3 research outputs found
Exploring an Alternative Cysteine-Reactive Chemistry to Enable Proteome-Wide PPI Analysis by Cross-Linking Mass Spectrometry
The
development of MS-cleavable cross-linking mass spectrometry
(XL-MS) has enabled the effective capture and identification of endogenous
protein–protein interactions (PPIs) and their residue contacts
at the global scale without cell engineering. So far, only lysine-reactive
cross-linkers have been successfully applied for proteome-wide PPI
profiling. However, lysine cross-linkers alone cannot uncover the
complete PPI map in cells. Previously, we have developed a maleimide-based
cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide
(BMSO)) that is effective for mapping PPIs of protein complexes to
yield interaction contacts complementary to lysine-reactive reagents.
While successful, the hydrolysis and limited selectivity of maleimides
at physiological pH make their applications in proteome-wide XL-MS
challenging. To enable global PPI mapping, we have explored an alternative
cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing
MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide
(DBrASO). Our results have demonstrated that DBrASO cross-linked peptides
display the same fragmentation characteristics as other sulfoxide-containing
MS-cleavable cross-linkers, permitting their unambiguous identification
by MSn. In combination with a newly developed
two-dimensional peptide fractionation method, we have successfully
performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated
its capability to complement lysine-reactive reagents and expand PPI
coverage at the systems-level
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Exploring an Alternative Cysteine-Reactive Chemistry to Enable Proteome-Wide PPI Analysis by Cross-Linking Mass Spectrometry
The
development of MS-cleavable cross-linking mass spectrometry
(XL-MS) has enabled the effective capture and identification of endogenous
protein–protein interactions (PPIs) and their residue contacts
at the global scale without cell engineering. So far, only lysine-reactive
cross-linkers have been successfully applied for proteome-wide PPI
profiling. However, lysine cross-linkers alone cannot uncover the
complete PPI map in cells. Previously, we have developed a maleimide-based
cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide
(BMSO)) that is effective for mapping PPIs of protein complexes to
yield interaction contacts complementary to lysine-reactive reagents.
While successful, the hydrolysis and limited selectivity of maleimides
at physiological pH make their applications in proteome-wide XL-MS
challenging. To enable global PPI mapping, we have explored an alternative
cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing
MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide
(DBrASO). Our results have demonstrated that DBrASO cross-linked peptides
display the same fragmentation characteristics as other sulfoxide-containing
MS-cleavable cross-linkers, permitting their unambiguous identification
by MSn. In combination with a newly developed
two-dimensional peptide fractionation method, we have successfully
performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated
its capability to complement lysine-reactive reagents and expand PPI
coverage at the systems-level
Exploring an Alternative Cysteine-Reactive Chemistry to Enable Proteome-Wide PPI Analysis by Cross-Linking Mass Spectrometry
The
development of MS-cleavable cross-linking mass spectrometry
(XL-MS) has enabled the effective capture and identification of endogenous
protein–protein interactions (PPIs) and their residue contacts
at the global scale without cell engineering. So far, only lysine-reactive
cross-linkers have been successfully applied for proteome-wide PPI
profiling. However, lysine cross-linkers alone cannot uncover the
complete PPI map in cells. Previously, we have developed a maleimide-based
cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide
(BMSO)) that is effective for mapping PPIs of protein complexes to
yield interaction contacts complementary to lysine-reactive reagents.
While successful, the hydrolysis and limited selectivity of maleimides
at physiological pH make their applications in proteome-wide XL-MS
challenging. To enable global PPI mapping, we have explored an alternative
cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing
MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide
(DBrASO). Our results have demonstrated that DBrASO cross-linked peptides
display the same fragmentation characteristics as other sulfoxide-containing
MS-cleavable cross-linkers, permitting their unambiguous identification
by MSn. In combination with a newly developed
two-dimensional peptide fractionation method, we have successfully
performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated
its capability to complement lysine-reactive reagents and expand PPI
coverage at the systems-level