Limited Proteolysis via Millisecond Digestions in Protease-Modified Membranes

Abstract

Sequential adsorption of poly(styrene sulfonate) (PSS) and proteases in porous nylon yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm3) 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 ms 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 kD) 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 kD–10 kD) 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 Arabidopsis 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.

Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis

“…55 These reactors are very active, because of the high local enzyme concentration in membrane pores. 50 The extent of digestion varies with the solution residence time in the membrane ( t res ), which is a function of the membrane thickness ( l ), the volumetric flow rate ( Q ), the exposed area at the faces of the membrane ( A ), and the membrane porosity (ε) (eq 1). $$t_{\text{res}}=\frac{lA\epsilon }{Q}$$ …”

“…Our prior study used a membrane with nominal 0.45 µm pores and a thickness of 170 µm, 50 whereas this work employs both a larger pore size (1.2 µm) and a lower thickness (110 µm) to further limit digestion and provide longer peptides. The lower thickness decreases the residence time for a given flow rate, and the larger pore size should give longer radial diffusion distances to immobilized enzymes.…”

“…outer diameter (OD) tubing via ferrules. 50 Pepsin from porcine gastric mucosa (lyophilized powder, 3200–4500 units/mg protein), iodoacetamide (IAM, ≥ 99%), polystyrene sulfonate (PSS, average molecular weight ~70 000), and acetonitrile (ACN, HPLC grade, ≥ 99.9%) were obtained from Sigma–Aldrich. Isopropyl alcohol (IPA, MACRON), sequencing-grade modified trypsin (Promega), and trifluoroacetic acid (TFA, purchased from EMD) were used as received.…”

A new sample preparation method for the absolute quantitation of a target proteome using 18O labeling combined with multiple reaction monitoring mass spectrometry

“…4 With the development of the proteomic technology, especially the application of ultrahigh-performance liquid chromatography equipped with long chromatographic columns (more than 50 cm in length), packed with sub-2 μm packing materials and capable of ultrahigh resolution, high accuracy and high scan speed mass spectrometry, the digests of protein mixtures can be efficiently separated, identified and quantified in approximately 12 h. [5][6][7] In these cases, the proteolytic steps account for most of the proteomic analysis time, which limits the application of proteomic technologies in real biological samples. 3,8,9 Various materials have been developed as enzyme carriers, including polymer membranes, 10,11 capillary columns, [12][13][14][15][16] microfluidic chips, [17][18][19][20][21] microparticles and nanoparticles, [22][23][24][25][26] porous materials, [27][28][29] graphene oxide [30][31][32][33][34][35][36][37][38][39] and fibers. 3 To address this issue, immobilized enzyme reactors (IMERs) appear to be a better choice for the most efficient and rapid protein digestion at high enzyme to substrate ratios.…”

Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry

“…The Bruening group previously demonstrated a similar concept using a nylon membrane electrostatically adsorbed with pepsin or trypsin. Pushing a protein solution (protein dissolved in 5% formic acid solution) through the membrane-based enzyme reactor in less than 1 s breaks the protein into large peptides that facilitate sequence mapping of horse apomyoglobin (17 kDa) and bovine serum albumin (66 kDa) by infusion electrospray ionization MS/MS (17). The advantages of their enzyme disc include simple preparation procedures, as well as the low back pressure in the thin disc that allows for rapid sample flow rate.…”