PCT MicroTube Adapter Kit for Pressure-Enhanced Enzymatic Proteolysis

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The PCT MicroTube Adapter Kit, in combination with the PCT SPS, can reliably and reproducibly control the enzymatic digestion of proteins while reducing the time of digestion from hours to minutes with the same or better quality as other, currently available techniques.

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PCT MicroTube Adapter Kit for Pressure-Enhanced Enzymatic Proteolysis

  1. 1. Pressure BioSciences, Inc.PCT MicroTubeTM Adapter Kit* forPressure-Enhanced Enzymatic Proteolysis 1 hr Trypsin Digest with PCT 12 hr Trypsin Digest No PCT Pressure Cycling Technology (PCT) Discovery Starts with Sample PreparationTM *Patent Pending
  2. 2. Pressure BioSciences, Inc. Pressure BioSciences, Inc. (NASDAQ: PBIO) is a life sciences company focused on the development and commercialization of a novel, enabling platform technology called Pressure Cycling Technology (“PCT”). BarocyclerPCT Sample Preparation SystemThe Pressure Cycling Technology Sample PreparationSystem (“PCT SPS”) employs rapid cycles of hydrostaticpressure, between ambient and ultra high levels, toprecisely control biomolecular interactions. The PCT SPScan be used to accelerate enzymatic reactions, such asprotein digestion with trypsin and other proteolytic enzymes,to prepare samples for analysis by mass spectrometry. Inaddition, the PCT SPS can be used to disrupt tissues andcells to extract cellular structures and components suchproteins, lipids, nucleic acids, and small molecules forfurther analysis. The PCT SPS is comprised of a Barocycler(a small, semi-automated bench top instrument used togenerate high, hydrostatic pressure) and specially designedsingle-use processing tubes. Temperature can becontrolled during the PCT-based sample preparationprocess by an external circulating water bath, which can bean important addition to the Pressure Cycling TechnologySample Preparation System. “PCT offers clear advantages over our standard method of sample prep, including shorter processing time, higher throughput, and reduced operating costs – and all without sacrificing quality. We found these advantages so significant that we recently incorporated PCT into our standard mass spec proteomics analysis workflow.” Dr. Shane Burgess Director, Life Science and Biotechnology Institute Mississippi State University PCT MicroTube Adapter Kit* Processing time, sample throughput, accuracy, efficiency, and standardization are key elements of the sample preparation process for mass spectrometry. The PCT MicroTube Adapter Kit, in combination with the PCT SPS, can reliably and reproducibly control the enzymatic digestion of proteins while reducing the time of digestion from hours to minutes with the same or better quality as other, currently available techniques. The PCT MicroTube Adapter Kit comes complete with an ergonomically designed, space-saving Work Station containing PCT MicroTubes and PCT MicroCaps, as well as tools and hardware, to enable the user to process from one to forty eight samples simultaneously in the PCT SPS. * Patent Pending PCT MicroTube Adapter Kit
  3. 3. PCT MicroTube* The PCT MicroTube was designed to meet the critical requirements of modern proteomic analysis by mass spectrometry. It is made from a fluoropolymer, fluorinated ethylene propylene (FEP). This polymer was selected because of its unique features. FEP is highly inert and retains its integrity within an extremely wide temperature range (-200oC to +100oC). The PCT MicroTube’s outstanding chemical resistance, non-wetting surface, and negligible protein and nucleic acid adsorption, help to ensure nearly complete sample recovery, features that are essential for high pressure-enhanced enzymatic proteolysis. * Patent Pending PCT MicroCap* PCT MicroCaps were also designed to meet the critical requirements of modern proteomic analysis by mass spectrometry. They are made from polytetraflouroethylene PCT M icroCaps (PTFE), a polymer selected for features that are ideal for high pressure applications. The PCT MicroCap is Indent for available in three designs, designated 50, 100, or 150. Picking G el Plug Each MicroCap design designates an approximate reaction volume. By selecting a particular MicroCap type, samples may be digested in reaction volumes of approximately 50, 100 and 150 mL. In addition, all PCT MicroCaps have the100 µ L added unique feature of being able to be used as gel spot pickers to excise and transport small protein spots from a 150 µ L gel. The PCT MicroCap holds the gel protein spot safely within the cap, while it is transferred to the PCT MicroTube, 50 µ L ready for processing by PCT. * Patent Pending“Incorporating PCT into our standard mass spec workflow has resulted in several significant advantages,including an improved number of unique peptides identified and a reduction in undesired proteinmodifications…and now with the new PCT MicroTube Adapter Kit, throughput will no longer be a bottleneck.”Dr. Paul PevsnerNew York University School of Medicine Suggested PCT Workflow
  4. 4. “PCT results in identification of more unique peptides than our standard sample preparation technique for mass spectrometry. It appears that PCT may also be highly successful in digesting and recovery of very complex samples, an area of great difficulty at the present time. These advantages, coupled with the unique characteristics of the recently designed PCT MicroTubes, offer researchers working in mass spectrometry-based proteomics a wonderful new tool for scientific discovery.” Dr. Alexander R. Ivanov Harvard School of Public HealthFigure 1. Quantitative Profiling of Peptide Abundance for In-SolutionDigested Model Proteins. Selected examples illustrate superiordigestion efficiency and peptide recovery using PCT.Data provided by Dr. Alexander Ivanov, Harvard School of Public Health Figure 2. PCT significantly increases digestion efficiency and peptide recovery following in-solution digestion. Digestion was performed using an array of conditions, as indicated. LC-MS was performed on C-18, 10 cm x 150 µm (VC-10-C18WS-150EU) column using a 30 minute 5-60% gradient (A: 0.1% formic acid; B: acetonitrile, 0.1% formic acid) Peptides were detected on an LTQ-Orbitrap XL mass spectrometer with ETD ionization source; Alternating ETD/CID spectra were collected and the data were analyzed using Proteome discoverer software with 1% FDR data filtering to determine sequence coverage and the total number of peptides. Peak areas for selected peptides were measured using LCquan package for each LC/MS run (9 runs per digest condition). Values were normalized to the average area obtained for each peptide (n=72). Median values from each set of 9 LC/MS runs for each peptide and each condition are presented. Data provided by Dr. Roger Biringer, Thermo Fischer Scientific
  5. 5. Figure 3. PCT-assisted digestion has been shown to increase efficiency and peptide recovery from gel-separated proteins. New consumable containers offer features which facilitate gel spot picking using a combination of disposable gel cutter tool combined with the tube closure. Data provided by Dr. Michail Alterman, CBER, FDA. “We believe that this new patent pending sample containment and processing system, combined with the unique power of PCT, gives the researcher extraordinary ability to gain new knowledge using today’s analytical identification techniques, such as mass spectrometry. Based on the results from our beta site testers, we believe that our PCT MicroTube Adapter Kit can benefit a large segment of the proteomics and genomics research community.” Dr. Edmund Ting Sr. VP of Engineering Pressure BioSciences, Inc Features and Benefits of the PCT SPS and PCT MicroTubes • Standardize Protein Digestion for Mass Spectrometry • Substantially Increase Protein Digestion Efficiency • Significantly Accelerate In-Solution and In-Gel Protein Digestion • Promote Accurate & Efficient Peptide Cleavage • Avoid Undesired Protein Modifications Due to Prolonged Digestion Time • Process Up to 48 Samples at a Time • Versatile Range of Sample Sizes (50-150 µL) • Compatible with a Variety of Proteolytic Enzymes • Inert, Non-Wetting, Single-Use MicroTube Processing Containers • Unique MicroCap Design for Protein Spot Transfer from Acrylamide Gels.
  6. 6. ReferencesPapersSchumacher R.T., et al., Automated Solution for Sample Preparation: Nucleic Acid and Protein Extraction from Cells and Tissues UsingPressure Cycling Technology (PCT). Am. Laboratory. 2002, 34, 38-43López-Ferrer D, et al., Application of pressurized solvents for ultra fast proteolysis: Proteomics on the fly. J Proteome Res. 2008;7(8).Gross V., et al., Tissue Fractionation by Hydrostatic Pressure Cycling Technology:The Unified Sample Preparation Technique for Systems Biology Studies. J Biomol Tech. 2008, 19(3)PostersA Comparison of Methods for Efficient Digestion of Protein TherapeuticsLorna L. Maheu, Heather M. Connelly, Adam G. Harder, and Steven L. CockrillAnalytical Sciences, Amgen Inc., Longmont, CO 80503 thPoster presented at the 56 ASMS Conference, 2008High Pressure Trypsin Digestion of Proteins for Proteomic AnalysisShane A. Wyatt; Timothy R. CroleyCommonwealth of Virginia, Richmond, VAPoster presented at the 56th ASMS Conference, 2008Application of Pressurized Solvents for Ultra Fast Proteolysis: Proteomics on the FlyDaniel Lopez- Ferrer; Konstantinos Petritis; Natacha M Lourette; Brian H. Clowers; Kim K. Hixson; Tyler H Heibeck; Eric A. Livesay; RyanKelly; David Prior; Ljiljana Pasa-tolic; David G. Camp; Mikhail Belov; Richard D. Smith Pacific Northwest National Laboratory, Richland, WAPoster presented at the 56th ASMS Conference, 2008IMAGING MALDI of Colorectal Carcinoma-Field Defects in Satellite TissueTiffany Remsen, M. Momeni, P. Kessler, F. Francois, A. Stern, S. Anand, and P. PevsnerNew York University School of MedicinePoster presented Biomarker World Congress, 2009Searching for efficient and high-throughput alternatives for essential sample preparation techniques in mass spectrometry-basedfunctional proteomicsEmily Freeman; Yelena Margolin; Alexander R. IvanovHarvard School of Public Health, Boston, MAPoster presented at the ABRF Conference, 2009Ultra-Rapid Pressure Digestion and Label-Free Quantitative Proteomics of Yersiniae Infected Mice TissuesKim K. Hixson1; Daniel Lopez Ferrer1; Matthew Bender2; Patricia L. Worsham3; Karl K. Weitz1; Nate Lawrence4; Amy Rasley5; Therese W.Clauss ; Ljiljana Pasa-tolic1; Richard D. Smith1; Mary S. Lipton11Pacific Northwest National Lab, Richland, WA; 2NBACC, Washington, DC; 13 USAMRIID, Frederick, MD; 4Pressure Biosciences, Inc., South Easton, MA; 5Lawrence Livermore National Lab, Livermore, CA thPoster presented at the 57 ASMS Conference, 2009Improved Protein Coverage and Throughput in Proteomics Using On-Line Multiplexed Enzyme Digestions and Targeted MS/MSwith a Modified LTQ-FTICRDaniel Lopez Ferrer1; Konstantinos Petritis1; Andrei Liyu1; Yufeng Shen1; Benito Canas2; Kim K. Hixson1; Richard D. Smith1; Mikhail Belov1Poster presented at the 57th ASMS Conference, 2009Application of pressurized solvents for ultra fast proteolysis: Proteomics on the fly.1 PNNL / Battelle Northwest, Richland, WA; 2Universidad Complutense de Madrid, Madrid, SpainPoster presented at the 57th ASMS Conference, 2009High-pressure assisted in-gel tryptic digest: qualitative and quantitative aspects 1 1 2 2Michail Alterman ; Melkamu Getie-Kebtie ; Alexander Lazarev ; Vera S. Gross1 FDA, CBER, Rockville, MD; 2Pressure Biosciences, Inc, Woburn, MA; thPoster presented at the 57 ASMS Conference, 2009Proteomics under pressure: Rapid extraction and digestion in a single tube 2 1 1 1 2 thAlexander Lazarev; Emily Freeman ; Vera S. Gross ; Greta Carlson ; Edmund Ting ; Alexander R. Ivanov Poster presented at the 57ASMS Conference, 2009 For more information please visit Pressure BioSciences website: www.pressurebiosciences.com For Research Use Only Not for Use in Diagnostic Procedures Barocycler®, PULSE ®Tubes, PCT MicroTubesTM, PCT MicroCapsTM , ProteoSolveTM and The PCT Shredder KitTM are trademarks of Pressure BioSciences, Inc. 14 Norfolk Avenue South Easton, MA 02375 PH:508-230-1828 Fax:508-230-1829 www.pressurebiosciences.com

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