Asp-N is a endoproteinase hydrolyzes peptide bonds on the N-terminal side of aspartic residues. The native form is isolated from Pseudomonas fragi. The majority of vendors currently provide a commercial product that consists of 2µg of lyophilized material in a flat bottom vial, and sold for $175–200 US. Formatting such a small amount of material in flat bottom vial can lead to inconsistent resuspension of the protease. Inconsistent working concentrations will lead to non-reproducible data. The current high price also prohibits large-scale use.
The new recombinant Asp-N protease is cloned from Stenotrophomonas maltophilia and expressed in E. coli. Recombinant Asp-N has similar amino acid cleavage specificity as compared to native Asp-N. Digestion of a yeast extract with native and recombinant Asp-N produces very similar results. Providing 10µg lyophilized material in V-shaped vial with a visible cake enables more consistent re-suspension resulting in reproducible data. Due to improved yields the list price is now approximately 40% less when compared to native enzyme.
Learn more about this new recombinant Asp-N protease.
Cellular stress is associated with global misfolding and aggregation of the endogenous proteome. Monitoring stress-induced abnormalities remains one of the major technical challenges facing established sensors. Misfolded monomers induced by mild stresses, however, remain largely invisible with current sensors.
In a recent publication (1) Fares and colleagues describe a new sensor based upon a fluorescent molecular rotor that is conjugated to a Halo mutant (AgHalo). In non-stressed cells, the AgHalo sensor remains largely folded, and is fluorescent when misfolded. The fluorescent molecular rotor, when conjugated to purified AgHalo to form the proteome stress sensor, is able to report on urea-induced partially unfolded (misfolded) conformations with a higher fluorescent increase than the previously reported fluorophore-based sensors. Heat-induced misfolding is also effectively monitored by the fluorescence change of the sensor that is based on fluorescent molecular rotor, but not the solvatochromic fluorophore. The unique feature of the fluorescent molecular rotor makes the new generation of the AgHalo proteome sensor more sensitive to misfolded conformations that are primarily induced by mild proteome stress. Further, the new sensor exhibits a higher fluorescence signal when detecting soluble and insoluble protein aggregates that are induced by more severe proteome stress. These data collectively suggest that thermo-labile Halo conjugated with a fluorescent molecular rotor serves as a suitable sensor to detect a wide range of proteome stress conditions.
Fares, M. et al. (2018) A Molecular Rotor-Based Halo-Tag Ligand Enables a Fluorogenic Proteome Stress Sensor to Detect Protein Misfolding in Mildly Stressed Proteome. Bioconjugate Chem 29, 215–24.
The art of brewing alcoholic beverages has existed for thousands of years. The process of beer brewing begins with barley grains, which are malted to allow partial germination, triggering expression of key enzymes. The germinated grains are then dried and milled. Next, starch, proteins, and other molecules are solubilized during mashing. During mashing, solubilized enzymes degrade starch to fermentable sugars, and digest proteins to produce peptides and free amino acids. Fermentable sugars and free amino acids are required for efficient yeast growth during fermentation.
After the mash, the wort is removed, and hops are added for bitterness and aroma, and the wort is boiled. After boiling, the wort is inoculated with yeast, and fermentation proceeds to produce bright beer. Typically this bright beer is then filtered, carbonated, packaged, and sold. Many proteins originating from the barley grain and the yeast are present in beer, and these have been reported to affect the quality of the final product. However, some of the biochemical details of this process remain unclear. To better understand what happens during the various steps of the brewing process, Schultz et al. used mass spectrometry proteomics to perform a global untargeted analysis of the proteins present across time during beer production and described this work in a recent paper (1). Samples analyzed included sweet wort produced by a high temperature infusion mash, hopped wort, and bright beer. Continue reading
Multi-subunit protein complexes control membrane fusion events in eukaryotic cells (1). CORVET and HOPS are two such multi-subunit complexes, both containing the Sec1/Munc18 protein subunit VPS33A (2). Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. Recent research suggests that VPS33B and VIPAR comprise two subunits of a novel multi-subunit complex analogous in configuration to CORVET and HOPS (3).
In a recent publication (4), Hunter and colleagues, further characterized the VPS33B and VIPAR complex. Using co-immunoprecipitation and proximity-based ligation assay, they identified two novel VPS33B-interacting proteins, VPS53 and CCDC22.
In vitro binding experiments, VPS33B and GST-VIPAR were co-expressed in Escherichia coli and purified by GSH affinity. The VPS33B/GSTVIPAR complex was used as bait in pulldown experiments, with myc-CCDC22 and myc-VPS53 expressed by cell-free in vitro transcription/translation in wheat germ lysate. Myc-CCDC22 was very efficiently pulled down by VPS33B/GST-VIPAR, whereas myc-VPS53 was not .The interaction between VPS53 and the VPS33B-VIPAR complex was either indirect, requires other proteins contribute to the interaction, or requires a post-translational modification not conferred in the plant cell-free expression system (wheat germ). Pull-down experiments with individual subunits or expressing as complexes, was inefficient and did not result in binding to VPS33B/GST-VIPAR.
To further understand how VPS33B-VIPAR may interact with CCDC22, Hunter and colleagues attempted to refine the region of CCDC22 that interacts with VPS33B/GST-VIPAR by generating a series of truncated forms of CCDC22. However, none of five CCDC22 truncations were able to bind to VPS33B/GST-VIPAR. The hypothesis was that truncated forms of CCDC22 are unstable and unable to fold correctly in this assay system.
Additional experiments noted that the protein complex in HEK293T cells which contained VPS33B and VIPAR was considerably smaller than CORVET/HOPS, suggesting that, unlike VPS33A, VPS33B does not assemble into a large stable multi-subunit protein complex.
- D’Agostino, M. et. al. (2017) A tethering complex drives the terminal stage of SNARE-dependent membrane fusion. Nature 551, 634–638.
- Balderhaar, H. J. K. and Ungermann, C. (2013) CORVET and HOPS tethering complexes – coordinators of endosome and lysosome fusion. J. Cell Sci. 126, 1307–16.
- Spang, A. (2016) Membrane Tethering Complexes in the Endosomal System. Front. Cell Dev. Biol. 4, 35.
- Hunter, M. et. al. (2017) Proteomic and biochemical comparison of the cellular interaction partners of human VPS33A and VPS33B. [Internet bioRxiv http://dx.doi.org/10.1101/236695 Accessed 3/12/2018]
DNA is organized by protein:DNA complexes called nucleosomes in eukaryotes. Nucleosomes are composed of 147 base pairs of DNA wrapped around a histone octamer containing two copies of each core histone protein. Histone proteins play significant roles in many nuclear processes including transcription, DNA damage repair and heterochromatin formation. Histone proteins are extensively and dynamically post-translationally modified, and these post-translational modifications (PTMs) are thought to comprise a specific combinatorial PTM profile of a histone that dictates its specific function. Abnormal regulations of PTM may lead to developmental disorders and disease development such as cancer.
Antibodies have been widely used to characterize histones and histone PTMs. However, antibody-based techniques have several limitations. Mass spectrometry (MS) has therefore emerged as the most suitable analytical tool to quantify proteomes and protein PTMs. The most commonly used strategy is still bottom-up MS, and the most widely adopted protocol includes derivatization of lysine residues in histones to allow trypsin to generate Arg-C like peptides (4–20 aa). However, samples such as primary tissues, complex model systems, and biofluids are hard to retrieve in large quantities. Because of this, it is critical to know whether the amount of sample available would lead to an exhaustive analysis if subjected to MS.
In a recent publication, Guo, et al. examined (1) the reproducibility in quantification of histone PTMs using a wide range of starting material: from 50,000 to 5,000,000 cells. They used four different cell lines: HeLa, 293T, human embryonic stem cells (hESCs), and myoblasts. Their results demonstrated that an accurate quantification of abundant histone PTMs can be efficiently obtained by using low-resolution MS and as low as 50,000 cells as starting material Low abundance histone marks showed more variability in quantification when comparing different amounts of starting material, so a larger amount of starting material (at least 500,000 cells) is recommended.
Guo, Q. et al. (2017) Assessment of Quantification Precision of Histone Post-Translational Modifications by Using an Ion Trap and down To 50,000 Cells as Starting Material. J. Proteome Res. 17, 234–42.
Recombinant erythropoietin (rhEPO) is often used as “doping agent” by athletes in endurance sports to increase blood oxygen capacity. Some strategies improve the pharmacological properties of erythropoietin (EPO) through the genetic and chemical modification of the native EPO protein. The EPO-Fcs are fusion proteins composed of monomeric or dimeric recombinant EPO and the dimeric Fc region of human IgG molecules. The Fc region includes the hinge region and the CH2 and CH3 domains. Recombinant human EPOs (rhEPO) fused to the IgG Fc domain demonstrate a prolonged half-life and enhanced erythropoietic activity in vivo compared with native or rhEPO.
Drug-testing agencies will need to obtain primary structure information and develop a reliable analytical method for the determination of EPO-Fc abuse in sport. The possibility of EPO-Fc detection using nanohigh-performance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS) was already demonstrated (1). However, the prototyping peptides derived from EPO and IgG are not selective enough because both free proteins are naturally presented in human serum. In a recent publication, researchers describe the effort to identify peptides covering unknown fusion breakpoints (later referred to as “spacer” peptides; 2). The identification of “spacer” peptides will allow the confirmation of the presence of exogenous EPO-Fc in human biological fluids.
A bottom-up approach and the intact molecular weight measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified.
Results indicated that “spacer peptides” could be used in the determination of EPO-Fc fusion proteins in biological samples using common LC−tandem MS methods.
- Reichel, C. et al. (2012) Detection of EPO-Fc fusion protein in human blood: screening and confirmation protocols for sports drug testing.
Drug Test. Anal. 4, 818−29.
- Mesonzhnik, N. et al. (2017) Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography−Mass Spectrometry.
J. of Proteome Res. 17, 689-97.
Illustration showing NanoLuc and firefly luciferase reporters.
The luciferase immunoprecipitation system (LIPS) assay is a liquid phase immunoassay allowing high-throughput serological screening of antigen-specific antibodies. The immunoassay involves quantitating serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Rluc) fused to an antigen of interest. The Rluc-antigen fusion protein is recognized by antigen-specific antibodies, and antigen-antibody complexes are captured by protein A/G beads that recognize the Fc region of the IgG antibody (1).
In a recent publication (2), this assay was used to assess the presence of autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in patients with atrophic body gastritis and in controls. Continue reading
Monoclonal antibodies (mAbs) have been widely used to eliminate undesired cells via various mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and programmed cell death (PCD). Unlike the Fc-dependent mechanism of ADCC and CDC, certain antibody–antigen interactions can evoke direct PCD via apoptosis or oncosis. Previously, researchers have reported the specific killing of undifferentiated human embryonic stem cells (hESC) by mAb84 (IgM) via oncosis (1)
In a recent publication (2), a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC). One of the many experimental tools used to characterize the mechanism of oncosis was the fragmention of the A1 antibody with IdeS and papain.
Papain digestion of IgG produces Fab fragments in the presence of reducing agent. F(ab)2 fragments of A1 were produced using IdeS Protease.
The results indicate that both Fab_A1 and F(ab)2_A1 bind to hESC but only F(ab)2_A1 retained hESC killing. Hence bivalency, but not Fc-domain, is essential for A1 killing on hESC.
- Choo, A.B. et al. (2008) Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1. Stem Cells 26, 1454.
- Zheng, J.Y. et al. (2017) Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis. Cell Death and Differentiation 24, 546–58.
Further reading about IdeS Protease is available here.
Several pharmaceutical companies have biosimilar versions of therapeutic mAbs in development. Biosimilars can promise significant cost savings for patients, but the unavoidable differences
between the original and thencopycat biologic raise questions regarding product interchangeability. Both innovator mAbs and biosimilars are heterogeneous populations of variants characterized by differences in glycosylation,oxidation, deamidation, glycation, and aggregation state. Their heterogeneity could potentially affect target protein binding through the F´ab domain, receptor binding through the Fc domain, and protein aggregation.
As more biosimilar mAbs gain regulatory approval, having clear framework for a rapid characterization of innovator and biosimilar products to identify clinically relevant differences is important. A recent reference (1) applied a comprehensive mass spectrometry (MS)-based strategy using bottom-up, middle-down, and intact strategies. These data were then integrated with ion mobility mass spectrometry (IM-MS) and collision-induced unfolding (CIU) analyses, as well as data from select biophysical techniques and receptor binding assays to comprehensively evaluate biosimilarity between Remicade and Remsima.
The authors observed that the levels of oxidation, deamidation, and mutation of individual amino acids were remarkably similar. they found different levels of C-terminal truncation, soluble protein aggregates, and glycation that all likely have a limited clinical impact. Importantly, they identified more than 25 glycoforms for each product and observed glycoform population differences.
Overall the use of mass spectrometry-based analysis provides rapid and robust analytical information vital for biosimilar development. They demonstrated the utility of our multiple-attribute monitoring workflow using the model mAbs Remicade and Remsima and have provided a template for analysis of future mAb biosimilars.
1. Pisupati, K. et. al. (2017) A Multidimensional Analytical Comparison of Remicade and the Biosimilar Remsima. Anal. Chem 89, 38–46.
Protein:protein interactions (PPIs) play a key role in regulating cellular activities including DNA replication, transcription,translation, RNA splicing, protein secretion, cell cycle control and signal transduction. A comprehensive method is needed to identify the PPIs before the significance of the protein:protein interactions can be characterized. Affinity purification−mass spectrometry (AP−MS) has become the method of choice for discovering PPIs under native conditions. This method uses affinity purification of proteins under native conditions to preserve PPIs. Using this method, the protein complexes are captured by antibodies specific for the bait proteins or for tags that were introduced on the bait proteins and pulled down onto immobilized protein A/G beads. The complexes are further digested into peptides with trypsin. The protein interactors of the bait proteins are identified by quantification of the tryptic peptides via mass spectrometry.
The success of AP-MS depends on the efficiency of trypsin digestion and the recovery of the tryptic peptides for MS analysis. Several different protocols have been used for trypsin digestion of protein complexes in AP-MS studies, but no systematic studies have been conducted on the impact of trypsin digestion conditions on the identification of PPIs. A recent publication used NFB/RelA and BRD4 as bait proteins and five different trypsin digestion conditions (two using “on beads” and three using “elution” digestion protocols). Although the performance of the trypsin digestion protocols changed slightly depending on the different bait proteins, antibodies and cell lines used, the authors of the paper found that elution digestion methods consistently outperformed on-beads digestion methods.
Zhang, Y. et al. (2017) Quantitative Assessment of the Effects of Trypsin Digestion Methods on Affinity Purification−Mass Spectrometry-based Protein−Protein Interaction Analysis
J of Proteome. Res. 16, 3068–82.