Bottom-up Proteomics: Need Help?

The use of mass spectrometry for the characterization of individual or complex protein samples continues to be one of the fastest growing fields in the life science market.

Bottom-up proteomics is the traditional approach to address these questions. Optimization of each the individual steps (e.g. sample prep, digestion and instrument performance) is critical to the overall success of the entire experiment.

To address issues that may arise in your experimental design, Promega has developed unique tools and complementary webinars to help you along the way.

Here you can find a summary of individual webinars for the following topics: Continue reading “Bottom-up Proteomics: Need Help?”

A BiT or BRET, Which is Better?

Now that Promega is expanding its offerings of options for examining live-cell protein interactions or quantitation at endogenous protein expression levels, we in Technical Services are getting the question about which option is better. The answer is, as with many assays… it depends! First let’s talk about what are the NanoBiT and NanoBRET technologies, and then we will provide some similarities and differences to help you choose the assay that best suits your individual needs. Continue reading “A BiT or BRET, Which is Better?”

Optimizing Pressure Cycling Sample Preparation for Bottom-Up Proteomics

Large-scale analyses of the proteome have revealed proteomic changes in response to disease, and these changes hold great promise for diagnostics and treatment of complex disease if proteomic analysis can be brought into the clinical laboratory. Successful and reliable large-scale proteomics requires sample preparation workflows that are reproducible, reliable and show little variability. To bring proteomics into the clinical laboratory, standardized procedures and workflows for sample prep and analysis are required to generate valid, actionable results on a time scale useful for the clinic.

The two most common sample types analyzed for clinical proteomics are body fluids and tissue biopsies. To process these kinds of samples, there are two initial steps: tissue solubilization, followed by proteolytic digestion. Solubilization of solid tissues is the most labor-intensive and produces the most variable results.

The introduction of pressure cycling technology (PCT) using Barocycler instrumentation has greatly improved both tissue solubilization and digestion consistency. The PCT-based sample preparation protocols generally utilize urea as a lysis buffer for protein denaturing and solubilization. Urea has several drawbacks including inhibiting trypsin activity and introducing  unwanted modifications like carbamylation.

Lucas and colleagues analyzed whether replacing urea with SDC would produce similar tissue digestion profiles and improve the PCT method.

SDC allowed the use of higher temperatures compared to urea, and hence the first step (lysis, reduction, and alkylation) was performed at 56 °C. The second digestion step in the Barocycler was optimized, and the third step was eliminated. To further reduce digestion time, they capitalized on Rapid Trypsin/Lys-C.  Rapid Trypsin/Lys-C maintains robust activity at 70 °C, and allowed Barocycler digestion to be performed in a single step, completing digestion in 30 cycles (approximately 30 min) rather than 105 minutes, streamlining the protocol.

The data presented an improved conventional tissue PCT approach in a Barocycler by replacing urea and proteolytic enzymes with SDC, N-propanol, and modified commercially available enzymes that have higher optimum temperatures.

Paper Referenced

Lucas, N. et al. (2019) Accelerated Barocycler Lysis and Extraction Sample Preparation for Clinical Proteomics by Mass Spectrometry. J of Proteome Res 18, 399–405.

Automated Approach for Multiomic Analysis

With the use of a suite of “-omics” technologies you can examine the way in which complex cellular processes work together across all molecular domains (i.e., proteomics, metabolomics, transcriptomics) in a single biological system. Several studies have been published across a wide range of fields illustrating the power of such a unified approach (1,2). Most studies however did not focus on the development of a high-throughput, unified sample preparation approach to complement high-throughput “omic” analytics.

A recent publication by Gutierrez and colleagues presents a simple high-throughput process (SPOT) that has been optimized to provide high-quality specimens for metabolomics, proteomics, and transcriptomics from a common cell culture sample (3). They demonstrate that this approach can process  16−24 samples from a cell pellet to a desalted sample ready for mass spectrometry analysis within 9 hours. They also demonstrated that the combined process did not sacrifice the quality of data when compared to individual sample preparation methods.

Literature Cited

1. Roume, H. (2013) Sequential Isolation of Metabolites, RNA, DNA, and Proteins from the Same Unique Sample. Methods Enzymol. 531, 219−236.
2. Lo, A. W. et al. (2017) ‘Omic’ Approaches to Study Uropathogenic Escherichia Coli Virulence. Trends Microbiol. 25, 729−740.
3. Gutierrez, D. et al. (2018)  An Integrated, High-Throughput Strategy for Multiomic Systems Level Analysis J. Proteome Res.

What’s In YOUR Protein? Optimizing Protease Digestions to Get the Inside Scoop

It’s time to analyze your protein and you are trying to decide where to begin. You are asking questions like: Which protease do I choose? How much enzyme should I use in my digest? How long should I perform my digest?

Unfortunately, there is no one-size fits all answer to this type of question other than… “well it depends.” All protease digests will be a balance between denaturing the protein sample to allow access to cleavage sites, optimizing conditions for the protease to function, and compatibility with your workflow and downstream applications. We provide general guidelines that work for most samples, but frequently you will need to optimize the conditions need for your specific sample and application.

Here, I use the example of a trypsin digest for downstream mass spectrometry to highlight key questions to ask and factors that can be optimized for any digest. Continue reading “What’s In YOUR Protein? Optimizing Protease Digestions to Get the Inside Scoop”

Of Mice and Microbes: The Science Behind Food Analysis

In general, people like to know that their food is what the label says it is. It’s a real bummer to find out that beef lasagna you just ate was actually horsemeat. Plus, there are many religious, ethical and medical reasons to be cognizant of what you eat. Someone who’s gluten intolerant and Halal probably doesn’t want a bite of that BLT.

Labels don’t always accurately reflect what is in food. So how do we confirm that we are in fact buying crab, and not whitefish with a side of Vibrio contamination?

For the most part, it comes down to separation science. Scientists and technicians use various chromatographic methods, such as gas chromatography, liquid chromatography, and mass spectrometry, to separate the complex mixture of molecules in food into individual components. By first mapping out the molecular profile of reference samples, they can then take an unknown sample and compare its profile to what it should look like. If the two don’t match up, an analyst would assume that the unknown is not what it claims to be. Continue reading “Of Mice and Microbes: The Science Behind Food Analysis”

Tetanus Neurotoxin: Potential Mechanism for Drug Delivery

Tetanus neurotoxin (TeNT), produced by Clostridium tetani, is one of the most potent neurotoxins in humans. TeNT causes tetanus, which is characterized by painful muscular contractions and spasms as well as seizure. TeNT is composed of a light chain and a heavy chain (TTH). The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons.

Based on these properties, a recent publication (1) considered that TTC could be a promising vehicle to deliver drug cargos to neurons. To explore this possibility, they engineered fusion proteins containing various TeNT fragments. They chose B-cell leukemia/lymphoma 2 protein (Bcl-2) as a partner protein, because Bcl-2 is one of the most potent anti-apoptotic proteins and has an appropriate size (26kDa) to act as a fusion partner.

They tested these fusion proteins in both cell-based and cell-free protein expression systems to determine whether the purified fusion products retained both anti-apoptotic and neuronal migration properties. One construct (Bcl2-hTTC) exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems.

  1. Watanbe, Y. et. al. (2018) Tetanus toxin fragments and Bcl-2 fusion proteins : cytoprotection and retrograde axonal migration. BMC Biotechnology 18, 39.

New Recombinant Asp-N Mass Spec Protease: Improved Format and Reduced Price

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.

Beer Is Complicated: Proteome Analysis via Mass Spectrometry

still life with a keg of beer and draft beer by the glass.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 “Beer Is Complicated: Proteome Analysis via Mass Spectrometry”

Mass Spec Analysis of PTMs Using Minimal Sample Material

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.

Reference

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.