3 research outputs found
Limited Proteolysis via Millisecond Digestions in Protease-Modified Membranes
Sequential adsorption of polyÂ(styrene sulfonate) (PSS)
and proteases in porous nylon yields enzymatic membrane reactors for
limited protein digestion. Although a high local enzyme density (∼30
mg/cm<sup>3</sup>) and small pore diameters in the membrane lead to
digestion in <1 s, the low membrane thickness (170 μm) affords
control over residence times at the millisecond level to limit digestion.
Apomyoglobin digestion demonstrates that peptide lengths increase
as the residence time in the membrane decreases. Moreover, electron
transfer dissociation (ETD) tandem mass spectrometry (MS/MS) on a
large myoglobin proteolytic peptide (8 kDa) provides a resolution
of 1–2 amino acids. Under denaturing conditions, limited membrane
digestion of bovine serum albumin (BSA) and subsequent ESI-Orbitrap
MS analysis reveal large peptides (3–10 kDa) that increase
the sequence coverage from 53% (2 s digestion) to 82% (0.05 s digestion).
With this approach, we also performed membrane-based limited proteolysis
of a large <i>Arabidopsis</i> GTPase, Root Hair Defective
3 (RHD3) and showed suitable probing for labile regions near the C-terminus
to suggest what protein reconstruction might make RHD3 more suitable
for crystallization
Galdieria_in_Richmond_Mine_FISH
Micrographs of two G. sulphuraria cells detected using Cya1208 rRNA probe in an A-drift biofilm from the Richmond Min
208_Genomes
List of 208 sequenced genomes used to generate protein families and phylogenetic tree