PCT MicroTube TM Adapter Kit*

Pressure BioSciences, Inc.

for

Pressure-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

PCT Sample Preparation System The Pressure Cycling Technology Sample Preparation System (“PCT SPS”) employs rapid cycles of hydrostatic pressure, between ambient and ultra high levels, to precisely control biomolecular interactions. The PCT SPS can be used to accelerate enzymatic reactions, such as protein digestion with trypsin and other proteolytic enzymes, to prepare samples for analysis by mass spectrometry. In addition, the PCT SPS can be used to disrupt tissues and cells to extract cellular structures and components such proteins, lipids, nucleic acids, and small molecules for further analysis. The PCT SPS is comprised of a Barocycler (a small, semi-automated bench top instrument used to generate high, hydrostatic pressure) and specially designed single-use processing tubes. Temperature can be controlled during the PCT-based sample preparation process by an external circulating water bath, which can be an important addition to the Pressure Cycling Technology Sample Preparation System.

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”).

“PCT offers clear advantages over our standard meth od of sample prep, including shorter processing tim e, higher throughput, and reduced operating costs – an d all without sacrificing quality. We found these advantages so significant that we recently incorporated PCT in to our standard mass spec proteomics analysis workf low.”

Dr. Shane Burgess Director, Life Science and Biotechnology Institute Mississippi State University

Barocycler

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

PCT MicroCap* PCT MicroCaps were also designed to meet the critical requirements of modern proteomic analysis by mass spectrometry. They are made from polytetraflouroethylene (PTFE), a polymer selected for features that are ideal for high pressure applications. The PCT MicroCap is available in three designs, designated 50, 100, or 150. 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 the added unique feature of being able to be used as gel spot pickers to excise and transport small protein spots from a gel. The PCT MicroCap holds the gel protein spot safely within the cap, while it is transferred to the PCT MicroTube, ready for processing by PCT. * Patent Pending

PCT MicroCaps

100 µµµµL

50 µµµµL

150 µµµµL

Indent for Picking Gel Plug

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

“Incorporating PCT into our standard mass spec work flow has resulted in several significant advantages , including an improved number of unique peptides ide ntified and a reduction in undesired protein modifications…and now with the new PCT MicroTube Ad apter Kit, throughput will no longer be a bottlenec k.”

Dr. Paul Pevsner New York University School of Medicine

Suggested PCT Workflow

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

“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 Health

Figure 1 . Quantitative Profiling of Peptide Abundance for In-Solution Digested Model Proteins. Selected examples illustrate superior digestion efficiency and peptide recovery using PCT. Data provided by Dr. Alexander Ivanov, Harvard School of Public Health

.

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.

Features and Benefits of the PCT SPS and PCT MicroT ubes • 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

“We believe that this new patent pending sample con tainment and processing system, combined with the u nique power of PCT, gives the researcher extraordinary ab ility to gain new knowledge using today’s analytica l identification techniques, such as mass spectrometr y. Based on the results from our beta site testers , we believe that our PCT MicroTube Adapter Kit can benefit a la rge segment of the proteomics and genomics research community.”

Dr. Edmund Ting Sr. VP of Engineering Pressure BioSciences, Inc

For more information please visit Pressure BioSciences website: www.pressurebiosciences.com

References Papers Schumacher R.T., et al., Automated Solution for Sample Preparation: Nucleic Acid and Protein Extraction from Cells and Tissues Using Pressure Cycling Technology (PCT). Am. Laboratory. 2002, 34, 38-43 López-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) Posters A Comparison of Methods for Efficient Digestion of Protein Therapeutics Lorna L. Maheu, Heather M. Connelly, Adam G. Harder, and Steven L. Cockrill Analytical Sciences, Amgen Inc., Longmont, CO 80503 Poster presented at the 56th ASMS Conference, 2008 High Pressure Trypsin Digestion of Proteins for Pro teomic Analysis Shane A. Wyatt; Timothy R. Croley Commonwealth of Virginia, Richmond, VA Poster presented at the 56th ASMS Conference, 2008 Application of Pressurized Solvents for Ultra Fast Proteolysis: Proteomics on the Fly Daniel Lopez- Ferrer; Konstantinos Petritis; Natacha M Lourette; Brian H. Clowers; Kim K. Hixson; Tyler H Heibeck; Eric A. Livesay; Ryan Kelly; David Prior; Ljiljana Pasa-tolic; David G. Camp; Mikhail Belov; Richard D. Smith Pacific Northwest National Laboratory, Richland, WA Poster presented at the 56th ASMS Conference, 2008 IMAGING MALDI of Colorectal Carcinoma-Field Defects in Satellite Tissue Tiffany Remsen, M. Momeni, P. Kessler, F. Francois, A. Stern, S. Anand, and P. Pevsner New York University School of Medicine Poster presented Biomarker World Congress, 2009 Searching for efficient and high-throughput alterna tives for essential sample preparation techniques i n mass spectrometry-based functional proteomics Emily Freeman; Yelena Margolin; Alexander R. Ivanov Harvard School of Public Health, Boston, MA Poster presented at the ABRF Conference, 2009 Ultra-Rapid Pressure Digestion and Label-Free Quant itative Proteomics of Yersiniae Infected Mice Tissu es Kim K. Hixson1; Daniel Lopez Ferrer1; Matthew Bender2; Patricia L. Worsham3; Karl K. Weitz1; Nate Lawrence4; Amy Rasley5; Therese W. Clauss1; Ljiljana Pasa-tolic1; Richard D. Smith1; Mary S. Lipton11Pacific Northwest National Lab, Richland, WA; 2NBACC, Washington, DC; 3USAMRIID, Frederick, MD; 4Pressure Biosciences, Inc., South Easton, MA; 5Lawrence Livermore National Lab, Livermore, CA Poster presented at the 57th ASMS Conference, 2009

Improved Protein Coverage and Throughput in Proteom ics Using On-Line Multiplexed Enzyme Digestions and Targeted MS/MS with a Modified LTQ-FTICR Daniel Lopez Ferrer1; Konstantinos Petritis1; Andrei Liyu1; Yufeng Shen1; Benito Canas2; Kim K. Hixson1; Richard D. Smith1; Mikhail Belov1

Poster presented at the 57th ASMS Conference, 2009 Application of pressurized solvents for ultra fast proteolysis: Proteomics on the fly . 1PNNL / Battelle Northwest, Richland, WA; 2Universidad Complutense de Madrid, Madrid, Spain Poster presented at the 57th ASMS Conference, 2009 High-pressure assisted in-gel tryptic digest: quali tative and quantitative aspects Michail Alterman1; Melkamu Getie-Kebtie1; Alexander Lazarev2; Vera S. Gross2

1FDA, CBER, Rockville, MD; 2Pressure Biosciences, Inc, Woburn, MA; Poster presented at the 57th ASMS Conference, 2009 Proteomics under pressure: Rapid extraction and dig estion in a single tube Alexander Lazarev; Emily Freeman2; Vera S. Gross1; Greta Carlson1; Edmund Ting1; Alexander R. Ivanov2 Poster presented at the 57th ASMS Conference, 2009

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