Abstract
O-GlcNAcylation is one of the most abundant metazoan nuclear-cytoplasmic post-translational modifications. Proteins modified by O-GlcNAc play key cellular roles in signaling, transcription, metabolism, and cell division. Mechanistic studies on protein O-GlcNAcylation are hampered by the lack of methods that can simultaneously quantify O-GlcNAcylation, determine its stoichiometry, and monitor O-GlcNAcylation kinetics. Here, we demonstrate that high-resolution native mass spectrometry can be employed to monitor the small mass shifts induced by modification by O-GlcNAc on two known protein substrates, CK2α and TAB1, without the need for radioactive labeling or chemoenzymatic tagging using large mass tags. Limited proteolysis enabled further localization of the O-GlcNAc sites. In peptide-centric MS analysis, the O-GlcNAc moiety is known to be easily lost. In contrast, we demonstrate that the O-GlcNAc is retained under native MS conditions, enabling precise quantitative analysis of stoichiometry and O-GlcNAcylation kinetics. Together, the data highlight that high resolution native MS may provide an alternative tool to monitor kinetics on one of the most labile of protein post-translational modifications, in an efficient, reliable, and quantitative manner.
Highlights
P ost-translational modifications are vital cell communication signals that can transfer messages between proteins enabling signaling pathways to be turned on or off
We show the advantages of native MS in monitoring O-GlcNAcylation, highlighting its ability to determine O-GlcNAcylation stoichiometry on proteins while simultaneously being able to quantify O-GlcNAcylation kinetics
We overexpressed and purified CK2α from E. coli, leading to a very clean native MS spectrum displaying a narrow charge state distribution (12+ and 13+ charge state ions) corresponding to a molecular weight of 43202.3 Da, which is within 0.002% of the calculated mass based on the sequence (43203.2 Da; Figure 1A, Supporting Information Figure 1)
Summary
GlcNAc transferase after 0 and 90 min (B). Native ESI-MS spectra of TAB1 (C) and upon incubation with the O-GlcNAc transferase after 5, 90, and 1440 min (D). The ratio of OGlcNAcylated versus unmodified TAB1 protein remained constant upon increasing the collision energy from 0 V (Figure 2A) to 175 V (Figure 2B and Supporting Information Figure 7) This is striking as already at 30 V collision energy over 40% of the O-GlcNAc moieties were released from the corresponding O-GlcNAcylated C-terminal peptide (Figure 2 and Figure S7). To further illustrate the ability of native MS in quantitatively monitoring O-GlcNAcylation kinetics, the percentage OGlcNAcylation of TAB17−402 (blue), TAB1Δ385−402 (orange), and TAB1Δ395−402 (green) incubated in a single vial with the O-GlcNAc transferase was determined and plotted as a function of the reaction time (Figure 3A). Upon plotting the % O-GlcNAcylation of the TAB1 peptide res[387−402] over time, very similar kinetics were observed compared with those when measured on the intact TAB17−402 protein by native MS (Figure 3, Supporting Information Figure 6B). Evidence suggests this is not the case considering the O-GlcNAc residue predominantly resides on Ser[395] in the TAB17−402 protein (Supporting Information Figure 8), and the signal contributing to the O-GlcNAcylated protein with two O-GlcNAcylation sites is less than 5% of the total signal intensity after 24 h (Supporting Information Figure 4)
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