Protein Analysis and Purification

Why Wait? Sample Prep/Protein Digestion in as Little as 30 Minutes!

Posted on by Gary Kobs

While many proteases are used in bottom-up mass spectrometric (MS) analysis, trypsin (4,5) is the de facto protease of choice for most applications. There are several reasons for this: Trypsin is highly efficient, active, and specific. Tryptic peptides produced after proteolysis are ideally suited, in terms of both size (350–1,600 Daltons) and charge (+2 to +4), for MS analysis. One significant drawback to trypsin digestion is the long sample preparation times, which typically range from 4 hours to overnight for most protocols. Achieving efficient digestion usually requires that protein substrates first be unfolded either with surfactants or denaturants such as urea or guanidine. These chemical additives can have negative effects, including protein modification, inhibition of trypsin or incompatibility with downstream LC-MS/MS. Accordingly, additional steps are typically required to remove these compounds prior to analysis.

Continue reading “Why Wait? Sample Prep/Protein Digestion in as Little as 30 Minutes!”

Making BRET the Bright Choice for In vivo Imaging: Use of NanoLuc® Luciferase with Fluorescent Protein Acceptors

Posted on by Kyle Hooper
13305818-cr-da-nanoluc-application_ligund

Live animal in vivo imaging is a common and useful tool for research, but current tools could be better. Two recent papers discuss adaptations of BRET technology combining the brightness of fluorescence with the low background of a bioluminescence reaction to create enhanced in vivo imaging capabilities.

The key is to image photons at wavelengths above 600nm, as lower wavelengths are absorbed by heme-containing proteins (Chu, J., et al., 2016 ). Fluorescent protein use in vivo is limited because the proteins must be excited by an external light source, which generates autofluorescence and has limited penetration due to absorption by tissues. Bioluminescence imaging continues to be a solution, especially firefly luciferase (612nm emission at 37°C), but its use typically requires long image acquisition times. Other luciferases, like NanoLuc, Renilla, and Gaussia, etc. either do not produce enough light or the wavelengths are readily absorbed by tissues, limiting their use to near-surface imaging.

The two papers discussed here illustrate how researchers have combined NanoLuc® luciferase with a fluorescent protein to harness bioluminescent resonance energy transfer (BRET) for brighter in vivo imaging reporters.

Continue reading “Making BRET the Bright Choice for In vivo Imaging: Use of NanoLuc® Luciferase with Fluorescent Protein Acceptors”

Widening the Proteolysis Bottleneck: A New Protein Sample Preparation Tool

Posted on by Kari Kenefick
The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.
The poster featured in this blog provides background information and data on the development of Rapid Digestion-Trypsin.

Improvements in Protein Bioprocessing

As more and more protein-based therapeutics enter research pipelines, more efficient protocols are needed. In particular, we need better protocols for the characterization of protein structure and function, as well as means of quantitation. One main step in this pipeline, proteolysis of these proteins into peptides, presents a bottleneck and can require optimization of multiple steps including reduction, alkylation and digestion time.

We have developed a new trypsin reagent, Rapid Digestion–Trypsin, that streamlines the protein sample preparation process. This new development significantly reduces the time to achieve proteolysis to about 1 hour, a remarkable improvement over existing overnight sample preparation times.

How Does it Work?

With this new trypsin product, proteolysis is performed at 70°C, incorporating both denaturation and rapid digestion. The protocol can be used with multiple protein types, including pure proteins and complex mixtures, and is compatible with digestion under native, reduced or nonreduced conditions.

Continue reading “Widening the Proteolysis Bottleneck: A New Protein Sample Preparation Tool”

Probing RGS:Gα Protein Interactions with NanoBiT Assays

Posted on by Kyle Hooper
gpcr_in_membrane_on_white2

When I was a post-doc at UT Southwestern, I was fortunate to interact with two Nobel prize winners, Johann Deisenhofer and Fred Gilman.  Johann once helped me move a -80°C freezer into his lab when we lost power in my building. I once replaced my boss at small faculty mixer with a guest speaker and had a drink with Fred Gilman and several other faculty members from around the university. Among the faculty, one professor had a cell phone on his belt, an odd sight in 1995. Fred Gilman asked him what it was and why he had it. It was so his lab could notify him of good results anytime of the day. Fred laughed and told him to get rid of it – if it’s good data, it will survive until morning.

I was reminded of this story when I read a recent paper by Bodle, C.R. et al (1) about the development of a NanoBiT® Complementation Assay (2) to measure interactions of Regulators of G Protein Signaling (RGS) with Gα proteins in cells. (Fred Gilman was the first to isolate a G protein and that led to him being a co-recipient of the Nobel Prize in 1994). The authors created over a dozen NanoBiT Gα:RGS domain pairs and found they could classify different RGS proteins by the speed of the interaction in a cellular context. The interactions were readily reversible with known inhibitors and were suitable for high-throughput screening due to Z’ factors exceeding 0.5. The study paves the way for future work to identify broad spectrum RGS domain:Gα inhibitors and even RGS domain-specific inhibitors. This is the second paper applying NanoBiT Technology to GPCR studies (3).

A Little Background…
A primary function of GPCRs is transmission of extracellular signals across the plasma membrane via coupling with intracellular heterotrimeric G proteins. Upon receptor stimulation, the Gα subunit dissociates from the βγ subunit, initiating the cascade of downstream second messenger pathways that alter transcription (4). The Gα subunits are actually slow GTPases that propagate signals when GTP is bound but shutdown and reassociate with the βγ subunit when GTP is cleaved to GDP. This activation process is known as the GTPase cycle. G proteins are extremely slow GTPases.

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Cell Free Expression Application: In vitro degradation assay

Posted on by Gary Kobs

A protein chain being produced from a ribosome.
A protein chain being produced from a ribosome.

Researchers and clinicians are fairly certain that all cervical cancers are caused by Human Papillomavirus (HPV) infections, and that HPV16 and HPV18 are responsible for about 70% of all cases. HPV16 and HPV18 have also been shown to cause almost half the vaginal, vulvar, and penile cancers, while about 85% of anal cancers are also caused by HPV16.

E6 is a potent oncogene of HR-HPVs, and its role in progression to malignancy continues to be explored. The E6 oncoprotein of HPV can promote viral DNA replication through several pathways. It forms a complex with human E3-ubiquitin ligase E6-associated protein (E6AP), which can in turn target the p53 tumor-suppressor protein, leading to its ubiquitin-mediated degradation. In particular, E6 from HR-HPVs can block apoptosis, activate telomerase, disrupt cell adhesion, polarity and epithelial differentiation, alter transcription and G-protein signaling, and reduce immune recognition of HPV-infected cells.

In a recent publication a new procedure generated a stable, unmutated HPV16 E6 protein (1). Continue reading “Cell Free Expression Application: In vitro degradation assay”

Improved Method for the Rapid Analysis of Monoclonal Antibodies Using IdeS

Posted on by Gary Kobs
ides_ab

Therapeutic monoclonal antibodies (MAbs) are inherently heterogeneous due to a wide range of both enzymatic and chemical modifications, such as oxidation, deamidation and glycosylation which may occur during expression, purification or storage. For identification and functional evaluation of these modifications, stability studies
are typically performed by employing stress conditions such as exposure to chemical oxidizers, elevated pH and temperature.

To characterize MAbs, a variety of analytical techniques are chosen, such as size exclusion chromatography and ion exchange chromatography. However, due to the large size of the intact MAbs, these methods lack structural resolution. Often, the chromatographic peaks resolved by SEC and IEC methods are collected and further analyzed by peptide mapping to obtain more detailed information. Peptide mapping, in which antibodies are cleaved into small peptides through protease digestion followed by LC–MS/MS analysis, is generally the method of choice for detection and quantitation of site-specific modifications. However sample preparation and lengthy chromatographic separation make peptide mapping impractical for the analysis of large numbers of samples. In contrast to peptide mapping analysis, the middle-down approach offers the advantage of high-throughput and specificity for antibody characterization.

Limited proteolysis of IgG molecules by the IdeS enzyme has been introduced for antibody characterization due to its high cleavage specificity and simple digestion procedure.

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Protein:DNA Interactions—High-Throughput Analysis

Posted on by Gary Kobs
Copyright: molekuul / 123RF Stock Photo
Copyright: molekuul / 123RF Stock Photo

Protein-DNA interactions are fundamental processes in gene regulation in a living cells. These interactions affect a wide variety of cellular processes including DNA replication, repair, and recombination. In vivo methods such as chromatin immunoprecipitation (1) and in vitro electrophoretic mobility shift assays (2) have been used for several years in the characterization of protein-DNA interactions. However, these methods lack the throughput required for answering genome-wide questions and do not measure absolute binding affinities. To address these issues a recent publication (3) presented a high-throughput micro fluidic platform for Quantitative Protein Interaction with DNA (QPID). QPID is an microfluidic-based assay that cam perform up to 4096 parallel measurements on a single device.

The basic elements of each experiment includes oligonucleotides that were synthesized and hybridized to a Cy5-labeled primer and extended using Klenow. All transcription factors that were evaluated contained a 3’HIS and 5’ cMyc tag and were expressed in rabbit reticulocyte coupled transcription and translation reaction (TNT® Coupled Reticulocyte Lysate). Expressed proteins are loaded onto to the QIPD device and immobilized. In the DNA binding assay the fluorescent DNA oligonucleotides are incubated with the immobilized transcription factors and fluorescent images taken. To validate this concept the binding of four different transcription factor complexes to 32 oligonucleotides at 32 different concentrations was characterized in a single experiment. In a second application, the binding of ATF1 and ATF3 to 128 different DNA sequences at different concentrations were analyzed on a single device.

Literature Cited

  1. Ren, B. et al. (2007) Genome-wide mapping of in vivo protein-DNA binding proteins. Science 316, 1497–502.
  2. Garner, M.M. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions. Nuc. Acids. Res. 9, 3047-60.
  3. Glick,Y et al. (2016) Integrated microfluidic approach for quantitative high throughput measurements of transcription factor binding affinities. Nuc. Acid Res. 44, e51.

Moving Out of the Cell: Advantages of Cell-Free Protein Expression

Posted on by Kelly Grooms

Cell-free protein expression is a simplified and accelerated avenue for the transcription and/or translation of a specific protein in a quasi cell environment. An alternative to slower, more cumbersome cell-based methods, cell-free protein expression methods are simple and fast and can overcome toxicity and solubility issues sometimes experienced in the traditional E. coli expression systems.

Cell-free protein expression offers significant time savings over cell-based expression methods.
Cell-free protein expression offers significant time savings over cell-based expression methods.

Cell-free protein expression offers a convenient method for generating small amounts of protein for a variety of applications (e.g., protein:protein interactions, protein: nucleic acid interactions, structural analysis, functional assays and toxicity screening). This approach lends itself to specific protein labeling with fluorescence, biotin, radioactivity or heavy atoms, via modified charged tRNA’s or amino acids. Cell-free protein expression systems provide quick access to proteins of interest and remain a staple in the collection of tools available for the elucidation of protein structure and function, understanding cellular pathways and mechanisms and high-throughput screening of compounds for drug discovery. There are a number of different cell-free expressions systems, each with different strengths. Deciding which one is right for you depends upon your research needs and goals.

Continue reading “Moving Out of the Cell: Advantages of Cell-Free Protein Expression”

Optimization of Alternative Proteases for Bottom-Up Proteomics

Posted on by Gary Kobs
Alternate Proteases Cover

Bottom-up proteomics focuses on the analysis of protein mixtures after enzymatic digestion of the proteins into peptides. The resulting complex mixture of peptides is analyzed by reverse-phase liquid chromatography (RP-LC) coupled to tandem mass spectrometry (MS/MS). Identification of peptides and subsequently proteins is completed by matching peptide fragment ion spectra to theoretical spectra generated from protein databases.

Trypsin has become the gold standard for protein digestion to peptides for shotgun proteomics. Trypsin is a serine protease. It cleaves proteins into peptides with an average size of 700-1500 daltons, which is in the ideal range for MS (1). It is highly specific, cutting at the carboxyl side of arginine and lysine residues. The C-terminal arginine and lysine peptides are charged, making them detectable by MS. Trypsin is highly active and tolerant of many additives.

Even with these technical features, the use of trypsin in bottom-up proteomics may impose certain limits in the ability to grasp the full proteome, Tightly-folded proteins can resist trypsin digestion. Post-translational modifications (PTMs) present a different challenge for trypsin because glycans often limit trypsin access to cleavage sites, and acetylation makes lysine and arginine residues resistant to trypsin digestion.

Are you looking for proteases to use in your research?
Explore our portfolio of proteases today.

To overcome these problems, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated when using these alternative proteases have not been systematically documented.

In a recent reference (2), optimized protocols for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, Lys-C, Lys-N, Asp-N, Glu-C and Arg-C have been created.

Data describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in approximately in 2 days.

Try a sample of high-efficiency Trypsin Platinum today!

Visit our website for more on Trypsin Platinum, Mass Spectrometry Grade, with enhanced proteolytic efficiency and superior autoproteolytic resistance.

Reference

  1. Laskay, U. et al. (2013) Proteome Digestion Specificity Analysis for the Rational Design of Extended Bottom-up and middle-down proteomics experiments. J of Proteome Res. 12, 5558–69.
  2. Giansanti, P. et. al. (2016) Six alternative protease for mass spectrometry based proteomics beyond trypsin. Nat. Protocols 11, 993–6

Related Posts

Optimizing Antibody Enrichment for Pharmacokinetic Assays

Posted on by Gary Kobs
Schematic showing immuno-enrichment using High Capacity Magne® Streptavidin Beads.
Schematic showing immuno-enrichment using High Capacity Magne® Streptavidin Beads.

During preclinical research and development of therapeutic antibodies, multiple variants of each antibody are assessed for pharmacokinetic (PK) characteristics across model systems such as rodents, beagles and primates. Ligand-binding assays (LBA) or liquid chromatography coupled to tandem mass spectrometry(LC–MS/MS)-based methods represent the two most common technologies used to perform the PK studies for mAb candidates(1,2).

Using either method it is essential to ensure accurate quantitative results that the initial enrichment of the target therapeutic antibody from serum or plasma be optimal. Biotinylated antibodies or antigens (against the therapeutic targets) immobilized onto high capacity streptavidin beads will enrich therapeutic antibody from serum or plasma samples. The affinity of biotin for streptavidin (Kd = 10–15) is one of the strongest and most stable interactions in biology therefore the biotin-streptavidin interaction cannot be reversed under non-denaturing conditions. Hence, it is possible to perform extensive washing to remove nonspecifically bound protein and elute therapeutic antibodies without also eluting the biotinylated component, thus improving the detection limit.

Magnetic based separation techniques have several advantages in comparison with standard separation procedures. This process is usually very simple, with only a few handling steps. All the steps of the purification procedure can take place in one single test tube. The magnetic separation techniques are also the basis of various automated procedures. Learn more about  the High Capacity Magne™ Streptavidin Beads (Cat # V7820) .

References