Sequential proteolysis and high-field FTICR MS to determine disulfide connectivity and 4-maleimide TEMPO spin-label location in L126C GM2 activator protein

Jeremiah D. Tipton, Jeffrey D. Carter, Jordan D. Mathias, Mark Emmett, Gail E. Fanucci, Alan G. Marshall

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The GM2 activator protein (GM2AP) is an 18 kDa nonenzymatic accessory protein involved in the degradation of neuronal gangliosides. Genetic mutations of GM2AP can disrupt ganglioside catabolism and lead to deadly lysosomal storage disorders. Crystallography of wild-type GM2AP reveals 4 disulfide bonds and multiple conformations of a flexible loop region that is thought to be involved in lipid binding. To extend the crystallography results, a cysteine construct (L126C) was expressed and modified with 4-maleimide TEMPO for electron paramagnetic resonance (EPR) studies. However, because a ninth cysteine has been added by site-directed mutagenesis and the protein was expressed in E. coli in the form of inclusion bodies, the protein could misfold during expression. To verify correct protein folding and labeling, a sequential multiple-protease digestion, nano-liquid chromatograph (LC) electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry assay was developed. High-magnetic field and robust automatic gain control results in subppm mass accuracy for location of the spin-labeled cysteine and verification of proper connectivity of the four disulfide bonds. The sequential multiple protease digestion strategy and ultra-high mass accuracy provided by FTICR MS allow for rapid and unequivocal assignment of relevant peptides and provide a simple pipeline for analyzing other GM2AP constructs.

Original languageEnglish (US)
Pages (from-to)7611-7617
Number of pages7
JournalAnalytical Chemistry
Volume81
Issue number18
DOIs
StatePublished - Sep 15 2009
Externally publishedYes

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G(M2) Activator Protein
Proteolysis
Spin Labels
Disulfides
Cysteine
Crystallography
Gangliosides
Peptide Hydrolases
Protein folding
Cyclotron resonance
Electrospray ionization
Mutagenesis
Proteins
Gain control
Accessories
Labeling
Escherichia coli
Mass spectrometry
Paramagnetic resonance
Conformations

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Sequential proteolysis and high-field FTICR MS to determine disulfide connectivity and 4-maleimide TEMPO spin-label location in L126C GM2 activator protein. / Tipton, Jeremiah D.; Carter, Jeffrey D.; Mathias, Jordan D.; Emmett, Mark; Fanucci, Gail E.; Marshall, Alan G.

In: Analytical Chemistry, Vol. 81, No. 18, 15.09.2009, p. 7611-7617.

Research output: Contribution to journalArticle

Tipton, Jeremiah D. ; Carter, Jeffrey D. ; Mathias, Jordan D. ; Emmett, Mark ; Fanucci, Gail E. ; Marshall, Alan G. / Sequential proteolysis and high-field FTICR MS to determine disulfide connectivity and 4-maleimide TEMPO spin-label location in L126C GM2 activator protein. In: Analytical Chemistry. 2009 ; Vol. 81, No. 18. pp. 7611-7617.
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