within a 5 ppm mass accuracy window.
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