Methylene blue active substances in plaque of Bacillus subtilis subsp. subtilis and enrichment by supplemental calcium in culture media

A series of experiments was conducted to identify the molecular species responsible for surface active emulsification (surfactant) bioactivity in Bacillus subtilis subsp. subtilis strain ATCC PTA-125135, and to describe culture conditions to support the enriched production of said bioactivity in cultured plaque of the strain. The assay for methylene blue active substances (MBAS) was found to be suitable for describing surfactant activity, where a solvent-extracted molecular fraction from the biofilm was found to retain surfactant activity and positively quantified as MBAS. Furthermore, an HPLC-refined protein fraction was found to quantify as MBAS with approximately 1·36-fold or greater surfactant activity per mol than sodium dodecyl sulphate, and a proteomic analysis of solvent extracted residues confirmed that biofilm surface layer protein BslA was a primary constituent of extracted residues. Surfactant bioactivity, quantified as MBAS, was enriched in cultured plaque by the supplementation of culture media with calcium chloride or calcium nitrate.     

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

Methodology for sequence analysis of ∼150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8 m urea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3–9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous post-translational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms of N-linked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion–ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides.Monoclonal antibodies (mAbs)¹ and related biological molecules constitute one of the most rapidly growing classes of human therapeutics. These large proteins (Fig. 1) have molecular weights near 150 kDa and are composed of two identical ∼50 kDa heavy chains (HC) and two identical ∼25 kDa light chains (LC) (1). They also contain at least 16 disulfide bonds that maintain three-dimensional structure and biological activity (2). Although sharing similar secondary protein structures, different mAbs differ greatly in the sequence of variable regions, especially in the complementarity determining regions (CDRs) which are responsible for the diversity and specificity of antibody-antigen binding. Changes to the mAb structure introduced during the manufacturing process or storage may influence the therapeutic efficacy, bio-availability and -clearance, and immunogenic properties and thus alter drug safety (3–5). Comprehensive characterization of mAbs primary structure, post-translational modifications (PTMs), and disulfide linkages is critical to the evaluation of drug efficacy and safety, as well as understanding the structure/function relationships (4, 6). Presented in this work is a novel protein analytical platform that consists of innovative methods for mass spectrometry (MS) characterization of mAbs. The methodology reported here will have a dramatic impact on the whole field of antibody characterization.Open in a separate windowFig. 1.Diagram of a murine monoclonal antibody structure.Typical MS characterization of proteins uses a “Bottom-Up” approach. This method involves tryptic digestion of the protein(s) into small peptides (mostly below 2500 Da) followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analyses of the resulting peptides (7). Although sensitive for MS analysis, small tryptic peptides often have issues such as weak retention in liquid chromatography, difficulties in assigning peptides to specific gene products, and loss of combinatorial PTM information (8). Recent years have seen developments in direct MS analysis of intact proteins (often called “Top-Down” MS). Despite increasing success in characterization of small to medium-sized proteins, MS analysis of intact proteins larger than 50 kDa, including mAbs, is still unsatisfactory because of inefficient gas-phase protein fragmentation and complex fragment ions that restrict efficient data interpretation (9, 10).A “compromise” between Bottom-Up and Top-Down approaches is the “Middle-Down” (or “Middle-Up”) method. Middle-Down analysis typically involves proteolysis using proteases (e.g. Lys-C) or chemicals that hydrolyze proteins at a single type of amino acid residue. This approach aims to generate 3–15 kDa peptides which are compatible with high resolution MS/MS analysis on a chromatographic time scale. The Middle-Down approach inherits some of the advantages of Top-Down analysis, yet has less demanding instrumental requirements compared with intact protein MS in achieving sufficient signal-to-noise ratio (S/N) of fragment ions for sequence mapping (11–15).However, limitations of currently available tools for Middle-Down protein analysis are also obvious. First, none of the twenty amino acids is evenly distributed along a polypeptide. Protein digestion at single-type amino acid residues can still produce very small (<1000 Da) or ultra large (>15 kDa) peptides, which deviates from the original intention of the Middle-Down approach (16). Second, the enzymatic digestion efficiency is often low for proteins with highly folded structure or low solubility. Although high concentrations of chaotropic agents such as 8 m urea are often used for protein denaturation, this harsh condition quickly deactivates many commonly used proteases. Third, traditional data-dependent ETD or electron-capture dissociation MS/MS analyses adopt a single reaction parameter for gas-phase dissociation and select only several abundant ions regardless of their charge states. As these methods were previously optimized for tryptic peptide ions that typically carry +2 or +3 charges, they are incompatible with the analysis of large, highly charged peptides that require optimized ETD to achieve high sequence coverage and PTM mapping (12).Herein we report a “time-controlled” proteolysis method for tailored Middle-Down MS analysis of mAb. To hydrolyze the 150 kDa mAb into large peptides for HPLC-MS analysis, we fabricated a capillary enzyme reactor column that contains a specified length of immobilized protease (supplemental Fig. S1 and S2A). Precise control of the sample flow rate leads to defined digestion time of the substrate protein in the reactor. A short digestion time results in a small number of “cuts” along the protein chain and consequently the formation of large peptides (supplemental Fig. S2B). 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. In our present work, we designed a more robust enzyme reactor that digests alkylated or native mAb into 3–12 kDa peptides in a buffer containing 8 m urea (a condition incompatible with most widely used proteases), and characterized their amino acid sequences, PTMs, as well as the disulfide linkages using HPLC-MS/MS.We chose a rarely used protease, aspergillopepsin I, for the enzyme reactor. Aspergillopepsin I, also known as Aspergillus saitoi acid proteinase, generally catalyzes the hydrolysis of substrate proteins at P1 and P1′ of hydrophobic residues, but also accepts Lys at P1 (18). There are several innovative aspects of employing this enzyme: (1) Aspergillopepsin I is active in 8 m urea at pH 3–4 for at least 1 h. This extreme chaotropic condition may disrupt the higher-order structure of proteins to a great extent and allows for easy access of the protease to most regions of the substrate protein once the disulfide bonds are reduced. (2) Compared with proteases with dual- or single-type amino acid specificity, aspergillopepsin I provides more cleavage sites along an unfolded substrate protein. Allowing limited time for the substrate protein to interact with immobilized aspergillopepsin I should generate large peptides with a relatively narrow size distribution because of similar numbers of missed cleavages on these peptides. (3) The enzyme reactor automatically “quenches” proteolysis as the sample flows out of the column. This is in great contrast to in-tube digestion using solubilized proteases that are active in acidic conditions. In the latter case, digestion is difficult to quench or control because of the sustained enzymatic activity in an acidic condition. (4) Compared with electrostatic or hydrophobic interactions for enzyme immobilization, covalent conjugation of the protease onto porous beads should prevent the replacement of enzymes by upcoming substrate proteins. (5) The enzyme beads can be stored at 4 °C for at least half a year once water is removed, allowing the production of hundreds of disposable enzyme reactors from one batch of beads. In addition, we introduced a new cysteine (Cys) alkylation reagent, N-(2-aminoethyl)maleimide (NAEM) for protein MS analysis. This reagent improves ETD (19) of peptides containing Cys residues by adding a basic, readily protonated side chain to thiol groups.The above features of our new strategy led to the generation of large, highly charged peptides that cover the entire murine mAb. Analyzing ETD and collisionally activated dissociation (CAD) fragments from the most abundant large peptides by ProSightPC revealed near complete sequence coverage of the mAb and multiple PTMs. Furthermore, we digested the native mAb into large fragments of disulfide-bonded peptides using time-controlled digestion. The ETD/ion-ion proton transfer (IIPT) technique (20) allowed facile identification of the N- and C-terminal sequences of two disulfide-bonded peptides and localization of the disulfide bond(s) within/connecting different mAb domains.