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.
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.
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.
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.
To shorten the time required to prepare samples for LC-MS/MS analysis, we have developed a specialized trypsin preparation that supports rapid and efficient digestion at temperatures as high as 70°C. There are several benefits to this approach. First, proteolytic reaction times are dramatically shortened. Second, because no chemical denaturants have been added, off -line sample cleanup is not necessary, leading to shorter preparation times and diminished sample losses.
The Rapid Digestion trypsin protocols are highly flexible. They can accommodate a variety of additives including reducing and alkylating agents. There are no restrictions on sample volume or substrate concentrations with these kits. Furthermore, the protocol is simple to follow and requires no laboratory equipment beyond a heat block. Digestion is achieved completely using an in-solution approach, and since the enzyme is not immobilized on beads, the protocol does not have strict requirements for rapid shaking and off -line filtering to remove beads.
In addition to the benefits of this flexibility, we also developed a Rapid Digestion–Trypsin/Lys-C mixture. Like the Trypsin/Lys-C Mix previously developed to prepare maximally efficiently proteolytic digests, particularly for complex mixtures, Rapid Digestion–Trypsin/Lys C is ideally suited for studies that require improved reproducibility across samples.
Brachylophosaurus was a mid-sized member of the hadrosaurid family of dinosaurs living about 78 million years ago, and is known from several skeletons and bonebed material from the Judith River Formation of Montana and the Oldman Formation of Alberta. Recent fossil evidence indicates structures similar to blood vessels in location and morphology, have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty network after degradation of original organic components (i.e., bacterial, slime mold or fungal in origin). Cleland et al. (1) tested the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resolution mass spectrometry and confirmed by immunofluorescence. Continue reading “Dino Protein: New Methods for Old (Very) Samples”
Asp-N, Sequencing Grade, is an endoproteinase that hydrolyzes peptide bonds on the N-terminal side of aspartic and cysteic acid residues: Asp and Cys. Asp-N activity is optimal in the pH range of 4.0–9.0. This sequencing grade enzyme can be used alone or in combination with trypsin or other proteases to produce protein digests for peptide mapping applications or protein identification by peptide mass fingerprinting or MS/MS spectral matching. It is suitable for in-solution or in-gel digestion reactions.
The following references illustrate the use of Asp-N in recent publications:
Protein sequence coverage
Jakobsson, M et al. (2013) Identification and characterization of a novel Human Methyltransferase modulating Hsp70 protein function through lysine methylation. J. Biol. Chem. 288, 27752–63.
Carroll, J. et. al. (2013) Post-translational modifications near the quinone binding site of mammalian complex I. J. Biol. Chem. 288, 24799–08.
Siguier, B. et al. (2014) First structural insights into α-L-Arabinofuranosidases from the two GH62 Glycoside hydrolase subfamilies. J. Biol. Chem. 289, 5261–73.
Vakhrushev, S. et al. (2013) Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells. Mol. Cell. Prot.12, 932–44.
Berk, J. et al. (2013) . O-Linked β-N- Acetylglucosamine (O-GlcNAc) Regulates emerin binding to autointegration Factor (BAF) in a chromatin and Lamin B-enriched “Niche”. J. Biol. Chem. 288, 30192–09.
Roux, P. and Thibault, P. (2013) The Coming of Age of phosphoproteomics –from Large Data sets to Inference of protein Functions. Mol. Cell. Prot.12, 3453–64.
The novel mass spectrometry compatible surfactant sulfonate-(sodium 3-((1-(furan-2-yl)undecyloxy) carbonylamino)-propane-1-sulfonate (i.e.ProteaseMAX) facilitates both in-gel and in-solution digestion applications by reducing the time required, enabling protein solubilization/denaturation and increasing peptide/protein identifications.
A new application was highlighted in a recent publication (1) which utilized ProteaseMAX to lyse cells prior to trypsin digestion and subsequent mass spec analysis. The composition of the buffer determines the overall efficiency of cell lysis, dissociation of protein complexes, protein solubility and ease of removal prior to LC/MS-MS analysis.
When compared to lysis buffers containing either urea or SDC, ProteaseMAX provided the optimal number of identified peptides/proteins.
In addition it can be easily removed from the lysate by acidic precipitation.
Pirmoradian, M. et al. (2013). Rapid and deep human proteome analysis by single-dimension shotgun proteomics. Mol. Cell. Prot.12, 3330–8.
In-gel digestion complete in only 1 hour.[/caption]Identification of proteins resolved by SDS-PAGE requires a lengthy in-gel digestion and extraction procedure. The publication, Mass Spectrometry Compatible Surfactant for the Optimized In-Gel Protein Digestion, Saveliev, S. et al. (2013) Anal. Chem.85, 907–14, addressed these obstacles by illustrating the technical benefits of the ProteaseMAX™ mass spectrometry surfactant. A recent webinar reviewed key aspects of that paper that included:
Improved peptide recovery from gels: ProteaseMAX™ improved identification of proteins by enhanced protein digestion, increased peptide extraction and minimized postdigestion peptide loss. This is a major benefit for the in-gel digestion of low abundant proteins and enables the use of minimal sample material.
The webinar also contained information regarding the how the structure of ProteaseMAX™ Surfactant enables it to compatible with mass spec and how it can be used for protein denaturation and solubilization.
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Efficient proteolysis is a major requirement for protein mass spectrometry analysis. Incomplete digestion has multiple ramifications including decreased number of identified proteins, compromised analytical reproducibility and protein quantitation, etc. Trypsin is one of the most robust proteases and is characterized by efficient proteolysis. Typical trypsin reactions do not digest proteins to completion, missing 15–30% of cleavage sites. Incomplete digestion affects protein identification, reproducibility of mass spectrometry analysis and accuracy of protein quantitation. Supplementing Trypsin with Lys-C compensates for the majority of missed cleavages. Continue reading “Enhanced Protein Mass Spectrometry Analysis with Trypsin/Lys-C Mix”