6 research outputs found
ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates
Protein
arginine methylations are important post-translational
modifications (PTMs) in eukaryotes, regulating many biological processes.
However, traditional collision-based mass spectrometry methods inevitably
cause neutral losses of methylarginines, preventing the deep mining
of biologically important sites. Herein we developed an optimized
mass spectrometry workflow based on electron-transfer dissociation
(ETD) with supplemental activation for proteomic profiling of arginine
methylation in human cells. Using symmetric dimethylarginine (sDMA)
as an example, we show that the ETD-based optimized workflow significantly
improved the identification and site localization of sDMA. Quantitative
proteomics identified 138 novel sDMA sites as potential PRMT5 substrates
in HeLa cells. Further biochemical studies on SERBP1, a newly identified
PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric
dimethylarginine in the central RGG/RG motif, and loss of either methylation
caused increased the recruitment of SERBP1 to stress granules under
oxidative stress. Overall, our optimized workflow not only enabled
the identification and localization of extensive, nonoverlapping sDMA
sites in human cells but also revealed novel PRMT5 substrates whose
sDMA may play potentially important biological functions
ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates
Protein
arginine methylations are important post-translational
modifications (PTMs) in eukaryotes, regulating many biological processes.
However, traditional collision-based mass spectrometry methods inevitably
cause neutral losses of methylarginines, preventing the deep mining
of biologically important sites. Herein we developed an optimized
mass spectrometry workflow based on electron-transfer dissociation
(ETD) with supplemental activation for proteomic profiling of arginine
methylation in human cells. Using symmetric dimethylarginine (sDMA)
as an example, we show that the ETD-based optimized workflow significantly
improved the identification and site localization of sDMA. Quantitative
proteomics identified 138 novel sDMA sites as potential PRMT5 substrates
in HeLa cells. Further biochemical studies on SERBP1, a newly identified
PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric
dimethylarginine in the central RGG/RG motif, and loss of either methylation
caused increased the recruitment of SERBP1 to stress granules under
oxidative stress. Overall, our optimized workflow not only enabled
the identification and localization of extensive, nonoverlapping sDMA
sites in human cells but also revealed novel PRMT5 substrates whose
sDMA may play potentially important biological functions
ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates
Protein
arginine methylations are important post-translational
modifications (PTMs) in eukaryotes, regulating many biological processes.
However, traditional collision-based mass spectrometry methods inevitably
cause neutral losses of methylarginines, preventing the deep mining
of biologically important sites. Herein we developed an optimized
mass spectrometry workflow based on electron-transfer dissociation
(ETD) with supplemental activation for proteomic profiling of arginine
methylation in human cells. Using symmetric dimethylarginine (sDMA)
as an example, we show that the ETD-based optimized workflow significantly
improved the identification and site localization of sDMA. Quantitative
proteomics identified 138 novel sDMA sites as potential PRMT5 substrates
in HeLa cells. Further biochemical studies on SERBP1, a newly identified
PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric
dimethylarginine in the central RGG/RG motif, and loss of either methylation
caused increased the recruitment of SERBP1 to stress granules under
oxidative stress. Overall, our optimized workflow not only enabled
the identification and localization of extensive, nonoverlapping sDMA
sites in human cells but also revealed novel PRMT5 substrates whose
sDMA may play potentially important biological functions
Evaluation of Different N‑Glycopeptide Enrichment Methods for N‑Glycosylation Sites Mapping in Mouse Brain
N-Glycosylation of
proteins plays a critical role in many biological
pathways. Because highly heterogeneous N-glycopeptides are present
in biological sources, the enrichment procedure is a crucial step
for mass spectrometry analysis. Five enrichment methods, including
IP-ZIC-HILIC, hydrazide chemistry, lectin affinity, ZIC-HILIC-FA,
and TiO<sub>2</sub> affinity were evaluated and compared in the study
of mapping N-glycosylation sites in mouse brain. On the basis of our
results, the identified N-glycosylation sites were 1891, 1241, 891,
869, and 710 and the FDR values were 3.29, 5.62, 9.54, 9.54, and 20.02%,
respectively. Therefore, IP-ZIC-HILIC enrichment method displayed
the highest sensitivity and specificity. In this work, we identified
a total of 3446 unique glycosylation sites conforming to the N-glycosylation
consensus motif (N-X-T/S/C; X ≠ P) with <sup>18</sup>O labeling
in 1597 N-glycoproteins. N-glycosylation site information was used
to confirm or correct the transmembrane topology of the 57 novel transmembrane
N-glycoproteins
Table_1_Role of N-Glycosylation in FcγRIIIa interaction with IgG.xlsx
Immunoglobulins G (IgG) and their Fc gamma receptors (FcγRs) play important roles in our immune system. The conserved N-glycan in the Fc region of IgG1 impacts interaction of IgG with FcγRs and the resulting effector functions, which has led to the design of antibody therapeutics with greatly improved antibody-dependent cell cytotoxicity (ADCC) activities. Studies have suggested that also N-glycosylation of the FcγRIII affects receptor interactions with IgG, but detailed studies of the interaction of IgG1 and FcγRIIIa with distinct N-glycans have been hindered by the natural heterogeneity in N-glycosylation. In this study, we employed comprehensive genetic engineering of the N-glycosylation capacities in mammalian cell lines to express IgG1 and FcγRIIIa with different N-glycan structures to more generally explore the role of N-glycosylation in IgG1:FcγRIIIa binding interactions. We included FcγRIIIa variants of both the 158F and 158V allotypes and investigated the key N-glycan features that affected binding affinity. Our study confirms that afucosylated IgG1 has the highest binding affinity to oligomannose FcγRIIIa, a glycan structure commonly found on Asn162 on FcγRIIIa expressed by NK cells but not monocytes or recombinantly expressed FcγRIIIa.</p
DataSheet_1_Role of N-Glycosylation in FcγRIIIa interaction with IgG.pdf
Immunoglobulins G (IgG) and their Fc gamma receptors (FcγRs) play important roles in our immune system. The conserved N-glycan in the Fc region of IgG1 impacts interaction of IgG with FcγRs and the resulting effector functions, which has led to the design of antibody therapeutics with greatly improved antibody-dependent cell cytotoxicity (ADCC) activities. Studies have suggested that also N-glycosylation of the FcγRIII affects receptor interactions with IgG, but detailed studies of the interaction of IgG1 and FcγRIIIa with distinct N-glycans have been hindered by the natural heterogeneity in N-glycosylation. In this study, we employed comprehensive genetic engineering of the N-glycosylation capacities in mammalian cell lines to express IgG1 and FcγRIIIa with different N-glycan structures to more generally explore the role of N-glycosylation in IgG1:FcγRIIIa binding interactions. We included FcγRIIIa variants of both the 158F and 158V allotypes and investigated the key N-glycan features that affected binding affinity. Our study confirms that afucosylated IgG1 has the highest binding affinity to oligomannose FcγRIIIa, a glycan structure commonly found on Asn162 on FcγRIIIa expressed by NK cells but not monocytes or recombinantly expressed FcγRIIIa.</p